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Development of lentiviral vector mediated gene transfer techniques as tools to overexpress/knockdown P2X4 receptors in vitro

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Development of lentiviral vector mediated gene transfer techniques as tools to overexpress/knockdown P2X4 receptors in vitro

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DEVELOPMENT OF LENTIVIRAL VECTOR MEDIATED GENE TRANSFER
TECHNIQUES AS TOOLS TO OVEREXPRESS/KNOCKDOWN P2X4 RECEPTORS
IN VITRO

by

Sneha Inamdar

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

August 2010

Copyright 2010 Sneha Inamdar

ii
ACKNOWLEDGEMENTS

I would like to thank my thesis advisor, Dr Daryl Davies for his constant
encouragement and guidance along with his valuable insights that helped me throughout
my graduate studies.
A special thanks to my co-advisor Dr Ronald Alkana and my committee member,
Dr Curtis Okamoto for their detailed review and constructive criticism which helped me
complete the writing of this dissertation.
I owe my deepest gratitude to my mentor, Dr Liana Asatryan for her significant
scientific contribution and good-natured support through my research endeavors.
My appreciation and gratitude goes to all fellow students in the laboratory for
their support and friendship; Dr Daya Perkins, Maya Popova and Letisha Wyatt. I also
wish to thank Miriam Fine for assisting me with laboratory techniques.
My deepest gratitude goes to my family and close friends for their unflagging
love and support throughout my studies.

iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF FIGURES v
ABBREVIATIONS vi
ABSTRACT viii
Chapter 1: INTRODUCTION 1
1.1 Alcohol and alcohol related issues 1
1.2 Ion Channels are important targets for Ethanol action 1
1.3 ATP-Gated Ion Channels form a third class of channels 2
1.4 P2X Channels have a novel structural organization 2
1.5 In vitro studies carried out on P2X4Rs to investigate sites for
ethanol action 3
1.6 Building evidence suggesting a role of P2X4Rs in ethanol
induced behaviors 3
Chapter 2: SPECIFIC AIMS 5
2.1 Develop rLV techniques to transduce the P2X4 gene into the
neuronal and HEK293 cells 6
2.2 Generate a mammalian cell line which demonstrates stable
expression of P2X4Rs achieved via rLV mediated transduction
technique 8
2.3 Develop rLV-shRNA techniques for knocking down
the P2X4Rs in microglial cells 9
Chapter 3: METHODS AND RESULTS 10
3.1 Develop rLV techniques to transduce the P2X4 gene into the
neuronal and HEK293 cells 10
3.1.1 Background information 10
3.1.2 Methods 13
3.1.3 Results 15
3.1.4 Discussion 19
3.2 Generate a mammalian cell line which demonstrates stable
expression of P2X4Rs achieved via rLV mediated transduction
technique 20
3.2.1 Background information 20
3.2.2 Methods 23

iv
3.2.3 Results 23
3.2.4 Discussion 27
3.3 Develop rLV-shRNA techniques for knocking down P2X4Rs
in microglial cells 28
3.3.1 Background information 28
3.3.2 Methods 40
Table 1: The target sequences of the rat P2X4R and shRNA
oligonucleotide designs 41
3.3.3 Results 44
3.3.4 Discussion 47

Chapter 4: CONCLUSION 48
REFERENCES 50

v
LIST OF FIGURES
Figure 1: Lentivirus production using Clontech’s Lenti-X HT Packaging
system and 293T cells. 12

Figure 2: rLV mediated P2X4 transduction in HEK293 cells 16

Figure 3: rLV mediated P2X4 transduction in rat hippocampal neurons 17

Figure 4: Western blot analysis of neuronal cells 18

Figure 5: rLV mediated stable P2X4 transduction in HEK293 cells. 25
Figure 6: Patch clamp recordings of ATP currents across P2X4Rs expressed
in a transiently and stably expressed 293HEK cell line 27

Figure 7: Mechanism of RNA interference in mammalian cells 30

Figure 8: Design of Small hairpin RNA (shRNAs) generated from a cloned
oligonucleotide DNA template to knockdown rat P2X4Rs. 39

