|UFDC Home||myUFDC Home | Help|
This item has the following downloads:
COMPARISON OF THE EFFECTS OF A PROCESSING SEQUENCE AND A
NUCLEAR EXPORT ELEMENT ON RIBOZYME ACTIVITY IN TRANSFECTED
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
I would like to thank Dr. Alfred Lewin for giving me the opportunity to join in his
laboratory and for his advice, support, and trust during these projects. I would like to
thank Dr. William Hauswirth for serving on my supervisory committee and for all of his
guidance during completion of this thesis. I would like to thank Dr. Paul Oh for serving
on my supervisory committee and for being an excellent mentor and confidant. I would
especially like to thank my family in Korea, my mom and younger sister, as well as my
mentors, Dr. Chang-Won Kang, Dr. Hoon-Taek Lee, Dr. Tae-Young Jung, Dr. Kil-Saeng
Jung, and Dr. Hong-Yang Park, and friends here and Korea for their continuous support,
cheer, and love over the years as I have pursued my dreams. I would especially like to
thank my boyfriend, Koo Yung Jung, for his encouragement and love during my study. I
would also like to thank all of my fellow laboratory colleagues for the advice, support,
knowledge, and wisdom that they have bestowed upon me throughout the time that I have
been in the master's program, especially Dr. Marina Gorbatyuk and James Thomas, who
supervised most of the work presented in this thesis.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S .................................................................................................. ii
LIST OF FIGURES ................................... ...... ... ................. .v
ABSTRACT .............. ..................... .......... .............. vii
1 INTRODUCTION ............... ................. ........... ................. ... ..... 1
2 M ATERIALS AND M ETHOD S ........................................ ......................... 15
ET-208 D design ........................................ ............. ........ .. ..............15
Rz448-NHP-OHP, Rz448-ET208-OHP, and Rz448-OHP Constructs.......................15
G el Purification of O ligonucleotides................................................................ 16
Annealing Phosphorylated Oligonucleotides .................. ..............................16
Digestion of the Circular AAV Packaging Vector with Proper Restriction
Enzymes ............................. ..... .................. 17
Gel Purification of Vector DNA ...................................... ............ ............... 17
Ligating Annealed DNA Oligonucleotide Insert and Vector DNA ..................17
T4 Polym erase Treatm ent......................................................... ............... 18
Bacterial Transform nations ........................ .................... ................... 18
Large-Scale Cesium Chloride (CsC1) DNA Prep ..................................................19
Plasmid Labeling Reaction with CyTM3 and CyTM5 Dyes ......................................21
Co-transfection of HEK 293 Cells with Plasmids Expressing Target and
R ibozym e .............. ..... ........ ..................................................... 21
Cell Preparation ................ ....... .......... ......... 21
Tm asfection .................................................. ........ ......... ........ 22
Harvest of Co-Transfected Cells ................................................... 23
RNA Extraction from Co-Transfected HEK 293 Cells ........................................23
Analysis of Ribozyme Activity by Quantitative Reverse Transcriptase-Polymerase
Chain Reaction (RT-PCR) .............................................................................. 24
3 R E S U L T S .............................................................................2 7
Construction of Rz448-ET208-OHP Packaging Plasmid ....................................27
Construct of Rz448-OHP Packaging Plasmid ............................... .....................28
The Co-Localization of Plasmids Expressing Target and Ribozyme within a Cell
after Co-Transfection of HEK 293 Cells ....... ........... ...........................28
Analysis of Ribozyme Activity by Quantitative RT-PCR ............... .....................28
R results from 1.5% A garose G els.................................... ......................... 29
At a molar ratio of 1:4 of target to ribozyme ................... ....... .........29
At a molar ratio of 1:1 of target to ribozyme ......................... ............30
At a molar ratio of 1:2.2 of target to ribozyme ....................................31
Results from 5% and 8% Polyacrylamide Gels Containing Urea....................31
4 DISCUSSION AND FUTURE STUDIES ...................................... ............... 44
L IST O F R E F E R E N C E S ........................................................................ .....................54
BIO GRAPH ICAL SK ETCH .................................................. ............................... 60
LIST OF FIGURES
1-1 A contemporary view of gene expression........ ............ ....................10
1-2 Model for splicing coupled mRNA export in Metazoans. ............. ..................11
1-3 A model for the regulation of cargo binding and release by Ran ..........................12
1-4 T h e R an cy cle ................................................... ................ 13
1-5 Hairpin and ham m erhead ribozym es................................... ......................... 14
2-1 Map of p21 NewHP packaging plasmid. ............................. ...........25
2-2 Map of Rz448 cloned into p21 NewHP packaging plasmid ...............................25
2-3 Map of ET-208 cloned into p21 NewHP Rz448 packaging plasmid....................26
3-1 The Rz448 activity on the reduction of dog rhodopsin mRNA levels in
transfected cells .................................... ............................... .........34
3-2 Map of Rz448 and ET208 cloned into p21 NewHP packaging plasmid. ...............34
3-3 ET-208 PCR product from Rz448-ET208-OHP clones 1-6. NC= negative
control ................ .... ....... ............... ............................35
3-4 Smal digest of Rz448-ET208-OHP clones 1-5 demonstrating presence of AAV
IT R s ............................................................................... 35
3-5 PCR amplification to confirm absence of ET-208 in Rz4448-OHP clones.............36
3-6 Plasmids containing ribozyme (Rz448-NHP-OHP, Rz448-ET208-OHP, and
Rz448-OHP) were labeled with CyTM3 (Red), while plasmids coding for target
w ere treated w ith CyTM 5 (Blue) ........................................ ......................... 36
3-7 1:4 ratio of target to Rz separated on agarose gels......... ... ............ ............... 37
3-8 Ratio of rhodopsin to beta-actin at a molar ratio of 1:4 (target: ribozyme) ............38
3-9 1:1 ratio of target to Rz separated on agarose gels............. ............ ............... 39
3-10 Ratio of rhodopsin to beta-actin at a molar ratio of 1:1 (target: ribozyme) ............40
3-11 Ratio of rhodopsin to beta-actin at a molar ratio of 1:2.2 (target: ribozyme) ..........40
3-12 Picture of polyacrylamide gel containing urea stained with SYBR Green I and
scanned by Im ageQ uant ................................................ ............................... 41
3-13 Ratio of rhodopsin to beta-actin at a molar ratio of 1:4 (target: ribozyme) ............41
3-14 Ratio of rhodopsin to beta-actin at a molar ratio of 1:1 (target: ribozyme) .............42
3-15 Ratio of rhodopsin to beta-actin at a molar ratio of 1:2.2 (target: ribozyme) ..........42
3-16 Picture of polyacrylamide gels containing urea stained with SYBR Green I and
scanned by ImageQuant at a molar ratio of 1:2.2 (target: ribozyme) ....................43
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
COMPARISON OF THE EFFECTS OF A PROCESSING SEQUENCE AND A
NUCLEAR EXPORT ELEMENT ON RIBOZYME ACTIVITY IN TRANSFECTED
Chair: Alfred S. Lewin
Major Department: Molecular Genetics and Microbiology
Ribozyme-mediated gene therapy is an anti-sense approach to inactivate gene
expression in order to reduce the accumulation of mutated proteins that cause diseases.
Rhodopsin-linked autosomal dominant retinitis pigmentosa (ADRP) has been an optimal
objective for ribozyme gene therapy for several reasons: 1) it is a slowly progressive
disease, 2) it is easy to access the target tissue, and 3) the eye is an immunologically
privileged site. Retinitis pigmentosa (RP) is a term that refers to a group of inherited
disorders in which abnormalities of the photoreceptor cells (rods and cones) of the retina
lead to blindness in approximately 1 in 3500 people in the U.S. In order for ribozyme
therapy to be effective, it is critical that the ribozyme is expressed at high levels.
Since efficient export of mRNAs from the nucleus into the cytoplasm plays an
important role in their expression, the nuclear export element (NEE) can be beneficial to
the field of ribozyme-mediated gene therapy. Transgene (i.e., ribozyme) expression can
be regulated by both the transcriptional regulatory elements including the promoter,
enhancer, intron, and poly(A) sequence and the post-transcriptional regulatory elements
such as splice signal (SS), constitutive RNA transport element (CTE), and the woodchuck
hepatitis virus post-transcriptional regulatory element (WPRE).
The results of one of the studies being performed in our laboratory show that
recombinant adeno-associated virus (rAAV)-delivered dog rod opsin-specific
hammerhead ribozyme 448 (DogRhoRz448), which is an allele-independently designed
ribozyme, can effectively reduce the levels of dog rhodopsin mRNA. The plasmid
encoding DogRhoRz448 used in this previous study contains two self-cleaving hairpin
ribozymes (new hairpin ribozyme [NHP] and old hairpin ribozyme [OHP]) as processing
sequences. The function of the hairpin ribozyme is to free the hammerhead ribozyme
448 from the long primary transcript, thereby making Rz448 available to cleave its target.
The matrix (M) protein of vesicular stomatitis virus (VSV) has been proposed to
effectively inhibit Ran-dependent nucleocytoplasmic bidirectional transport of both RNA
and proteins in Xenopus laevis oocytes. By using this inhibitor, the newly selected ET-
RNAs (Exceptional Transport RNAs) were discovered. In this current study, we have
replaced a self-cleaving hairpin ribozyme (NHP) in our Rz448 construct with ET-208,
which is one of the ET-RNAs, to examine whether ET-208 could promote better
expression of Rz448, resulting in greater knock down of dog rhodopsin expression. We
found that ET-208 was able to enhance the ability of the Rz448 plasmid to reduce the
levels of rhodopsin mRNA within co-transfected cells.
Many advances have been made in understanding the steps in the pathway of gene
activation to synthesis of functional proteins. The contemporary view of gene expression
proposes that several processes occurring in the nucleus and the cytoplasm are connected
(1). According to this model, this pathway can be divided into 5 steps: transcription
initiation (the beginning of RNA synthesis), transcription elongation (the extension of
transcript), transcription termination, polyadenylation (the cleavage of RNA and the
addition of a polyadenosine tail to the 3' end of the transcript) and translation (the
synthesis of a protein from RNA). Growing evidence has revealed that these distinct
stages of gene expression are functionally coupled (2-4) (Figure 1-1).
The export of mRNA is coupled to other steps in gene expression involving
splicing (5), transcription, and 3'-end formation (2). For example, both splicing-
dependent recruitment of the mRNA export machinery and exon-junction complex (EJC)
formation are conserved in eukaryotic cell (2) (Figure 1-2). Spliced mRNAs are
assembled into an individual spliced mRNP (ribonucleoprotein) complex that directs the
mRNA for export (5,6). In addition to these export proteins, EJC contains numerous
proteins involved in the cytoplasmic localization of mRNAs (2). EJC is made from that
conserved mRNA export machinery and other components of the spliced mRNP are
specifically recruited to a position 20 nucleotides upstream of exon-exon junctions (7).
Once formed, the entire EJC is exported and then dissociates from the mRNA in the
cytoplasm (8). Splicing is not essential for export but promotes the efficiency and/or
fidelity of the process by increasing the more efficient recruitment of export factors (2)
and generating a specific nucleoprotein complex (5). The mRNA export machinery and
some components are also co-transcriptionally recruited (2). They interact with
nucleoporins of the nuclear pore complex that are required for mRNA export and/or
several mRNA export factors (2). 3'-end formation is thought to be critical in co-
transcriptional loading of the mRNA export machinery onto mRNAs (2).