Figure 9: Generation of DNA fragments from shRNA1- pLVX vector 44

Figure 10: Protein expression of P2X4Rs and P2X7Rs in microglial cells
transduced with shRNA-rLV vector 46

vi
ABBREVIATIONS
ATP: Adenosine triphosphate
ATTC: American Type Culture Collection
BSL2: Biosafety Level 2
CMV: Cytomegalovirus
CNS: Central Nervous System
CO
2
: Carbon Dioxide
DHFR: Dihydrofolate reductase
DMEM: Dulbecco’s Modified Eagle Medium
DNA: Dioxyribonucleic acid
FACS: Fluorescence Activated Cell Sorting
FITC: Fluorescein isothiocyanate
GABA: Gamma amino butyric acid
GFP: Green Fluorescent Protein
GOI: Gene of Interest
HBV: Hepatitis B virus
HEK 293: Human Embryonic Kidney 293 cell line
HIV: Human Immunodeficiency Virus
LGICs: Ligand Gated Ion Channels
LTR: Long Terminal Repeats
NCBI: National Centre for Biotechnology Information.

vii
ORF: Open Reading Frames
P2X: Purinergic receptors
P2X4: Purinergic receptors type 4
P2X4Rs: P2X4 receptors
P2Y: Metabotropic
HCV: Hepatitis C virus
RISC: RNA-induced silencing complex
rLV: recombinant Lentiviral
RNAi: RNA interference
RRE: Rev-responsive element
RT-PCR: Real Time Polymerase Chain Reaction.
SARS: Severe acute respiratory syndrome
shRNA: Short hairpin RNA
TM2: Transmembrane 2
VSV-G: Vesicular stomatitis virus-glycoprotein
VTA: Ventral tegmental area
WPRE: Woodchuck hepatitis Post-transcriptional Regulatory Element

viii
ABSTRACT
The current study focused on the development of recombinant lentiviral (rLV)
mediated gene transfer techniques that would aid in testing the hypothesis that the
purinergic P2X4 receptors (P2X4Rs) play an important role in behavioral effects of
alcohol. Ethanol inhibits ATP-induced currents in P2X4Rs recombinantly expressed in
oocytes and HEK293 cells. Building evidence suggests that P2X4Rs play a role in
alcohol consumption. P2X4Rs are widely expressed in neurons and glial cells, however
little is known about the contribution of neuronal and/or glial P2X4Rs in alcohol-induced
behaviors. In order to study the action of ethanol on P2X4Rs in these cells, it is necessary
to develop tools to over-express/knockdown the expression of the receptors. The
recombinant rLV gene delivery techniques were developed to express P2X4Rs in
neuronal and HEK293 cells (Aim 1); to generate HEK293 cell line stably expressing
P2X4Rs (Aim 2) and to inhibit P2X4Rs in microglial cells (Aim 3).
To address this issue, Lentivirus encoding P2X4R-GFP fusion construct was
produced and was used to transduce neuronal and HEK293 cells. Fluorescence
microscopy and western immunoblotting results confirmed the successful transduction of
the P2X4Rs in the cells. The lentivirus encoding the P2X4R-GFP was then used to
generate a HEK293 cell line stably expressing the P2X4Rs. Fluorescence microscopy and
patch clamp technology was used to observe the stable expression of the P2X4Rs in
HEK293 cells over a period of 47 days. A rLV containing shRNA oligonucleotides
targeting the P2X4Rs was generated. Microglial BV-2 cells were transduced with the

ix
recombinant lentivirus and the knockdown of P2X4Rs was tested using fluorescence
imaging and western immunoblotting. rLV infection of BV-2 cells with two different
shRNA oligonucleotides resulted in more than 50% reduction of P2X4R protein
expression per shRNA tested. Infection of BV2 cells with the combination of the two
rLV shRNA oligonucleotides almost completely inhibited the expression of P2X4Rs
(upto 85 %). The results suggest that the recombinant rLV vector can be successfully
used for efficient gene delivery of P2X4Rs in neuronal and HEK293 cells as well as for
knocking down P2X4Rs in microglial cells. This work sets the stage for the use of rLV
technology to investigate the physiological and cellular roles of neuronal and microglial
P2X4Rs in ethanol-induced behaviors.

1

CHAPTER 1
INTRODUCTION

1.1 Alcohol and alcohol related issues
Alcohol abuse is one of the major problems in our society affecting approximately
14 million people in the United States (Volpicelli 2001;McGinnis and Foege 1999) and
the costs for alcohol related disorders reach $ 185 billion (Grant et al. 2004) every year.
Alcoholism is a disabling addictive disorder that interferes with one’s mental and
physical health as well as with social, family and job responsibilities.
Alcohol acts on numerous receptors. A limited knowledge of the targets and the
mechanism of ethanol action in the CNS is a major challenge in development of effective
treatment.

1.2 Ion Channels Are Important Targets for Ethanol Action
Ligand-gated ion channels (LGICs) are considered primary targets for causing the
behavioral effects of ethanol action (Deitrich et al. 1989;Davies and Alkana 2001;Betz
1990). Two large superfamilies of LGICs have been widely studied. The nicotinic
acetylcholine-receptor superfamily (Cys loop) includes nicotinic acetylcoline-receptors
(nAchRs), 5-hydroxytryptamine type 3, GABA and glycine receptors (Betz 1990;North
and Surprenant 2000) and the glutamate superfamily (Monaghan et al. 1989;Sommer and
Seeburg 1992). A relatively new superfamily of LGICs; the ATP gated purinergic P2X

2

receptors have acquired significance as potential targets for ethanol action in recent times
(Xiong et al. 2000;Davies et al. 2002;Davies et al. 2005;Asatryan et al. 2008;Popova et
al. 2010;Asatryan L. et al. 2010).

1.3 ATP-Gated Ion Channels Form a Third Class of Channels
P2XRs are a distinct superfamily of LGICs that are unrelated to Cys-loop or the
glutamate superfamily. P2XRs are gated by synaptically released extracellular adenosine
triphosphate (ATP) and are fast acting, cation -permeable ion channels which are widely
distributed in the central and peripheral nervous system (Khakh 2001;Chizh and Illes
2001).

1.4 P2X Channels Have a Novel Structural Organization
At this time, seven subtypes of the P2X family of LGICs have been identified
(P2X1-P2X7). All the subunit proteins have been found in the CNS (Khakh et al.
2001;Khakh 2001;North 2002;Chizh and Illes 2001;Deuchars et al. 2002). P2XRs form
homomeric (e.g P2X2, P2X4) channels as well as functional heteromeric receptors (e.g
P2X2/3, P2X4/6). Three subunits of the P2XRs assemble to form the heteromeric
receptors. They are functional when expressed in Xenopus oocytes or mammalian cell
lines (Khakh et al. 2001;Khakh 2001;North 2002;Chizh and Illes 2001;Deuchars et al.
2002). It has been shown that P2X2R, P2X3R and P2X4R expressed in Xenopus oocytes
are sensitive to ethanol at intoxicating concentrations (Davies et al. 2002;Davies et al.

3

2005;Davies et al. 2005;Xiong et al. 2000). The subunits consist of two transmembrane
(TM) domains, a large extracellular domain (ectodomain) and the intracellular amino (N)
and carboxy (C) terminals. The ectodomain contains an ATP- binding site and the TM1
and TM2 membrane spanning domains are involved in ion channel gating and lining of
the ion pore (Burnstock 2004;Deuchars et al. 2002). The crystal structure of the zebrafish
P2X4Rs was reported at 3.1 Å resolution which confirmed the trimeric construction of
P2X4Rs (Kawate et al. 2009).

1.5 In vitro studies carried out on P2X4Rs to investigate sites for ethanol action
Recently it was shown that the ectodomain-transmembrane (TM) 2 interface of
P2X4Rs consists of residues that are important for ethanol action (Popova et al. 2010).
Xenopus oocytes were used for expressing wild type and mutant P2X4Rs. Residues in the
ectodomain-TM1 interface (positions 50-61) were substituted by alanine which resulted
in minimal changes in ethanol response. However when similar alanine substitutions
were made at the ectodomain-TM2 interface (positions 321-337), two residues, D331 and
M336 showed significantly reduced ethanol inhibition of ATP-gated currents (Popova et
al. 2010).