The nucleus and the cytoplasm are physically and functionally separated by the
nuclear envelope in the eukaryotic cell. Genetic information is stored and transcribed
from DNA to RNA in the nucleus, whereas the transfer of information from RNA to
protein occurs in the cytoplasm (9). Therefore, the export of RNA to the cytoplasm is an
important process. Additionally, the intracellular localizations of RNAs are essential for
their correct processing as well as function (10).
All major classes of RNA (mRNA, 5S rRNA, tRNA, snRNA, and rRNA) exit from
the nucleus to the cytoplasm as RNA-protein complexes (9,11-13) via the nuclear pore
complexes (NPCs) perforating the double-membraned nuclear envelope (14-16) using
energy-dependent (17), carrier-mediated transport systems (18,19). The nuclear pore
complex (125,000 kDa) is composed of 50-100 nucleoporin subunits. The subunits are
arranged to produce a large central channel of 28nm and eight smaller peripheral
channels of 9nm. The larger channel is used for active nucleocytoplasmic transport of
large particles such as RNA-containing proteins and the smaller channels provide routes
for passive diffusion of small molecules such as ions and metabolites (20,21).
The distribution of RNA between the nucleus and the cytoplasm results from the
interactions between RNAs and nuclear export elements (NEEs) or nuclear retention
elements (NREs) as well as specific nuclear transport or localization factors (9,10,18,22-
25). The export of RNA occurs in 3 steps. First, the RNA is transported to the nuclear
envelope along the nuclear matrix and docked to specific nucleoporins at the nuclear
entrance of the nuclear pore complex (15,18,26). Second, it is translocated through the
central channel of the nuclear pore complex (27), and third it is transported along the
cytoskeletal matrix (14). In addition, the Ran system is required for export of many
RNAs dependent on Ran-GTPase and its associated binding, exchange, and activation
factors (9,13,24,28,29) (Figure 1-3). The major guanine-nucleotide-exchange factor of
Ran is the nuclear protein RCC 1, whereas its main GTPase-activating protein, RanGAP1,
is almost exclusively found in the cytoplasm (9) (Figure 1-4). This asymmetric
distribution offers an attractive model to explain how direction could be achieved in
nuclear transport (9). Binding of exportins to mRNAs is dependent on the presence of
Ran-GTP. Export cargo release is accomplished by RanGTP hydrolysis, which is
triggered in the cytoplasmic compartment by the RanGAP protein and additional factors
(9). The Ran system is also needed for import of RNA export factors that must shuttle
between the nucleus and the cytoplasm (18,30,31). Thus, mutation or inhibition of this
system leads to obstruction of the export of most RNAs.
Previous experiments have suggested that the matrix (M) protein of vesicular
stomatitis virus (VSV) effectively inhibits Ran-dependent nucleocytoplasmic
bidirectional transport of both RNA and proteins in Xenopus laevis oocytes (32) when it
is in the nucleus and associated with the nuclear pore complexes (33). James E.
Dahlberg's group used this inhibitor to select novel RNA sequences that overcome this
block. Unlike typical transport of most RNAs, the newly selected ET-RNAs (exceptional
transport RNAs) are exported efficiently even when the Ran system and related processes
are disrupted (10). It means that export of these RNAs does not require protein factors
and export pathways thought to be essential for export of most RNAs. The ET-RNAs
were selected from a collection of RNAs containing 20 random nucleotides. Those that
served as nuclear export elements fall into one of three sequences (10). In this study, we
used ET-208, the strongest exporter of the selected ET-RNAs, in an attempt to enhance
the expression of a ribozyme previously tested for gene therapy.
Ribozymes are RNA molecules with enzymatic activities such as sequence-specific
cleavage, ligation, and polymerization of nucleotides (34). Naturally occurring
ribozymes have been grouped into several classes (34). Two of the more characterized
ribozymes, which are from the group of self-cleaving viral agents, are the hairpin and
hammerhead that naturally function as self-cleaving agents in viral replication (34,35).
Their substrate specificity and small size provide therapeutic benefits for ribozyme-
mediated gene therapies (34,36). Both ribozymes catalyze sequence-specific cleavage of
RNA that results in products with 2', 3'-cyclic phosphate and 5'-hydroxyl termini
(34,37). The hairpin ribozyme consists of a 34 nucleotide catalytic core, comprised of
stems A and B that include all the nucleotides essential for hairpin ribozyme function
(37), and four helices, of which helices I and II are responsible for target recognition (34)
(Figure 1-5). The hammerhead ribozyme has a catalytic core of 11 nucleotides (37,38)
that is stabilized by a hairpin structure and is flanked by two helices that are responsible
for substrate recognition (34) (Figure 1-5). Both hairpin and hammerhead ribozymes can
be designed to recognize substrates having a disease-causing point mutation (34,35). The
hairpin ribozymes target gene transcripts containing a quintuple sequence 5'YNGUC3',
where N is any nucleotide and Y is a pyrimidine, while hammerhead ribozymes
recognize less restricted sequences of substrate RNA containing a 5'NUX3' triplet, where
X is any nucleotide except guanosine (34-36).
Ribozyme-mediated therapy is an anti-sense approach to inactivate gene expression
in order to reduce the accumulation of mutated proteins that cause diseases such as
rhodopsin-linked autosomal dominant retinitis pigmentosa (ADRP). Ribozymes have
also been tested to block replication of RNA viruses or retroviruses and to inactivate
dormant oncogenes such as ras or bcr-abl (36). There have been two kinds of therapeutic
ribozyme design. One is that a ribozyme specifically targets and cleaves an mRNA
substrate containing the nucleotide sequence of a point mutation, but fails to cleave an
mRNA substrate containing the wild-type nucleotide sequence (allele-specific ribozymes)
(35). The other is that a ribozyme targets the defective mRNA at site that is not altered
by mutation (allele-independent ribozymes) (35). Such ribozymes could reduce the
expression of both toxic (mutant) proteins and normal homologues encoded by the
partner chromosome. This allele-independent approach is used as part of an RNA
replacement strategy in which the ribozyme is accompanied by a ribozyme resistant
mRNA encoding the wild-type protein.
Retinitis pigmentosa (RP) is a term that refers to a group of inherited disorders in
which abnormalities of the photoreceptor cells (rods and cones) of the retina (39) lead to
blindness in approximately 1 in 3500 people in the U.S. (36). The disease is typically
detected in the second decade of life as night blindness and loss of peripheral vision. The
retinal degeneration is usually slowly progressive and may take decades before leading to
legal blindness. Mutations affect the rod cells specifically, but later in the course of the
disease, cone cells also undergo apoptosis and central vision is lost. Retinitis pigmentosa
is caused by one of three types of a genetic defect: autosomal dominant inheritance,
autosomal recessive inheritance, and X-linked inheritance. About 25% of RP is
dominantly inherited (36). One of the causes of ADRP is a dominant negative mutation
in rhodopsin where a histidine is substituted for a proline at codon 23 (P23H) in
rhodopsin gene (39,40). This leads to the accumulation of abnormal rhodopsin proteins
that ultimately results in the apoptotic death of photoreceptor cells (39) causing loss of
vision (34,36). While P23H rhodopsin is most prevalent in North America, over 100
disease-causing mutations in rhodopsin lead to ADRP. In addition, mutations in over 25
other genes can lead to retinitis pigmentosa (40).
One of the studies being conducted in our laboratory is to examine the therapeutic
potential of recombinant adeno-associated virus (rAAV)-delivered hammerhead
ribozymes targeting retinal mRNAs associated with ADRP. It is our hypothesis that
ribozymes can diminish the production of mutated rhodopsin protein by selectively
cleaving mRNA molecules encoding the aberrant forms of the proteins (34,36). The
effectiveness of ribozyme-mediated reduction of mutant mRNAs that cause ADRP has
been demonstrated in vivo in a P23H transgenic rat model. Eyes of rats that were
injected with P23H specific ribozymes showed significant reduced rate of photoreceptor
degeneration and vision loss (36,41,42).
In order for ribozyme therapy to be effective, it is critical that the ribozyme is
expressed at high levels. Since efficient export of mRNAs from the nucleus into the
cytoplasm plays an important role in their expression, the nuclear export element (NEE)
can be beneficial to the field of ribozyme-mediated gene therapy.
The results of another study being preformed in our laboratory have shown that dog
rod opsin-specific hammerhead ribozyme 448 (DogRhoRz448) can effectively reduce the
levels of dog rhodopsin mRNA. This is a part of an RNA replacement strategy in which
endogenous rhodopsin mRNA would be digested by ribozymes and replaced with a
ribozyme resistant form of wild type mRNA. The ability of DogRhoRz448 to knock-
down dog rod opsin was assessed in tissue cultured cells by co-transfecting HEK 293
cells with target (dog rhodopsin) cDNA and ribozyme 448 inserted in plasmids under the
control of the chicken beta-actin (CBA) promoter coupled with the cytomegalovirus
(CMV) enhancer. Ribozyme activity was assayed by quantitative reverse transcriptase-
polymerase chain reaction (RT-PCR) 48 hours post transfection. At a molar ratio of 1:4
and 1:6 of target to ribozyme, the level of dog rhodopsin mRNA was reduced by
approximately 83% and 94% respectively compared to control.
The significance of the study about the effectiveness of DogRhoRz448 is that dogs
with a point mutation at T4R exhibit a retinal phenotype that closely imitates that in
humans with rhodopsin mutations (43). Naturally occurring hereditary retinal
degeneration in dog, which is caused by this mutation, is referred to progressive retinal
atrophies (PRAs) (43). Therefore, using this nonhuman rhodopsin mutant large animal
provides an inestimable tool to evaluate ribozyme-mediated therapies for ADRP before
beginning of human clinical trials.
Many examples of recombinant proteins whose expression in mammalian cells
requires the presence of an intron (5,44,45). Especially, mRNAs that are transcribed
from cDNAs are expressed poorly compared to the same mRNAs transcribed from a gene
containing introns (5). For this reason, many available vectors contain an intron (5). The
supporting evidence for a link between splicing and efficient mRNA export explains this
intron requirement (5). Insertion of an intron into CMV expression cassettes greatly
promotes gene expression in the cases that have been examined (5,46-48). In the absence
of an intron within expression vectors, polyadenylation might target the mRNA lacking
an intron for efficient export (5).
The plasmid encoding dog rod opsin-specific hammerhead ribozyme 448
previously used in our research contains the ubiquitous chicken beta-actin promoter
coupled with the CMV-enhancer driving high-level and stable expression of the ribozyme
from AAV vector in vivo (49). Immediately downstream of the promoter is an intron,
which should help in the accumulation and localization of ribozyme to cytoplasm (5).
Two self-cleaving hairpin ribozymes are found immediately downstream of the ribozyme
448 which henceforth will be referred to as new hairpin ribozyme (NHP) and old hairpin
ribozyme (OHP). The function of the hairpin ribozyme is to free the hammerhead
ribozyme 448 from the long primary transcript, thereby making ribozyme 448 available
to cleave its target. In addition, the plasmid contains two 145bp inverted terminal repeats
(ITRs) of AAV2 that are absolutely required for packaging DNA into rAAV.