1.6 Building evidence suggesting a role of P2X4Rs in ethanol induced behaviors
It has been found that P2XR channels are sensitive to ethanol at intoxicating
concentrations (Li et al. 1998;Li et al. 2000). Thus ethanol could exert behavioral

4

changes, modifying the function of P2XRs. Pre-synaptic P2XRs have been shown to
assist the release of other neurotransmitters which are major targets for ethanol induced
behavioral effects (e.g. GABA, glycine and glutamate) (Papp et al. 2004;Mori et al.
2001;Chizh and Illes 2001;Deuchars et al. 2002).
Thus any ethanol induced change brought about in the function of the presynaptic
ATP-activated P2XRs may affect the strength of the neurochemical signal from
GABAergic, glycinergic and/or glutamatergic terminals.
Using a genomic analysis, the P2x4 gene which is responsible for the expression
of P2X4R was identified as a potential candidate that may affect the predisposition to
voluntary alcohol consumption (Tabakoff et al. 2009).
Among all P2XR subtypes, the P2X4Rs are the most abundantly expressed
subtype (Buell et al. 1996;Soto et al. 1996) and show the highest magnitude of response
to ethanol (Davies et al. 2002;Davies et al. 2005).

5

CHAPTER 2
SPECIFIC AIMS

This thesis project focuses on the development of the rLV mediated gene delivery
technique as a potential means to test the hypothesis that P2X4Rs play an important role
in causing behavioral effects of ethanol. In order to study gene function it is necessary to
develop efficient gene transfer techniques to introduce the gene of interest into the target
cells and to knockdown or overexpress the target genes.
The Specific Aims and the rationale underlying these aims are described below.
.
Aim 1: Develop rLV techniques to transduce the P2X4 gene into the neuronal and
HEK293 cells.
Aim 2: Generate a mammalian cell line which demonstrates stable expression of
P2X4Rs achieved via rLV mediated transduction technique.
Aim 3: Develop rLV-shRNA techniques for knocking down the P2X4Rs in microglial
cells.
The rationale and background for the specific aims listed above are described in
subsequent pages

6

2.1 Aim 1: Develop rLV techniques to transduce the P2X4 gene into the neuronal and
HEK293 cells
Gene transfer technology using various chemical, lipid and physical methods is a
powerful tool that can be used to study gene function in vitro. The process of introducing
nucleic acids particularly plasmid DNA into eukaryotic cell lines is referred to as
transfection. The different transfection reagents, reporter gene systems and selection
marker genes for stable expression have widened the scope for efficient transfection.
These factors set the stage for studying mammalian promoters and enhancer sequences,
trans-acting proteins, mRNA processing and protein-protein interactions (Groskreutz and
Schenborn 1997).
Viral vectors are used to deliver genetic material into cells. This process is being
used in vivo and in vitro. The term given to the delivery of genes into cells via a viral
vector is called transduction. The advantage of transduction over transfection is that
transduction can nearly infect all cells with lower levels of cytotoxicty. Viruses possess
specialized molecular mechanisms to efficiently carry out transduction. Viral vectors
share a few key properties like cell type specificity, safety, low toxicity and stability.
Most viral vectors possess wide tropism however the viral vector can be modified to
infect specific kinds of cells. Viral vectors are formed from pathogenic viruses however
they do not have a part of the viral genome which is required for viral replication. Many
viruses are unstable genetically and can rearrange their genes.

7

Sophisticated delivery systems such as the use of the rLV technology to deliver
recombinant DNA molecules into host cells have many applications. They have been
used for successful transduction of non-dividing and hard to transfect cells. rLV vectors
are of particular interest in long term expression studies as they integrate with the host
genome.
In earlier work we used Xenopus oocytes as an expression system to study the
effects of ethanol on different P2X4R subtypes. However, in vitro systems with
heterologous expression of receptors do not mimic certain pathways or processes that are
important in the post-translational modification of mammalian ion channels. Hence, it is
important to study these receptors in vivo.
To address this issue, Aim 1 studies focus on the development of rLV techniques
to transduce the P2X4 gene into the neuronal and HEK293 cells. Development of this
technique will allow us to translate the ethanol-related P2XR findings from recombinant
expression systems to neurons.

8

2.2 Aim 2: Generate a mammalian cell line, which demonstrates stable expression of
P2X4Rs achieved via rLV mediated transduction technique
Stable transfection refers to the integration of the gene of interest into the target
genome. The goal of Aim 2 is to generate a mammalian cell line which demonstrates
stable expression of P2X4Rs using the rLV vector. The reasons for doing this are briefly
outlined below.
• A stably transfected mammalian cell line expressing high levels P2X4Rs would
serve as a good model for functional assays including overexpression and
knockdown studies.
• To avoid the discrepancies with day to day transient transfection procedures.
• Currently, mammalian cells that stably express P2X4Rs are not commercially
available. There are few reports which state the use the of cell lines stably
expressing P2X4Rs (Priel and Silberberg 2004;Shinozaki et al. 2009), however
these cells lines were not generated with the use of rLV gene delivery methods.

9

2.3 Aim 3: Develop rLV-shRNA techniques for knocking down the P2X4Rs in
microglia cells
Glial cells which include the astrocytes, oligodendrocytes and microglia are
important in regulating glutamatergic and GABAergic neurotransmission, conduction of
nerve impulses, neurotransmitter metabolism and supply of energy metabolites for
synaptic function (Miguel-Hidalgo 2009). Microglial cells are resident macrophages in
the CNS and contribute to 5-10% of the total population of the glia. Microglia express
many types of P2 purinoreceptors including P2X ionotropic receptors and P2Y
metabotropic receptors (Inoue 2008). Recent evidence supports a role for P2X4Rs in
alcohol consumption, however, the contribution of neuronal and microglial P2X4Rs in
alcohol-induced behaviors is poorly understood (Tabakoff et al. 2009).
The goal of this Aim is to develop rLV-shRNA techniques for knocking down the
P2X4Rs in microglia cells. This new methodology will provide a method for efficiently
silencing the endogenous expression of the P2X4Rs in microglial cells. Once this goal
achieved, we can use the rLV-shRNA system to knockdown the P2X4Rs in vivo and
study the behavioral effects of ethanol. A similar knockdown of P2X4Rs in microglia
using the rLV approach was developed previously to study microglial chemotaxis
(Ohsawa et al. 2007).

10

CHAPTER 3

METHODS AND RESULTS

3.1 Develop rLV techniques to transduce the P2X4 gene into the neuronal and
HEK293 cells

3.1.1 BACKGROUND INFORMATION
We used the pLVX-Puro rLV vector along with the virus packaging mix to
deliver the P2X4 DNA into 293 HEK cells. The resulting virus was used to infect
hippocampal and cortical neurons expressing endogenous P2X4Rs. The Lenti-X
expression vector contains rLV LTRs, rLV packaging signal and a woodchuck hepatitis
post-transcriptional regulatory element (WPRE) which enhances vector packaging by
nuclear export of viral genomic transcripts (Zufferey et al. 1999). The Rev-responsive
element (RRE) renders a higher titer of the virus by increasing the nuclear exportation of
the viral genomic RNA (Cochrane and Chen 1990).
The Lenti-X packaging system provided by Clontech rendered high amount of
titers of a non-replicating, safe and a VSV-G pseudotyped lentivirus. The Lenti-X
packaging mix contains Pol, Tat, Rev, Gag, the VSV-G envelope protein in optimum
ratios (Wu et al. 2000) and the Tet–Off transcriptional activator (Gossen and Bujard
1992). This activator increases the expression of certain viral proteins to higher levels.
The system also provide high levels of biosafety as the resulting recombinant virus lacks

11

essential viral coding sequences which are required for replication. This helps in
generation of a replication-incompetent virus (Wu et al. 2000). A schematic
representation demonstrating the production of the lentivirus is given bellow (Fig 1).

12

Figure 1: Lentivirus production using Clontech’s Lenti-X HT Packaging System and
293T cells.
1. Lenti-X vector and a Lenti-X packaging mix are cotransfected into 293T cells.
2. Transcription and translation results in the production of recombinant rLV genomic
RNA and viral packaging proteins.
3. A packaging sequence on the viral RNA is recognized by the packaging proteins
which form viral cores.
4. These viral cores are transported to the cell membrane.
5. The cores are enveloped by cellular membrane containing VSV-G envelope proteins.
The infectious virions then bud from the cell and are collected in the medium.

13

3.1.2 METHODS
• Isolation of embryonic E18 cortical and hippocampal neurons
In order to obtain neurons, pregnant rats were sacrificed by CO2 inhalation. After
euthanasia, the cutaneous skin of the maternal rats was sterilized with 70 % alcohol for
10 mins. The abdominal area was opened surgically without disturbing underlying organs
and the fetuses from the uterine tissue were removed. The fetuses were decapitated. The
hippocampal and cortical regions were dissected in cold Hank’s Buffer solution. The
tissue is dissociated with mechanical force by using polished Pastuer pipettes. Cells were
plated on poly- D-lysine or polyethylenimine-coated dishes. As glutamate helps better
growth of primary neurons, 25 µM glutamate was added to the cultured neurons. The
incubating medium was changed every 3-4 days and the neurons were allowed to mature
for at least 5 days after which they were used for rLV transductions.