Adeno-associated viruses (AAVs) contain a single-stranded, relatively small
(-4.7kb) genome (50) which enables its manipulation in a plasmid to produce
recombinant AAV in high titers (51). AAV is a good candidate for the ribozyme delivery
with several features: 1) it has been shown to be able to infect a wide range of tissues
including both dividing and non-dividing cells such as photoreceptors, 2) it does not
induce an inflammatory immune response and has not been associated with any diseases
in humans or animals, 3) it has a broad cell tropism, and 4) it can be integrated stably into
the host cell genome or remain stable as an episome n non-dividing cells, leading to long-
term expression of the delivered gene (52,53,54). It has been demonstrated that
recombinant AAV injected subretinally was able to transduce all layers of the neuroretina
as well as cells of the retinal pigment epithelium of adult nude mice (55). rAAV also has
been shown to have no toxicity when injected subretinally into the eyes of rodents (54).
rAAV vectors are deleted of both rep that encodes four regulatory proteins and cap that
encodes three structural proteins genes (56), thus limiting its natural spread because both
wild-type AAV and adenovirus would be required for its propagation (57).
In this current study, we examined whether ET-208 could promote better
expression of ribozyme 448, resulting in greater knock down of dog rod opsin expression.
We have replaced a self-cleaving hairpin ribozyme (NHP) in our dog rod opsin-specific
ribozyme 448 construct with ET-208. We discovered that this element enhanced the
ability of the ribozyme plasmids to reduce accumulation of rhodopsin mRNA.
-l &r PIn loidhM 0
Icc, o ,
z I r--w .-RN-U
ti cnmrary viwn of geei c
1-5 -'f i r"^ M -
this contemporary view of gene expression, each stage is physically and
i~~ ~~~~ cl'Ja^1,-ri~oTAo
Nup2 1 4
V ";'^~-- UAP55
sp IICeoome^ A- -':
Figure 1-2. Model for splicing coupled mRNA export in Metazoans. (A) UAP56 and Aly
associate with the splicesome. Simplified pre-mRNA with a 5' cap, 2 exons
and an intron, and a poly(A) tail, hnRNP proteins package the pre-mRNA, and
SR proteins associate with exons. (B) The Tap-pl5 heterodimer targets the
mRNP to the nuclear pores. Aly acts as bridging protein between the exon-
junction complex (EJC) and Tap-pl5. (C) mRNA export factors dissociate
from the mRNP after export to the cytoplasm. Factors involved in NMD (e.g.
Upf3, Y14, and RNPS1) remain bound to the mRNP (Source: Reference 4).
RaniGTP-hydror*ys and sxplri cargo release
Figure 1-3. A model for the regulation of cargo binding and release by Ran. The
asymmetric distribution of the two different Ran forms can be used to regulate
the binding and release reactions between different carriers and their cargos.
Binging of exportin to their export substrates is dependent on the presence of
Ran-GTP. Cargo release is achieved by RanGTP hydrolysis, which is
triggered in the cytoplasmic compartment by the RanGAP protein and
additional factors (Source: Reference 5).
.... :1 ,^ tr .DP --T
Pi GTPF RGrEF:
Figure 1-4. The Ran cycle. The small nuclear GTPase Ran switches between a GDP- and
a GTP-bound state. Ran GTP/GDP cycle is regulated by Ran-guanine-
Nucleotide-exchange factors (RanGEFs) and by Ran-GTPase-activating
proteins (RanGAPs). The major RanGEF is RCC1, whereas the major
RanGAP is RanGAP1 (Source: Reference 5).
3'A U-ANNNG NNNNNN 5'
G-C II AG
TRENDS in Molecular Medicine
G U S
A. A U
Figure 1-5. Hairpin and hammerhead ribozymes. Schematic diagram of hairpin and
hammerhead ribozymes hybridized to their target sequences. Short bars
represent conventional base pairs, and dots represent non-standard base
pairings. Target sequences are shown in gray shading, and arrows indicate
cleavage sites. Helices are numbered with roman numerals (Source:
MATERIALS AND METHODS
Sense and antisense DNA oligonucleotides coding for ET-208 and including a
region of 20 nucleotides (5' TTGAGGGCCCTCATTGCCGC 3') that served as NEE
were designed with flanking restriction sites, Spel and Nsil and ordered from Invitrogen.
The length of the sense and antisense strands are 45 and 37 nucleotides respectively, and
the 5' ends of both oligonucleotides were phosphorylated.
Rz448-NHP-OHP, Rz448-ET208-OHP, and Rz448-OHP Constructs
The construct, Rz448-NHP-OHP (Figure 2-2) containing dog rod opsin-specific
hammerhead ribozyme 448, was designed by inserting the sequence for Rz448 between
the HindIII and Spel restriction sites in the P21NHP plasmid (Figure 2-1). This plasmid
contains the CMV enhancer coupled with the CBA promoter driving the expression of
the ribozyme, an intron, two self-cleaving hairpin ribozymes (NHP and OHP) that are
found immediately downstream of the RZ448, and two ITRs. The Rz448-ET208-OHP
construct (Figure 2-3) was designed by removing the NHP ribozyme with an Spel and
Nsil digest and inserting the ET-208 sequence between those sites. The Rz448-OHP
construct was made by removing the ET-208 sequence from the Rz448-ET208-OHP
plasmid with an Spel and Nsil digestion. The Spel and Nsil cohesive ends were then
treated with T4 DNA polymerase to blunt the ends and ligated to seal the plasmid.
Gel Purification of Oligonucleotides
The cloning efficiency of Rz448 and ET-208 can be improved by polyacrylamide
gel purification of the oligonucleotides before using them in a ligation reaction. 900
picomoles of each sense and antisense oligonucleotide were mixed with 4ul of formamide
loading dye [90% (w/v) formamide, 50mM EDTA, 0.4% (w/v) xylene cyanol, and 0.4%
(w/v) bromphenol blue] and incubated at 900C for 1 min, and then loaded on the
acrylamide gel. The oligos were separated on a 10% (w/v) acrylamide gel run in IX TBE
buffer [89mM Tris borate, pH 8.3, and 20mM EDTA]. The gel was run at 500-600V and
no more than 40 mA. After the bromphenol blue tracking dye had run about two-thirds
the length of a 20cm gel, the gel was transferred to a 20x20 cm Fluor-coated TLC plate.
In a dark room, a short-wavelength UV hand lamp was used to visualize the DNA band.
The bands were then excised with a scalpel, crushed in 200ul of elution solution [1M
ammonium acetate, 50mM Tris HC1 (pH 7.5), 20mM EDTA, 0.5% (w/v) SDS] in a
sterile 1.5ml microcentrifuge tube, and eluted overnight at 370C. The elution solution
was then removed from the gel slices, and the DNA was ethanol (EtOH) precipitated,
resuspended in 10ul of dH20. Its concentration was determined by the absorbance at
260nm and stored at -200C.
Annealing Phosphorylated Oligonucleotides
In a sterile 1.5ml microcentrifuge tube, the following contents were combined 20ul
of each oligonucleotide (sense and antisense), 10ul of 10X buffer (for restriction enzyme
HindIII from Promega), and dH20 to 100ul. The final volume of reaction solution was
100ul. A reaction tube was then heated to 900C for 5 min and slow-cooled at 370C for
another 10 min. The 10ul of the annealed DNA oligonucleotides, which was diluted in
990ul of dH20 (diluted at 1:100). This dilution was then used for the subsequent cloning
Digestion of the Circular AAV Packaging Vector with Proper Restriction Enzymes
The reaction solution was composed of 1Oul of resuspended circular plasmid, lul of
a desired restriction enzyme (here, HindIII, Spel, and Nsil), 2ul of 10X enzyme buffer,
2ul of 10X BSA, and dH20 to 20ul in a sterile 1.5ml microcentrifuge tube. The reaction
tube was then incubated at 370C for 1 hr. The linearized AAV packaging plasmid DNA
was ethanol precipitated, and then the second digestion was performed. Finally, the
vector plasmid was ready for gel purification.
Gel Purification of Vector DNA
Before ligation, agarose gel purification of the linearized AAV packaging vector
was performed by running the digest on a 1% agarose gel and staining with ethidium
bromide (EtBr) [10mg/ml]. The proper band was then excised after visualization with
low-intensity UV light. The excised band was then crushed in a sterile 1.5 ml
microcentrifuge tube and mixed with an equal volume of phenol (-pH 8.0) to elute the
DNA. The contents were mixed well by vortexing and then incubated at -700C for 1 hour
and centrifuged for 5 minutes at 13,200 rpm. Next, the aqueous phase was extracted with
an equal volume of phenol: chloroform: isoamyl alcohol (50:50:1, v/v/v) to eliminate all
protein contamination. The linearized DNA was precipitated by the addition of two
volumes of 100% EtOH. Finally, the sample is resuspended in lOul of dH20.
Ligating Annealed DNA Oligonucleotide Insert and Vector DNA
For different sized plasmid DNAs, the amount of oligonucleotide and linearized
plasmid vector DNA should be adjusted to maintain an appropriate ratio of
oligonucleotide: vector ends. Here, we used the ratio of annealed oligonucleotide and
linearized vector plasmid DNA that is 3:1. The following components were combined in
a sterile 1.5ml microcentrifuge tube: 3ul of DNA oligonucleotide inserts that had been
phosphorylated and annealed, lul of vector DNA that has been linearized with the proper
enzymes, lul (10units) of T4 DNA ligase, 2ul of 10 ligase buffer [500mM Tris-HCl (pH
7.5), 100mM MgC12] (from BioLabs), and dH20 up to 20ul. The reaction is incubated at
room temperature for 2 hr or 160C overnight. After incubation, the ligated plasmid
DNAs (Rz448-NHP-OHP and Rz448-ET208-OHP) were ethanol precipitated,
resuspended in 10ul of dH2O.
T4 Polymerase Treatment
The function of T4 DNA polymerase is to blunt cohesive ends on double-stranded
DNA molecules with 5'- or 3'- protruding termini by 3' overhang removal and 3'
recessed end fill-in. In a 1.5ml microcentrifuge tube, the following contents were
combined: 10ul of digested DNA, lul of 2mM dNTPs, 2ul of T4 DNA polymerase, 2.5ul
of 10X T4 DNA polymerase buffer, and dH20 to 25ul. A tube was then incubated at
220C for 30min and heated to 700C for 5 min to inactivate T4 DNA polymerase. Rz448-
OHP was than ethanol precipitated and resuspended in 10ul of dH2O.
We use one of several recombination deficient strains ofEscherichia coli, such as
Sure cells (Stratagene), in order to prevent loss of the AAV inverted terminal repeat
(ITR) sequences present in the AAV packaging vector. ITRs are critical for packaging
plasmid inserts as recombinant AAV vectors (34). 2ul of the ligation reaction were used
to transform electro-competent E.coli cells according to Sambrook and Russel (58). 20ul
of Sure cells were mixed with 2ul of the ligated plasmids and electronically charged at
1.5V. Transformed cells were suspended in Iml of autoclaved LB media are incubated in
a thermomixer at 370C shaking at 750 rpm for 1 hr to allow cell growth. Growing cells
were then plated on LB/ ampicilin (Amp) plates for selection of colonies containing the
ligated plasmid DNAs, and grown at 300C overnight to a low density to help preserve
ITRs. After growing overnight, several colonies were picked out and inoculated in 5ml
of LB media with ampicilin followed by another growing at 300C overnight with
shaking. After overnight incubation, increased amounts of three constructs were
extracted by using Perfectprep Plasmid Mini kit from Eppendorf The Eppendorf mini
prep kit was performed according to the manufacturer's recommendations. All clones are
screened for ITR retention by an Smal digest followed by running 0.7% or 0.6% agarose
gels. The plasmids were then sequenced for proper Rz448 and ET208 orientation.
Large-Scale Cesium Chloride (CsCI) DNA Prep
This procedure allows us to increase the amount of the three constructs and to
accomplish a highly purified preparation of DNA for transfection. After sequencing the
plasmids, Sure cells were transformed with these plasmids and spread on and LB/Amp
plate. A colony was inoculated in 5ml of LB/Amp media, and then 2 ml of this
preculture was inoculated and grown in 1L of LB/Amp media at 300C. After incubation
for 14 hours, the culture was transferred to a bottle and centrifuged at 4000 rpm for 10
min to pellet the bacterial cells. The supernatant was decanted, making sure not to
disturb the cell pellet. 30 ml of Tris-EDTA buffer was added to the cell pellet and
completely resuspended. Cell lysis solution [0.2M NaOH, 1% SDS] is then added to the
resuspension and mixed well. This resuspended mixture was incubated for 5 min at room
temperature until the lysate is relatively clear with no visible clumps of cell material.
After the addition of 30 ml of neutralizing solution [3M sodium acetate (NaAc) pH 5.2]
to the lysate, it was mixed well and placed on ice for 10 min. The neutralized lysate was
centrifuged at 10,000 rpm for 15 min and the aqueous phase was poured off through 3
layers cheesecloth. The volume was then determined, and 0.6 volumes of 2-isopropanol
was added. The mixture was incubated on ice for 1 hour. After centrifugation at 10,000
rpm for 30 min, the supernatant was removed. The pellet was gently washed 1 time with
70% EtOH and air dried for 30 min. The DNA pellet was then resuspended in 8 ml of
Tris-EDTA buffer. 8.4 g of CsCl was dissolved in the DNA, and 150ul of ethidium
bromide (EtBr) [10mg/ml] was added to the CsCl-DNA mixture. The DNA was then
transferred to an ultracentrifuge tube, and spun overnight (at least 16 hours) in NVT 65 at
55,000 rpm, 200C, and under vacuum. On the next day, the plasmid DNA band (lower
band) was extracted using a short-wavelength UV hand light to visualize the DNA band
stained with EtBr. Plasmid DNAs were transferred to a 15ml conical tube and the EtBr
was removed from the DNA with a series of isoamyl alcohol extractions. 2.5 volumes of
dH20 were added to DNA, followed by an addition of 2 volumes of 100% ethanol in
20ml Corex tubes. Tubes were incubated on ice for 1 hour. After incubation, plasmid
DNAs were pelleted at 12,000 rpm for 15 minutes at 40C. The supernatant was removed,
the DNA pellet was washed with 70% EtOH, and was dried in a speed vac. The DNA
pellet was resuspended in 400ul of Tris-EDTA buffer, and then transferred to a sterile
microcentrifuge tube. 40ul of neutralizing buffer [3M NaAc pH 5.2] and Iml of 100%
EtOH were added to the DNA and mixed well. The DNA is then incubated at room
temperature for 10-15 minutes. The plasmid DNA was pelleted for 5 min at 13,200 rpm.
The supernatant was decanted and washed with 70% EtOH followed by drying it in a
speed vac. The DNA was resuspended in 400ul of dH20 and its concentration is
determined by absorbance at 260nm. Plasmid DNAs were stored at -200C.
Plasmid Labeling Reaction with CyTM3 and CyT5 Dyes
The use of the CyTM3 and CyTM5 dyes allows us to monitor the intracellular
localization of plasmid DNA in cells following the transfection. According to
manufacture's description, the Label IT Reagents covalently attach marker molecules to
nucleic acids in a chemical reaction. Prepare at least 25% more plasmid DNA in weight
than needed to account for pipetting/transfer errors. In this study, three different
constructs (Rz448-NHP-OHP, Rz448-ET208-OHP, and Rz448-OHP) were labeled with
CyTM3, which gives you red color, and target plasmid DNA encoding dog rhodopsin are
labeled with CyTM5, blue color. In a sterile 1.5ml microcentrifuge tube, the following
components were combined for a 50ul plasmid labeling reaction: an appropriate volume
of plasmid DNA in weight is diluted in dH20 to bring the DNA to the proper volume of
labeling reaction, 2.5ul of Label IT TrackerTM Reagent (CyTM3 or CyTM5 dye), and 5ul Of
10X labeling buffer A. The non-enzymatic Label IT TrackerTM Intracellular Nucleic
Acid Localization Kit from Mirus was performed according to the manufacturer's
recommendations. After this, the concentration of labeled plasmid DNA is re-determined
by absorbance at 260nm.
Co-transfection of HEK 293 Cells with Plasmids Expressing Target and Ribozyme
We chose to use human embryonic kidney (HEK) 293 cells, because they do not
express our target (dog rhodopsin) which should eliminate target background. For our
experiments, we used that under passage 41. Cells were grown and maintained in
Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Co.) supplemented with
10% fetal bovine serum (FBS, Sigma-Aldrich Co.), 100U/ml of penicillin, 100mg/ml of
streptomycin (10X PenStrep, Mediatech, Cellgrow, VA). The day before transfections,
the cells were trypsinized and counted on an hemacytometer so that they will be 90-95%
confluent on the day of transfection.
Transfections were preformed in triplicate or quadruplicate. To account for
pipetting/transfer errors, all were prepared at least 25% more reaction volume than
needed. DMEM media were removed from cells plated on day before transfection and
replaced with fresh media lacking antibiotics (9ml for a 10cm dish or 1.5ml for 6-well
[35mm] plate). Opti-Mem I Reduced Serum Medium (Sigma-Aldrich Co.) without
serum is used to dilute plasmids carrying target and ribozymes and transfection
LipofectamineTM 2000 Reagent (Invitrogen). For multiple dishes, make a bulk mix of
plasmids. For each 10cm tissue-culture dish, 10ug of total plasmid DNA (target +
ribozyme) were diluted in lml of Opti-Mem. 4ug of total plasmid DNA were dissolved
in 250ul of Opti-Mem for each well of a 6-well plate. The diluted target and ribozyme
plasmid DNA were incubated separately for 5min at room temperature, and then mixed
together and incubated for an additional 10 minutes. The diluted LipofectamineTM 2000
Reagent was incubated at room temperature for 10 min. 3ul of LipofectamineTM 2000
Reagent was used per lug of DNA. Once LipofectamineTM 2000 Reagent was diluted, it
was combined with the diluted plasmid DNA and incubated for 20 min at room
temperature. The complexes were added directly to each dish or well containing cells
and medium [DMEM with 10% FBS (without antibiotics)] and gently rocked. The plates
were incubated with transfected cells in a 5% C02 incubator at 370C to maintain growing
cells for 48 hours.
Harvest of Co-Transfected Cells
48 hours post-transfection, cells were trypsinized to remove them from culture
plates and pelleted by centrifugation at 4000 rpm for 5 min at 40C. The trypsin
supernatant was removed and the cell pellet was then washed with 500ul of IX PBS
RNA Extraction from Co-Transfected HEK 293 Cells
RNA was extracted by using GenEluteTM Mammalian Total RNA kit from Sigma.
The GenEluteTM Mammalian Total RNA kit was performed according to the
manufacturer's recommendations. After RNA extraction, the eluted RNA is treated with
DNase (DNA-freeTM from Ambion) to remove contaminating DNA from RNA
preparation. According to the product description, contaminating DNA is digested to
levels below the limit of detection by routine PCR by using DNA-free. The
concentration of DNase treated RNA was determined by absorbance at 260nm. PCR was
conducted to verify that no DNA contaminants have been eliminated from the RNA
preparation. In a sterile PCR tube, the following contents were combined: an appropriate
volume of lug of RNA, lul of primer I [15 pmol/ul] (sense strand of dog rhodopsin
oligonucleotide), lug of primer II [15pmol/ul] (antisensestrand of dog rhodopsin
oligonucleotide), 2.5ul of 1OX Mg free buffer, 2ul of MgC2, lul of dNTPs [10mM],
0.5ul of Taq polymerase, and dH20 to bring the PCR solution to 25ul. After 20 or 30
cycles, PCR products are run on a 1.5% agarose gel, and then compared to the negative
control. If nothing is amplified, we consider our RNA preparation to be free of DNA.
Analysis of Ribozyme Activity by Quantitative Reverse Transcriptase-Polymerase
Chain Reaction (RT-PCR)
This allows for generating first-strand cDNA from an RNA template by using
proper primers. We used First-Strand cDNA Synthesis kit from Amersham Bioscience to
reverse transcribe RNA to cDNA. The reverse transcriptase reaction was set up as
described the protocol of the kit to synthesize first-strand cDNAs of dog rhodopsin and
beta-actin (2 ug of RNA used for 15ul reaction). To avoid pipetting variation, the reverse
transcription reaction for both genes was done in the same tube using sequence-specific
antisense primers [40 pmol/ul]. PCR reaction for dog rhodopsin and beta-actin control
was set up separately, since their optimized amplification conditions (annealing
temperature and extension time) are different. For detection of PCR products, 5ul or 10ul
of each reaction was loaded on an 1.5% agarose gel or an polyacrylamide gel
respectively. 5% and 8% polyacrylamide gels were used for beta-actin and dog
rhodopsin respectively. For quantitating PCR products run on the 1.5% agarose gel, the
Gel Quantification program from BioRad was used to quantitate. After the bromphenol
blue tracking dye has run about two-thirds the length of a 30cm gel at 400V/ no more
than 40mA, the gels were stained with a nucleic acid attain, SYBR Green I (Molecular
Probes), which is diluted 1:10,000, for 15-20 min. The stained gels are analyzed to
determine the volume of each PCR product using the Phospholmager and Image Quant
program. Numerical values were obtained and ratios of rhodopsin to beta-actin were
isB I (_9s)
CMV ie enhancer
Shicken b-actin promoter
p2I NewH HinLdIII (1921)
KtC2 Jp (1931)
&iwal('(263 '7.26) ^Extra Ha\pi n RZP
TR Cli/ l(2105)
SW l3715>) PYF441 enianWer
Figure 2-1. Map of p21 NewHP packaging plasmid.
CMV ie enhancer
chicken b-actin promoter
dog opsin 448
6681 bp HindIIl(1921)
Small (3755) r extra hairpin Rz
Figure 2-2. Map of Rz448 cloned into p21 NewHP packaging plasmid.
CMV ie enhancer
/ chicken b-actin promoter
6 dog opsin 448
6643 bp /
H3 bpindm (1921)
S- Spel (1960)
targeting seq 208
SmaI(3717) nR extra hairpin Rz
Figure 2-3. Map of ET-208 cloned into p21 NewHP Rz448 packaging plasmid.
As discussed in the introduction, Dr. Marina Gorbatyuk had already tested a highly
active ribozyme against dog rhodopsin mRNA. Ribozyme activity was assayed by
quantitative RT-PCR 48 hours post transfection with HEK 293 cells. At a molar ratio of
1:4 and 1:6 of target to ribozyme, the level of dog rhodopsin mRNA was reduced by
approximately 83% and 94% respectively compared to control (Figure 3-1). Therefore,
we used this ribozyme (Rz448) to test the effects of the targeting sequences.
Construction of Rz448-ET208-OHP Packaging Plasmid
After inserting the ET-208 and dog rhodopsin Rz448 sequences into the
p21NewHP plasmid, the presence of the ET-208 and Rz448 sequences in the cloned
vector was examined by performing a 25 cycle PCR amplification of the sequences
between Spel and Nsil (Figure 3-2). PCR products were run on 1.5% agarose gel and
stained with EtBr (Figure 3-3). In addition, the clones were screened for ITRs that are
absolutely necessary for packaging of AAV. ITR retention was determined by
performing a Smal digest, and running the digested plasmid on a 0.6% agarose gel
(Figure 3-4). Each AAV ITR contains two Smal restriction sites within its sequence. If
both ITRs are present, a Smal digest yields two bands that are 3608bp and 3013bp in size
(Figure 3-4). To verify proper orientation and correct sequence of ET208 and RZ448, all
clones were examined by sequencing analysis.