• HEK 293 cell culture
HEK 293 cells (ATCC, Manassas, VA) were grown in DMEM supplemented with
10% fetal bovine serum. Cells were passaged every 3-4 days as the confluence reached
50-80%.

• Lentivirus production for transductions of target cells
The rat P2X4 gene was subcloned into the pLVX rLV vector which resulted in a
P2X4-GFP fusion construct. Lentivirus encoding GFP alone or P2X4-GFP fusion was

14

produced using Lenti-X
TM
HT proprietary packaging mix, Clontech (Mountain View,
CA) consisting of viral, nonviral packaging components and 293T cells. The Lenti-X
Packaging Mix from Clontech provided all the necessary agents for generation of the
virus. 3µg at 0.5 µg/ µl of the plasmid DNA was added to the Lenti-X packaging mix.
The cells were incubated in tetracycline-free Tet System approved FBS. The rLV
supernatants were collected after 48 hours of incubation. The virus was filtered through
cellulose acetate filters (0.45 µm). The virus stock was stored in cryotubes at -80 °C.
The multiplicity of infection (MOI) of the viral stocks was determined using HT-1080
fibroblastoma cells and/or RT-PCR of viral RNAs and appeared to be at least 10(6)/ml.
Fluorescence imaging was used to observe the transduction of target cells.

• Cell lysis and western Immunoblotting
Cells were lysed in lysis buffer containing protease inhibitor (1:100). P2X4 and actin
proteins equivalent to 5-10 µg protein separated by 10 % SDS-PAGE and detected by
western blot analysis with 1 µg/ml anti-P2X4R antibody, 1 µg/ml anti-P2X4R antibody,
and anti-actin antibody (diluted 1:1000) from Chemicon International (Temekula, CA).

15

3.1.3 RESULTS
• rLV mediated transduction of the P2X4R gene into neuronal and HEK 293cells
First, we infected HEK 293 cells with lentivirus encoding GFP or P2X4-GFP fusion
construct to validate the successful production of the lentivirus. HEK 293 cells were
seeded in single 35mm culture plates the day before the infections. Infections were
initiated by addition of 5-20 µl lentivirus (MOI of 10(6)-10(7)/ml). Florescence was
detected 3-10 days post infection. As shown on Fig. 2, transduction efficiency appeared
to be over 90 % for lentivirus encoding GFP as well as P2X4-GFP fusion protein. GFP
fluorescence was long-lasting ( >10 days). In addition, the staining for P2X4-GFP
fluorescence appeared to be punctate and localized to the membranes as opposed to the
cytoplasmic localization of GFP. This resulted in dimmer fluorescence of cells expressing
P2X4-GFP fusion protein. A more magnified image would show a distinct localization of
the P2X4Rs on the plasma membrane. The data validated the successful production of
lentivirus encoding P2X4Rs.

16

Next, we performed rLV transductions of E18 embryonic hippocampal neuronal
cultures. Fig. 3 illustrates fluorescence imaging results for hippocampal neurons
transduced using GFP and P2X4-GFP fusion rLV particles. Transduction efficiency with
P2X4-GFP was lower (50-60%) compared to that of GFP only (> 90%). The P2X4-GFP

Figure 2: rLV mediated P2X4 transduction in HEK293 cells
A, B: FITC and Phase contrast image of HEK293 cells with lentivirus encoding GFP five
days post infection respectively. C, D: FITC and Phase contrast image of HEK293 cells
with lentivirus encoding the rat P2X4-GFP five days post transfection respectively.
50 µm
50 µm

50 µm

50 µm

17

fluorescence demonstrated typical punctate distribution suggesting membrane
localization of P2X4Rs.

Figure 3: rLV mediated P2X4 transduction in rat hippocampal neurons.
A, B: FITC and Phase contrast image of rat hippocampal neurons with lentivirus encoding
GFP five days post infection respectively. C, D: FITC and Phase contrast image of rat
hippocampal neurons with lentivirus encoding the rat P2X4-GFP five days post transfection
respectively. The punctuate pattern is typical of the P2X4R distribution in the cells.