Construct of Rz448-OHP Packaging Plasmid
The Rz448-OHP plasmid, which lacks both the NHP ribozyme and ET-208, was
constructed to serve as a control plasmid in our comparison experiments. Since this
construct is based on the Rz448-ET208-OHP plasmid, the absence of ET-208 was
confirmed by performing a 30 cycle PCR with a sequence-specific sense primer for p21
NHP and the antisense strand of ET-208. Plasmids containing the ET-208 were also
amplified to serve as positive controls (PC). Amplified PCR products were run on a
1.5% agarose gel and stained with EtBr (Figure 3-5). Rz448-OHP clones 1 and 2 do not
have bands corresponding to the ET-208 band in the PC lanes (Figure 3-5). The Rz448-
OHP clones were also screened for ITR and sequenced to confirm the absence of ET208
(Data not shown).
The Co-Localization of Plasmids Expressing Target and Ribozyme within a Cell
after Co-Transfection of HEK 293 Cells
To visualize the co-localization of plasmids expressing target and ribozyme in co-
transfected HEK 293 cells, plasmids were labeled with fluorescent dyes before
transfection. Dyes were visualized with a confocal microscope (BioRad) at 48 hours
post-transfection. In Figure 3-6 (X20), cells bearing plasmids encoding ribozyme are
seen as a red dot and cells including a plasmid containing target are shown as a blue spot.
In the case of cells holding both plasmids expressing target and ribozyme are indicated as
a spot of violet color.
Analysis of Ribozyme Activity by Quantitative RT-PCR
The activity of ribozyme 448 targeting dog rod opsin RNA was assayed by
quantitative RT-PCR 48 hours post transfection. For this, we extracted total RNA and
compared the levels of target rhodopsin mRNA to an unchanged cellular mRNA, beta-
actin. The mRNAs for dog rhodopsin and beta-actin were reverse transcribed and their
cDNAs were amplified. The PCR products from both templates were run on a 1.5%
agarose and 5-8% polyacrylamide gel to quantitate the amount of beta-actin and dog
rhodopsin mRNA levels.
Results from 1.5% Agarose Gels
At a molar ratio of 1:4 of target to ribozyme
Cotransfection of HEK 293 cells was conducted in triplicate. On a 1.5% agarose
gel, rhodopsin ran as a single band corresponding to -350bp (Figure 3-7a), whereas a
band corresponding to -750bp was obtained from beta-actin (Figure 3-7b). A brighter
band indicates that the lane has more rhodopsin cDNA than a faint band. In a faint band,
the level of rhodopsin cDNA was decreased by the ribozyme activity. The control cells
did not have a plasmid expressing a dog rhodopsin-specific ribozyme. As shown in
Figure 3-7a, the control samples loaded in last three lanes are much brighter than the
other variants. This suggests that the other three variants (Rz448-NHP-OHP, Rz448-
ET208-OHP and Rz448-OHP from left to right) expressing ribozyme caused the
significant reduction in rhodopsin mRNA. On the other hand, in the case of beta-actin,
the intensity of each band is similar between experimental and control variants (Figure 3-
7b). Since beta-actin is a should not be affected by the ribozyme in HEK 293 cells, its
expression level should be unchanged and can therefore be used as an internal control for
these experiments. Using Excel (Microsoft), the ratio of rhodopsin to beta-actin was
averaged and indicated in a bar graph (Figure 3-8). According to these calculations, the
level of dog rhodopsin mRNA from all experimental variants was equally reduced by
approximately 90% as compared to the control cells lacking ribozyme. This finding
confirms the previous results that the levels of dog rhodopsin mRNA in transfected cells
can be decreased by the activity of dog opsin-specific ribozyme 448. However, since we
could not see a difference among the three plasmids expressing the ribozyme, we could
not ascertain whether ET-208 could promote better expression of ribozyme 448, and
cause a resulting in greater knock-down of dog rod opsin mRNA expression at this ratio
(1:4, target: ribozyme).
At a molar ratio of 1:1 of target to ribozyme
As stated above, since a significant decrease was observed in all experimental
variants tested (NHP-OHP, ET-208-OHP, or OHP) (Figure 3-7a), we could not judge
which one was best in reducing the levels of dog rhodopsin mRNA. For this reason, we
decided that decreasing the molar ratios of target to ribozyme was necessary to see
differences among the three experimental variants. To accomplish this, we designed a
second transfection experiment done in quadruplicate, at a molar ratio of 1:1 (target to
ribozyme). These cells were processed using the same procedures for post-transfection
incubation times and RNA isolation. RT-PCR was performed and equal volumes of DNA
products were loaded. Representative results for rhodopsin and beta-actin are shown in
Figures 3-9a and 3-9b, respectively. Based on the intensity of the rhodopsin bands
relative to the beta-actin bands, we can asses a greater or lesser efficacy in ribozyme
function. Of the three plasmids tested, Rz448-ET208-OHP resulted in the highest
reduction of rhodopsin mRNA. In contrast, with the Rz448-OHP plasmid, virtually the
same level of rhodopsin mRNA was observed compared to untreated control (Figure 3-
9a). As see in lanes 1-4 (Figure 3-9a), the plasmid having Rz448-NHP-OHP showed
higher levels of rhodopsin as compared to lanes 9-12, Rz448-OHP used as a control for
NHP and ET-208 (Figure 3-9a). Levels of beta-actin as seen of the agarose gel, mRNAs
were determined to be equal from all variants and controls (Figure 3-9b). These findings
demonstrated that ET-208 elevated the expression of the ribozyme 448 and resulted in the
best knock-down of dog rhodopsin mRNA among the experimental variants. As
illustrated in Figure 3-10, the averaged mRNA ratio of rhodopsin to beta-actin for Rz448-
ET208-OHP was reduced by approximately 73% compared to control. Using Rz448
with just the old hairpin ribozyme (Rz448-OHP) the level of dog rhodopsin mRNA was
decreased by approximately 36%. However, in the case of Rz448-NHP-OHP, almost no
difference (reduction of -9%) was observed. As a result of these findings, we propose
that the ET-208 sequence can significantly reduce the amount of dog rhodopsin mRNA
by the expression of ribozyme 448 at a high level.
At a molar ratio of 1:2.2 of target to ribozyme
An intermediate ratio of 1:2.2 was also examined to confirm our findings in the 1:1
ratio experiment. Cotransfections were performed in quadruplicate. The results are
shown in Figure 3-11. Approximately 55% reduction in mRNA levels was obtained
using Rz448-ET208-OHP, whereas an approximately 32% decrease was observed using
Rz448-OHP. Although the reduction difference was not as high as in the 1:1 ratio
experiment, ET-208 was still the most efficient in reducing levels of dog rhodopsin
mRNA among the three experimental variants. This finding confirms the previous results
that the most decreased level of dog rhodopsin mRNA was shown in cells transfected
with plasmids containing the ET-208 element.
Results from 5% and 8% Polyacrylamide Gels Containing Urea
A 10ul portion of the same cDNA created for the agarose gel experiment was used
for polyacrylamide gel electrophoresis. An 8% polyacrylamide gel was used to resolve
the -350bp rhodopsin PCR product, while an 5% gel was used for the ~750bp beta-actin
product. These gels were stained with a nucleic acid stain, SYBR Green I (Molecular
Probes). The fluorescence of the DNA bands was measured on the Storm
Phosphorimager (Molecular Dynamics) using the program ImageQuant. Numerical
values were obtained and ratios of rhodopsin to beta-actin were calculated (Figure 3-12).
A graphical representation of the averaged ratios of rhodopsin to beta-actin using
the three molar ratios of target to ribozyme (1:4, 1:1, and 1:2.2) is illustrated in Figure 3-
13, 3-14, and 3-15, respectively. In the case of molar ratio 1:4, the pattern of reduction of
all three experimental variants was similar to the results obtained from agarose gel.
However the rate of reduction was slightly decreased from 90% to 75% (Figure 3-13). At
a molar ratio of 1:1 (Figure 3-14), the amount of dog rhodopsin mRNA that was reduced
is the same as that observed in agarose gel. The levels of rhodopsin mRNA were reduced
by approximately 58% for both NHP and ET-208, while a reduction rate of about 31%
was observed using OHP alone. Strikingly, there was no significant difference in the
reduction between NHP and ET208 (Figure 3-14). The results based on the agarose gel
demonstrated approximately 73% and 9% reduction rate for ET-208 and NHP
respectively compared to control.
Unexpected results were also obtained at the molar ratio of target: ribozyme, 1:2.2
and are shown in Figure 3-15. NHP showed a greater reduction of rhodopsin mRNA
levels than ET-208. There was a decrease in the detected cleavage for the 1:2.2 ratio as
compared to the 1:1. While the 1:1 ratio showed a decrease of 58% for both ET-208 and
NHP, the 1:2.2 ratio showed a decrease of 20% and 43% respectively. From these
observations, we concluded that ET-208 promotes the expression of Rz448 more
efficiently when the target and ribozyme are expressed at equal molar ratios. This
combination of 1:1 (target: ribozyme) results in the greatest reduction of dog rhodopsin
rhod sin+Rz397 (1:6)
rhodopsin + Rz397(1:8)
rhodopsin + Rz 448 (1:4)
rhodopsin + Rz 448 (1:6:
Figure 3-1. The Rz448 activity on the reduction of dog rhodopsin mRNA levels in
transfected cells (Courtesy of Dr. M. Gorbatyuk).
CMV ie enhancer
(9" chicken b-actin promoter
dog opsin 448
"', 5pe (1960)
targeting seq 208
extra hairpin Rz
Figure 3-2. Map of Rz448 and ET208 cloned into p21 NewHP packaging plasmid.
NC std 1 2 3 4
Figure 3-3. ET-208 PCR product from Rz448-ET208-OHP clones 1-6. NC= negative
< No ITRs
std 1 2 3 4 5
Figure 3-4. Small digest of Rz448-ET208-OHP clones
1-5 demonstrating presence of
PC PC std 1 2 3
Figure 3-5. PCR amplification to confirm
PC= positive control.
absence of ET-208 in Rz4448-OHP clones.
Figure 3-6. Plasmids containing ribozyme (Rz448-NHP-OHP, Rz448-ET208-OHP, and
Rz448-OHP) were labeled with CyTM3 (Red), while plasmids coding for
target were treated with CyTM5 (Blue) (A confocal microscope by BioRad
C NHP ET208 OHP
C std 1 2 3 4 5 6 7 8 9 10 11 12
C NHP ET208 OHP Con
C std 1 2 3 4 5 6 7 8 9 10 11 12
Figure 3-7. 1:4 ratio of target to Rz separated on agarose gels. A) Rhodopsin cDNA
(-350 bp). Rz448-NHP-OHP clones 1-3, Rz448-ET208-OHP clones 4-6,
Rz448-OHP clones 7-9, and control 10-12. C= control containing dog
rhodopsin DNA for PCR. B) Beta-actin cDNA (-750 bp). Rz448-NHP-OHP
clones 1-3, Rz448-ET208-OHP clones 4-6, Rz448-OHP clones 7-9, and
control 10-12. C= control containing beta-actin DNA for PCR.
At a Molar Ratio of 1:4
Figure 3-8. Ratio of rhodopsin to beta-actin at a molar ratio of 1:4 (target: ribozyme)
I I I I
C NHP ET208 OHP Con
C std 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
C NHP ET208 OHP Con
C std 1 2 3 4 5 6 7 8 9 10 11 1213 1415 16
Figure 3-9. 1:1 ratio of target to Rz separated on agarose gels. A) Rhodopsin cDNA
(-350 bp). Rz448-NHP-OHP clones 1-4, Rz448-ET208-OHP clones 5-8,
Rz448-OHP clones 9-12, and control 13-16. C= control containing dog
rhodopsin DNA for PCR. B) Beta-actin cDNA (-750 bp). Rz448-NHP-OHP
clones 1-4, Rz448-ET208-OHP clones 5-8, Rz448-OHP clones 9-12, and
control 13-16. C= control containing beta-actin DNA for PCR.
At a Molar Ratio of 1:1
R- 44-e. HP
Figure 3-10. Ratio of rhodopsin to beta-actin at a molar ratio of 1:1 (target: ribozyme)
At a Molar Ratio of 1:2.2
Figure 3-11. Ratio of rhodopsin to beta-actin at a molar ratio of 1:2.2 (target: ribozyme)
Figure 3-12. Picture of polyacrylamide gel containing urea stained with SYBR Green I
and scanned by ImageQuant
At a Molar Ratio of 1:4
(Target to Ribozyme)
n Rz448+NHP Rz448+ET208 Rz448
0 (OHPi (OHPi (OHPi
Figure 3-13. Ratio ofrhodopsin to beta-actin at a molar ratio of 1:4 (target: ribozyme)
At a Molar Ratio of 1:1
(Target to Ribozyme)
Figure 3-14. Ratio of rhodopsin to beta-actin at a molar ratio of 1:1 (target: ribozyme)
At a Molar Ratio of 1:2.2
(Target to Ribozyme)
Pz-4.-..E TI --.
Figure 3-15. Ratio of rhodopsin to beta-actin at a molar ratio of 1:2.2 (target: ribozyme)
NHP ET208 OHP Con
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
NHP ET208 OHP Con
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Figure 3-16. Picture of polyacrylamide gels containing urea stained with SYBR Green I
and scanned by ImageQuant at a molar ratio of 1:2.2 (target: ribozyme). A)
Beta-actin (5%, -750 bp). Rz448-NHP-OHP clones 1-4, Rz448-ET208-OHP
clones 5-8, Rz448-OHP clones 9-12, and control 13-16. B) Rhodopsin (8%,
-350 bp). Rz448-NHP-OHP clones 1-4, Rz448-ET208-OHP clones 5-8,
Rz448-OHP clones 9-12, and control 13-16.
DISCUSSION AND FUTURE STUDIES
Two of the best characterized ribozymes that have been used in gene therapy are
the hairpin and the hammerhead that act naturally as viral self-cleaving agents (34,35).
Rhodopsin linked autosomal dominant retinitis pigmentosa (ADRP) has been an ideal
target of ribozyme-mediated therapy for a number of reasons: (1) it is a slowly
progressive disease; (2) it is easy to access the target tissue, and (3) the eye is an
immunologically privileged site (34,36). There are over 100 mutations in the rhodopsin
gene, leading to ADRP with the P23H mutation of rhodopsin being the most widespread
in the U.S. One of the studies being performed in our laboratory is rAAV-mediated
hammerhead ribozyme gene therapy for the T4R point mutation in dog rhodopsin that is
associated with progressive retinal atrophies (PRAs) (43). The importance of PRAs is
that dogs with this mutation display a retinal phenotype that mimics that in humans with
dominant rhodopsin mutations (43).
In order for gene therapy of genetic disorders to be effective, it is crucial that the
transgene (i.e., ribozyme) is expressed at high levels. Transgene expression can be
changed at both the transcriptional and post-transcriptional levels. In numerous previous
studies, the transcriptional regulatory elements have been optimized for efficient
transgene expression, including the promoter, enhancer, intron, and poly(A) sequence
(59). To fulfill this, some researchers have begun to focus on the NEEs (nuclear export
elements). Recent studies have investigated the post-transcriptional regulatory elements
such as splice signals (SS), constitutive RNA transport elements (CTE), and the
woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). All of these
signals are known to increase RNA export from the nucleus.
The best known example of stimulation at post-transcriptional level is the inclusion
of an intron within the expression cassette (5,46,47,60). It has been reported that the
addition of an intron can facilitate gene expression by causing the cytoplasmic
accumulation of mRNA in vitro and in vivo (60). In this regard, splicing, which is
biochemically linked to RNA export, promotes the efficiency of the process by formation
of a specific nucleoprotein complex (2,61). However, both CTE and WPRE have the
advantages of not requiring splicing events (60). These elements compensate for the lack
of an intron in the transgene expression (61). Several studies have demonstrated the
utility of these cis-acting RNA elements for increasing transgene expression in context of
either plasmid or viral gene vectors (5,62,63).
Retroviral replication requires the nuclear export unspliced forms of viral RNAs
(61,64-67), because eukaryotic cells encode factors that inhibit the nuclear export of
incompletely spliced mRNAs (67). To avoid the requirement for splicing prior to export,
retroviruses have evolved mechanisms that allow their intron-containing RNAs to exit the
nucleus (66). The constitutive transport element (CTE) of the simian type D retroviruses
is cis-acting element and stem-loop structure (67) that promotes the nuclear export and
cytoplasmic accumulation of incompletely spliced mRNAs (61). The CTE-mediated
nuclear export involves the interaction between CTE and the host-encoded RNA binding
protein TAP, a host factor which is thought to be required for export for cellular RNAs
(63,66-69). Some studies have tested the utility of CTE in the creation of an antisense
system (48,65,70). The CTE has also been proposed as an RNA motif that has the ability
to interact with intracellular RNA helicase that unwinds target mRNAs and facilitates
cleavage by ribozymes (48,65). Futami and coworkers connected the constitutive
transport element (CTE) to an antisense sequence (65). According to their results, a
hybrid antisense oligonucleotide (ODN) containing CTE and targeted to the bcl-2 gene
suppressed the expression of this gene more effectively than did the antisense ODN alone
(65). Warashina's group attached the CTE to their ribozyme (70). They hypothesized
that an RNA helicase coupled to a ribozyme might efficiently guide the ribozyme to its
target site by resolving any inhibitory mRNA structures, thereby leading to efficient
substrate cleavage (70). This modification significantly enhanced ribozyme activity in
vivo and permitted cleavage of sites previously found to be inaccessible, resulting in
suppression on the expression of genes (70). From the findings from Liu et al., the CTE
addition did not alter in vitro ribozyme activity (48). However, in vivo, Rz-coupled-
CTEs were more than 20% more active than non-CTE Rz (48). In this experiment, the
CTE probably assists ribozymes through RNA transport and not through RNA unwinding
Besides the SS and the CTE, the third RNA element that can significantly enhance
transgene expression is the post-transcriptional regulatory element of hepadnaviruses,
such as hepatitis B virus or woodchuck hepatitis virus (WPRE) (62), the latter being more
efficient (48,71). Other post-transcriptional elements, such as SS and CTE, depend on
the transgene of interest for their enhancing effect (48). On the other hand, the
stimulatory ability of the cis-acting WPRE (63), when it is inserted into the 3'
untranslated region of coding sequences (71), is transgene-, promoter-, and vector-
independent (48,61). The 600 bp size of the WPRE contains at least three distinct cis-
acting sub-elements required for maximal function (48,62). The WPRE seems to act
independently on transcription and splicing and may improve gene expression by
facilitating RNA export (48,62). Since woodchuck hepatitis virus (WHV) encodes
intronless messages (62,63), WPRE evolved to stimulate the expression of intronless
viral messages (71). This element has been shown to enhance the transgene expression,
which requires no viral proteins to function (63), from vectors of adeno-associated virus
(AAV), lentivirus, and retrovirus (48,61,71) both in vitro and in vivo (60). To investigate
the efficacy of the WPRE in enhancing transgene expression from adeno-virus vectors,
some studies have tested the level and distribution of the green fluorescent protein (GFP)
(71) and the luciferase expression encoded by a cDNA (60,72). Increases in the
efficiency of AAV-directed expression both in vivo and in vitro were demonstrated
(60,71,72). In an experimental rat glaucoma model, incorporation of WPRE into AAV
improved GFP expression in retinal ganglion cells (RGCs) (64). Work done by Paterna's
group proposed that the WPRE, which was inserted into rAAVs, improved the production
of certain neuroprotective proteins for human neurodegenerative diseases (73).
As discussed above, efforts aimed at increasing the transgene expression have been
developed the post-transcriptional enhancer elements associated with optimized
transcriptional regulatory elements in several ways. Many researchers have used the
CTE and WPRE to enhance the expression of mRNA and ribozymes. However, the CTE
addition did not change in vitro ribozyme activity (48). Unpublished observations from
Alan White, a graduate student from our laboratory, demonstrated that there was no
significant improvement made by insertion of WPRE in a ribozyme expressing AAV
vector. Despite all of these efforts, there still room for improvement in nuclear transport
of ribozymes for gene therapy.
Since efficient export of mRNAs from the nucleus into the cytoplasm plays an
important role in their expression, the NEE can be beneficial. One of the AAV2
ribozyme vectors, p21NHP RZ448, previously used in our studies uses two self-cleaving
hairpin ribozymes found immediately downstream of dog rhodopsin ribozyme 448 as a
processing sequence to free ribozyme 448 from the polyA tail of the primary transcript.
Additionally, expression of the ribozyme in this plasmid is under the control of CBA
promoter paired with the CMV-enhancer and has an intron located immediately
downstream of the promoter as well as two ITRs required for AAV packaging.
As discussed in the introduction, it has been found that the matrix (M) protein of
VSV effectively hinders Ran-dependent nucleocytoplasmic bidirectional transport of
RNA and proteins in Xenopus laevis oocytes (32). In one of the studies conducted by
James E. Dahlberg, they selected new RNA sequences that overcome the M protein
mediated RNA transport inhibition. (10). These new RNA sequences function as NEE
and fall into one of three sequences in a part of a collection of RNAs containing 20
random nucleotides (10). Of these ET-RNAs, ET-208 was recommended by James E.
Dahlberg for our experiment. Here, we investigated whether ET-208 could increase
export of ribozyme 448 to the cytoplasm, leading to greater knock-down of dog
We tested this by constructing three different plasmids (Rz448-NHP-OHP, Rz448-
ET208-OHP, and Rz448-OHP) for our comparison experiments. The presence or
absence and correct orientation of the ET-208 and the NHP ribozyme within these
constructs were verified with PCR and sequencing analysis. The plasmids were also
screened for ITRs. Large-scale cesium chloride (CsC1) preps were made of the constructs
to get highly purified and large amount of DNA for transfection. Plasmids containing
target and ribozyme sequences were labeled with fluorescent dyes. After the co-
transfection of the plasmids, the localization of the two within the transfected cells was
visualized with a confocal microscope.
Ribozyme activity leading to the reduction of dog rhodopsin mRNA was analyzed
by quantitative RT-PCR. To assay reduced levels of dog rod opsin mRNA, two kinds of
gel (agarose gel and polyacrylamide gel containing urea) were used and stained with EtBr
and SYBR Green I, respectively.
First, rhodopsin and beta-actin RT-PCR products were run on 1.5% agarose gel.
The ratio of rhodopsin to beta-actin was averaged. Transient co-transfetion at a molar
ratio of 1:4 of target to three different ribozyme constructs, all showed a greater than 90%
reduction compared to a control plasmid lacking ribozyme (Figure. 3-8). As described in
the results, since we could not see a difference among the plasmids expressing ribozyme;
we could not determine whether ET-208 could promote better expression of ribozyme
448. To overcome this obstacle, we tried decreasing target to ribozyme ratios during
transfection to determine at which ratio a difference might appear among the variants. At
a molar ratio of 1:1, a slightly decreased rate of reduction of the variants was observed
(Rz448-ET208-OHP: -73%, Rz448-OHP: -36%) (Figure 3-10), but these results are
consistent with findings from a ratio of 1:4 (Figure 3-8). Rz448-NHP-OHP variant
showed a decreased amount of reduction (9%) (Figure 3-10). An interesting result was
obtained at a ratio of 1:2.2. The level of target following transfection by Rz448-NHP-
OHP construct was not different than transfection with the control plasmid not carrying
ribozyme (Figure 3-11). Approximately a 55% and 32% reduction was observed for
Rz448-ET208-OHP and Rz448-OHP, respectively (Figure 3-11). In view of the results
from the 1.5% agarose gel, which demonstrated that the levels of dog rhodopsin mRNA
are markedly reduced by plasmid carrying ET-208 as its NEE, we can propose that the
ET-208 can enhance the activity of ribozyme at a low ratio of target to ribozyme.
However, at a high ratio, the extent of reduction is not significantly different between
NHP and ET-208.
Polyacrylamide gels were also used to quantitate the levels of dog rhodopsin target
after treatment our various Rz448 constructs. The results based on this analysis were
consistent with those obtained previously by 1.5% agarose gel assay. As expected, as
ratios of target to ribozyme declined, the amount of reduction was also decreased. About
75% reduction was observed from all experimental variants at a molar ratio of 1:4 (Figure
3-12). In the case of a ratio of 1:1, around 58% reduction rate was obtained by both NHP
and ET-208, whereas that of Rz448-OHP was 31%. Interestingly, there was no
significant difference in reduction between NHP and ET208 at this ratio. But the error
bars which represent the differences on data for each sample for NHP is greater than that
for ET-208 (Figure 3-13). For this reason, although there was a small difference between
NHP and ET-208, this assay system does not allow us to conclude that ET-208 promotes
greater activity than the NHP ribozyme. Two results above provided direct evidence
supporting our hypothesis that the Rz448-ET208-OHP construct works as well or better
than Rz448-NHP-OHP. On the other hand, it was surprising to find that NHP resulted in
the greatest knock down of dog rod opsin expression at a molar ratio of 1:2.2 (Figure 3-
14). The opposite was observed from the agarose gel. The decreased reduction rate of
each was 43% for NHP and 20% for ET-208, respectively.
There are several factors that could account for the observed differences. First,
analyses by an agarose gel and a polyacrylamide gel containing urea are done by different
methods. The agarose gels and polyacrylamide gels were stained with EtBr [10mg/ml]
and SYBR Green I (diluted 1:10,000), respectively. The increased sensitivity of SYBR
green increased the background fluoresnce in the gels and influenced the detection of
bands, possibly resulting in incorrect estimation of RNA levels. Second, as shown in
Figure 3-16, the bands were not as tightly formed as expected at a ratio of 1:2.2.
Especially in the case of rhodopsin, all bands were very faint. As a result of this, the
levels of rhodopsin cDNA might not be correctly detected. Finally, at a ratio of 1:2.2 of
target to ribozyme, the error bars for NHP were much greater than those for ET-208
(Figure 3-15). Therefore, there is not enough direct evidence for us to interpret our data
to mean that the NHP ribozyme is better than the NEE ET-208.
In conclusion, we found that ET-208 was able to enhance the ability of ribozyme
448 to reduce the levels of rhodopsin mRNA within co-transfected cells. In order to truly
compare the NEE ET-208 to the hairpin ribozyme processing sequence, we need to
remove the OHP ribozyme in all of our constructs used in this experiment. This
experiment will enable us to obtain direct evidence on whether the ET208 acting as an
NEE is better on promoting high expression of our hammerhead ribozymes than the
hairpin ribozyme that functions as a processing sequence in the separation of the
hammerhead ribozyme from the long transcript. Since splicing is known to enhance
nuclear-cytoplasmic transport of mRNA (2,5,44-47), we should also test ribozyme
constructs that lack an intron sequence, to determine if RNA export is enhanced by
ET208 in the absence of an intron.
In addition, some issues regarding methods used for this experiment will be
examined to get more information from the results. First, here, we used quantitative RT-
PCR to assay the activity of Rz448 targeting dog rhodopsin RNA determined by the
reduction of dog rhodopsin mRNA levels. Since RT-PCR is highly reproducible only in
the exponential phase of amplification, it is necessary to collect quantitative data at a
point in which every sample is in this phase to obtain the accuracy and precision. After
the reaction rate ceases to be exponential, it enters a linear phase of amplification. The
number of cycles of PCR used for this experiment was determined by being performed
for a greater or lesser number of cycles 15 -30 (M. Gorbatyuk, personal communication).
Real-time PCR automates this process by quantitating reaction products for each sample
in every cycle (74). The advantage of real-time PCR is that the result is a broad dynamic
range, with no user intervention required. Data analysis, including standard curve
generation and copy number of calculation, is performed automatically. Because of this
advantage, in the future experiments we will use real-time PCR to determine rhodopsin
RNA levels in transfected cells. Second, when we took pictures of co-transfected cells by
using a confocal microscope, we did not count the number of cells visualized as different
colors (blue or red) depending on that they contain which plasmids (target or ribozyme).
For this reason, we could not determine the efficiency of transfection from these pictures.
In the future experiment, we will sort cells depending on their colors labeled with
different fluorescent dyes to see the efficiency of our transfection.
Lastly, in the field of antisense approaches to knock down the expression of
mutated genes that cause diseases, small interfering RNA (siRNA) has also held promise
for the development of therapeutic gene silencing. RNA interference (RNAi) is a post-
transcriptional process triggered by the introduction of double-stranded RNA (dsRNA)
which leads to gene silencing in a sequence-specific manner (75). siRNA typically
consists of two 21-23 nucleotide single-stranded RNAs that form a 19 bp duplex with 2
nucleotide 3' overhangs (76). siRNAs are an intermediate of RNA interference the
process by which double-stranded RNA inactivates homologous genes(76). Since siRNA
target less specific sequences than ribozyme, it can be easily designed to recognize RNAs
encoding aberrant proteins. Although ribozymes are also able to elicit strong and specific
suppression of gene expression, they have limited target sequences, a quintuple sequence
for hairpin ribozymes and a triplet sequence for hammerhead ribozymes (34-36). For this
reason, siRNA has become popular in studying the biology of organisms ranging from
unicellular protozoans to mammals (77). However, the effect of siRNA delivered as
RNA is only transient and this fact severely limits the applications of siRNAs (78). To
overcome this limitation, the construction of plasmids for better expression of siRNA
within cells is necessary. With our findings from this experiment, we will examine
whether ET-208 could promote better expression of siRNA, resulting in greater reduction
of dog rhodopsin expression, by replacing Rz448 with siRNA sequence in our Rz448-
LIST OF REFERENCES
1. George O, Danny R (2002) A unified theory of gene expression. Cell. 108:439-451.
2. Robin R (2003) Coupling transcription, splicing and mRNA export. Current
Opnion in Cell Biology. 15:326-331.
3. Hammell CM, Gross S, Zenklusen D, Heath CV, Stutz F, Moore C, Cole CN
(2002) Coupling of termination 3' processing, and mRNA export. Mol Cell Biol.
4. Robin R, Ed H (2002) A conserved mRNA export machinery coupled to pre-
mRNA splicing. Cell. 108:523-531.
5. Ming-juan L, Robin R (1999) Splicing is required for rapid and efficient mRNA
export in metazoans. PNAS. 96(26):14937-14942.
6. Reed R, Hurt E (2002) A conserved mRNA export machinery coupled to pre-
mRNA splicing. Cell. 108:523-531.
7. Reichert VL, Le Hir H, Jurica MS (2002) 5' exon interactions within the human
splicesome establish a framework for exon junction complex structure and
assembly. Genes Dev. 16:2778-2791.
8. Lejeune F, Shitakes Y, Li X (2002) The exon junction complex is detected on
CBP80-bound but not elF4E-bound mRNA in mammalian cells: dynamics of
mRNP remodeling. EMBO J. 21:3536-3545.
9. Karsten W (1998) Importins and exportins: how to get in and out of the nucleus.
10. Christian G, Elsebet L, James ED (1997) Selection and nuclear immobilization of
exportable RNAs. Proc Natl Acad Sci USA. 94:10122-10127.
11. Michael WM, Choi M, Dreyfuss G (1995) A nuclear export signal in hnRNP Al: a
signal-mediated, temperature-dependent nuclear protein export pathway. Cell.
12. Gideon D, V Narry Kim, Naoyuki K (2002) Messenger-RNA binding proteins and
the messages they carry. Mol Cell Biol. 3:195-205.
13. Elissa PL, Pamela AS (2002) Protein and RNA export from the nucleus.
Developmental Cell. 2:261-272.
14. Steven ID, Carl MF (1998) Translocation of RNA-coated gold particles through the
nuclear pores of oocytes. The Journal of Cell Biology. 106:575-584.
15. Katharine SU, Maureen AP (1997) Nuclear export receptors: From importin to
exportin. Cell. 90:967-970.
16. Mattaj IW, Englmeier L (1998) Nucleocytoplasmic transport: the soluble phase.
Annu Rev Biochem. 67:265-306.
17. Charles NC, Claudio S (1997) Export of mRNA from microinjected nuclei of
Xenopus laevis oocytes. Cell & Developmental Biology. 8:71-78.
18. James ED, Elsebet L (1997) Coupling of nuclear RNA export and protein import in
vertebrate cells. Cell & Developmental Biology. 8:65-70.
19. ZasloffM (1983) tRNA transport from the nucleus in a eukaryotic cell: carrier-
mediated translocation process. Proc Natl Acad Sci USA. 80(21):6436-6440.
20. Hinshaw JE, Carragher BO, Milligan RA (1992) Architecture and design of the
nuclear pore complex. Cell. 69(7): 1133-1141.
21. Davis LI (1995) The nuclear pore complex. Annu Rev Biochem. 64:865-896.
22. Charles NC, Claudio S (1997) Regulation of the export of RNA from the nucleus.
Cell & Developmental Biology. 8:71-78.
23. Mutsuhito O, Alexandra S, Scott K, Iain WM (2002) Identity elements used in
export of mRNAs. Molecular Cell. 9:659-671.
24. Fabre E, Hurt EC (1994) Nuclear transport. Curr Opin Cell Biol. 6(3):335-342.
25. Jarmolowski A, Boelens WC, Izaurralde E, Mattaj IW (1994) Nuclear export of
different classes of RNA is mediated by specific factors. J Cell Biol. 124(5):627-
26. Fischer T, Strasser K, Racz A, Rodriguez-Navarro S, Oppizzi M, Ihrig P, Lechner
J, Hurt E (2002) The mRNA export machinery requires the novel Sac3p-Thplp
complex to dock at the nucleoplasmic entrance of the nuclear pores. EMBO J.
27. Izaurralde E (2002) A novel family of nuclear transport receptors mediates the
export of messenger RNA to the cytoplasm. Eur J Cell Biol. 81(11):577-584.
28. Gorlich D, Mattaj IW (1996) Nucleocytoplasmic transport. Science. 271:1513-1518.
29. Dahlberg JE, Lund E (1998) Functions of the GTPase Ran in RNA export from the
nucleus. Curr Opin Cell Biol. 10(3):400-408.
30. Lee MS, Henry M, Silver PA (1996) A Protein that shuttles between the nucleus
and the cytoplasm is an important mediator of RNA export. Genes Dev.
31. Gorlich D, Pante N, Kutay U, Aebi U, Bischoff FR (1996) Identification of
different roles for RanGDP and RanGTP in nuclear protein import. EMBO J.
32. Her LS, Lund E, James ED (1997) Inhibition of Ran guanosine triphosphatase-
dependent nuclear transport by the matrix protein of vesicular stomatitis virus.
33. Jeannine MP, Lu-Shuin H, Virgil V, Elsebet L, James ED (2000) The matrix
protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is
in the nucleus and associated with nuclear pore complexes. Molecular and cellular
34. Jason JF, D Alan W, Alfred SL, William WH (2002) Designing and characterizing
hammerhead ribozymes for use in AAV vector-mediated retinal gene therapies.
Methods in Enzymology. 346:358-377.
35. Jason JF, Marina G, Alfred SL, William WH (2003) Design and validation of
therapeutic hammerhead ribozymes for autosomal dominant disease. Methods in
Molecular Biology. 252:221-236.
36. Alfred SL, William WH (2001) Ribozyme gene therapy: applications for molecular
medicine. TRENDS Mol Med. 7(5):221-227.
37. Adrian R, Ferre D (2004) The hairpin ribozyme. Biopolymers. 73:71-78.
38. Anastasia K, Aurelie L, Eric W, Sumedha DJ (2003) Sequence elements outside the
hammerhead ribozyme catalytic core enable intracellular activity. Nature Structural
39. Naash MI, Hollyfield JG, al-Ubaidi MR (1993) Simulation of human autosomal
dominant retinitis pigmentosa in transgenic mice expressing a mutated murine
opsin gene. Proc Natl Acad Sci USA. 90(12):5499-5503.
40. Stephen PD, Lori SS, Sara JB (1996) RetNet: Retinal information network. The
University of Texas Health Science Center. http:// www. sph.uth.tmc.edu/Retnet/.
41. Lewin AS, Drenser KA, Hauswirth WW, Nishikawa S, Yasumura D, Flannery JG,
LaVail MM (1998) Ribozyme rescue of photoreceptor cells in a transgenic rat
model of autosomal dominant retinitis pigmentosa. Nat Med. 4(9):967-971.
42. Hauswirth WW, Lewin AS (2000) Ribozyme rescue of photoreceptor cells in P23H
transgenic rats: long-term survival and late-stage therapy. Prog Retinal Eye Res.
43. James WK, Artur VC, Tomas SA, Michael JP, Susan EP, Brian JM, Samuel GJ,
Gustavo DA, Gregory MA (2002) Naturally occurring rhodopsin mutation in the
dog causes retinal dysfunction and degeneration mimicking human dominant
retinitis pigmentosa. PNAS. 99(9):6328-6333.
44. Jonsson JJ, Foresman MD, Wilson N, Mclvor RS (1992) Intron requirement for
expression of the human purine nucleoside phosphorylase gene. Nucleic Acids Res.
45. Rafiq M, Suen CK, Choudhury N, Joannou CL, White KN, Evans RW (1997)
Expression of recombinant human ceruloplasmin an absolute requirement for
splicing signals in the expression cassette. FEBS Lett. 407(2):132-136.
46. Simari RD, Yang ZY, Ling X (1998) Requirement for enhanced transgene
expression by untranslated sequences from the human cytomegalovirus immediate-
early gene. Mol Med. 4(11):700-706.
47. DeYoung MB, Zamarron C, Lin AP, Qiu C, Driscoll RM, Dichek DA (1999)
Optimizing vascular gene transfer of plasminogen activator inhibitor 1. Hum Gene
48. Liu J, Timmers AM, Lewin AS, Hauswirth WW (2004) In vivo somatic
knockdown of PDE RNA in normal mice using AAV-vectored ribozyme is
enhanced by addition of a constitutive transport element (CTE). Presentation by the
Association for Research in Vision and Ophthalmology. Inc.
49. Lingfei XU, Thomas D, Cuihua G, Terence RF, Sihong S, Barry JB, Mark SS,
Katherine PP (2001) CMV-beta-actin promoter directs higher expression from an
adeno-associated viral vector in the liver than the cytomegalovirus or elongation
1 alpha promoter and results in therapeutic levels of human factor X in mice.
Human Gene Therpy. 12:563-573.
50. Alexander P, Inder MV (2001) Gene therapy: Promises and problems. Annu Rev
Genomics Hum. Genet. 2:177-211.
51. Carter PJ, Samulski RJ (2000) Adeno-associated viral vectors as gene delivery
vehicles. Int J Mol Med. 6(1):17-27.
52. Bartlett J, Samulski RJ (1995) Genetics and biology of adeno-associated virus.
Viral vectors: Gene therapy and neuroscience applications. Academic Press. 55-73.
53. Jean B, Albert MM, Maguire AV (1999) Stable transgene expression in rod
photoreceptors after recombinant adeno-associated virus-mediated gene transfer to
monkey retina. PNAS. 96:9920-9925.
54. Grant CA, Ponnazhagan S, Wang X (1997) Evaluation of recombinant adeno-
associated virus as a gene transfer vector for the retina. Curr Eye Res. 16:949-956.
55. Ali RR, Reichel MB, Thrasher AJ (1996) Gene transfer into the mouse retina
mediated by an adeno-associated viral vector. Human Molecular Genetics.
56. Berns KI (2001) Parvoviridae: the viruses and their replication, In B N Fields. DM
Knipe, PM Howley (ed.) Fundamental virology. 4th ed. vol.2. Lippincott-Raven
Publishers. Philadelphia. PA. 1007-1041.
57. Muzyczka N (1992) Use of adeno-associated virus as a general transduction vector
for mammalian cells. Curr Top Microbiol Immunol. 158:97-129.
58. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manua. 3rd ed.
Cold Spring Harbor Laboratory Press. New York. 184.
59. Xu ZL, Mizuguchi H, Mayumi T, Hayakawa T (2003) Woodchuck hepatitis virus
post-transcriptional regulation element enhances transgene expression from
adenovirus vectors. Biochim Biophys Acta. 1621:266-271.
60. Zufferey R, Donello JE, Trono D, Hope TJ (1999) Woodchuck hepatitis virus
posttranscriptional regulatory element enhances expression of transgenes delivered
by retroviral vectors. J Virol. 73:2886-2892.
61. Schambach A, Wodrich H, Hildinger M, Bohne J, Krausslich HG, Baum C (2000)
Context dependence of different modules for posttranscriptional enhancement of
gene expression from retroviral vectors. Mol Ther. 2:435-445.
62. Donello JE, Loeb JE, Hope TJ (1998) Woodchuck hepatitis virus contains a
tripartite posttranscriptional regulatory element. J Virol. 72:5085-5092.
63. Popa I, Harris ME, Donello JE, Hope TJ (2002) CRM1-dependent function of a
cis-acting RNA export element. Mol Cell Biol. 22:2057-2067.
64. Martin KR, Quigley HA, Zack DJ, Levkovitch-Verbin H, Kielczewski J, Valenta
D, Baumrind L, Pease ME, Klein RL, Hauswirth WW (2003) Gene therapy with
brain-deliverd neurotrophic factor as a protection: Retinal ganglion cells in a rat
glaucoma model. Invest Ophthalmol Vis Sci. 44:4357-4365.
65. Foams T, Miyagishi M, Iwai S (2003) Stimulatory effect of an indirectly attached
RNA helicase-recruiting sequence on the suppression of gene expression by
antisense oligonucleotides. Antisense Nucleic Acid Drug Dev. 13:9-17.
66. Pasquinelli AE, Ernst RK, Lund E (1997) The constitutive transport element (CTE)
of mason-pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway.
EMBO J. 16:7500-7510.
67. Braun IC, Rohrbach E, Schmitt C, Izaurralde E (1999) TAP binds to the
constitutive transport element (CTE) through a novel RNA-binding motif that is
sufficient to promote CTE-dependent RNA export from the nucleus. EMBO J.
68. Bear J, Tan W, Zolotukhin AS, Tabernero C, Hudson EA, Felber BK (1999)
Identification of novel import and export signals of human TAP, the protein that
binds to the constitutive transport element of the type D retrovirus mRNAs. Mol
Cell Biol. 19:6306-6317.
69. Bogerd HP, Echarri A, Ross TM, Cullen BR (1998) Inhibition of human
immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but
not mason-pfizer monkey virus constitutive transport element activity, by a mutant
human neucleoporin targeted to Crml. J Virol. 72:8627-8635.
70. Warashina M, Kuwabara T, Kato Y, Sano M, Taira K (2001) RNA-protein hybrid
ribozymes that efficiently cleave any mRNA independently of the structure of the
target RNA. Proc Natl Acad Sci USA. 98:5572-5577.
71. Loeb JE, Cordier WS, Harris ME, Weitzman MD, Hope TJ (1999) Enhanced
expression of transgenes from adeno-associated virus vectors with the woodchuck
hepatitis virus posttranscriptional regulatory element: implications for gene
therapy. Hum Gene Ther. 10:2295-2305.
72. Lipshutz GS, Titre D, Brindle M (2003) Comparison of gene expression after
intraperitoneal delivery of AAV2 or AAV5 in utero. Mol Ther. 8:90-98.
73. Paterna JC, Moccetti T, Mura A, Feldon J, Bueler H (2000) Influence of promoter
and WHV post-transcriptional regulatory element in AAV-mediated trasgene
expression in the rat brain. Gene Ther. 7:1304-1311.
74. TechNotes from Ambion, (2004) Real-time PCR goes prime time. Ambion Inc.
http:// www. ambion.com/techlib/tn/81/813.html. July 2004.
75. Cheng JC (2003) RNA interference and human disease. Mol Genet Metab. 80(1-
76. Michael TM, Phillip AS (2002) Gene silencing in mammals by small interfering
RNAs. NATURE REVIEWS. 3:737-747.
77. Gregory JH (2002) RNA interference. NATURE. 418:244-251.
78. Overview from InvivoGen (2004) Small interfering RNA (siRNA): A revolution in
functional genomics. InvivoGen. http:// www.
invivogen.com/siRNA/siRNAoverview.htm. July 2004.
Eun-Jung Choi was born November 21, 1976, in Seoul, Korea. She was educated
in Korea from elementary to undergraduate schools. She obtained her bachelor's degree
in animal and life sciences from KonKuk University in February 2001. She was awarded
scholarships and a research assistantship during her time in the college. She worked at a
nutrition lab during her sophomore and junior years. Then, she worked at a genetic
engineering lab. She spent about 1 year in the states for English training at the University
of Tennessee. Then, she worked at the National Livestock Research Institute.
She was admitted to the University of Florida, College of Medicine, for fall 2002 as
a master's student. She majored in molecular genetics and microbiology and worked in
Dr. Alfred Lewin's lab with a focus on ribozyme-mediated gene therapy for eye diseases.
Upon graduation from the master's program, she will attend the Interdisciplinary
Program in Biomedical Sciences at the University of Florida to attain her degree of
doctor of philosophy.