100µm
100µm
100µm

100µm

18

To determine the presence of P2X4R protein, the transduced neuronal lysates were
analysed using western blotting. Antibodies against P2X4R and GFP proteins
demonstrated the presence of a band at a molecular weight of 90 kDa (Fig. 4). P2X4R
protein is commonly detected at a molecular weight of 60 kDa and the weight for GFP
protein is ~ 28 kDa. Therefore, as expected the detected band at 90 kDa represents the
P2X4-GFP fusion protein. These results demonstrated the successful expression of
P2X4R protein in neuronal cells after rLV-mediated delivery of P2X4R gene.

Figure 4: Western blot analysis of neuronal cells.
Lane 1: Non infected rat hippocampal neuronal cells
Lane 2: Neurons infected with rLV encoding GFP
Lane 3: Neurons infected with rLV encoding GFP-P2X4

19

3.1.4 DISCUSSION
It is well recognized that achieving successful transduction in highly
differentiated cells such as the neurons is very challenging. Gene transfer into these cells
using lipid-based reagents has shown low transfection efficiencies (< 10% for LGICs). In
our studies, the use of an alternative rLV-mediated gene transfer in HEK 293 and
neuronal cells demonstrated significantly higher transduction efficiencies. In addition,
typical membrane localization of P2X4Rs was achieved. Taken together, we demonstrate
that rLV delivery technique is an efficient way to deliver vector constructs for
overexpression of P2X4R not well known in primary neuronal cultures. Furthermore, our
findings indicate that rLV delivery of P2X4R gene into neurons can be further used for in
vivo experiments for manipulations of P2X4R expression and subsequent behavioral
tests.

20

3.2 Generate a mammalian cell line which demonstrates stable expression of P2X4Rs
achieved via rLV mediated transduction technique

3.2.1 BACKGROUND INFORMATION
Stable transfection refers to the integration of the gene of interest into the target
genome. Commonly a vector is used to transfer the gene of interest and hence the
expression of the target gene is influenced by the type of vector and the method of
transfection. On the other hand, transient transfection is applicable for a shorter and faster
analysis of gene expression studies. Stable transfections have applications in gene
function, regulation (Grimm 2004), gene therapy (Glover et.al 2005) and protein
production (Wurm 2004). The gene of interest has to travel from the cell to the nucleus
and integrate with the host chromosomal DNA for the efficient transfection to occur. A
vector generally a cationic lipid or a virus is used. The type and time of expression
depends on the type of promoter used. Different promoters are used for constitutive and
regulated expression. For example; CMV promoters are used for constitutive expression
(Worthington et.al 2005). Sometimes the site at which the gene of interest integrates in
the host genome is important. Negative effects due to random integration can be avoided
by the use of transposons which are site specific and homologous (Ivics et.al 1997;Keng
et.al 2005). Selection of stably transfected cell lines can be achieved by including a
marker gene which provides the cells resistance against an antibiotic. When the antibody

21

is added to the cells, only the cells that have incorporated the gene of interest carrying the
marker gene will survive while the non-transfected ones will die.
The variety of systems used for selection includes genetic markers resistant to
dihydrofolate reductase (DHFR), glutamine synthetase, puromycin, neomycin
phosphatase. For instance, Neo, a bacterial gene which confers resistance to neomycin-
kanamycin antibiotics is inserted into a SV40 hybrid plasmid vector. When this vector is
used to transfect mammalian cells, only the cells that have incorporated the neo gene tend
to survive and grow in the presence of antibiotic G418 whereas the normal non-
transfected ones die (Southern et.al 1982).
A stable population can be sorted out by FACS (Fluorescence Activated Cell
Sorting) in order to determine the percentage of transfected cells and to exclude the non-
transfected cells.
A number of factors play an important role in influencing the efficiency of stable
transfection. They include the number of freeze-thaw cycles, number of passages, cell
confluency, contaminants or cell health and the quantities of DNA, green fluorescent
protein and the viral titer. It is important that the cells grow in a suitable medium which
provides serum and growth factors required for cell survival. The media or any other
component that comes in contact with the cells should be free of contamination. The
incubator at 37˚C should be supplied with CO
2
and the relative humidity should be
maintained at 100%. The American Type Culture Collection (ATCC) web site should be
referred to select the type of culture, media and the CO
2
levels. The confluency of the cell

22

line is an important point to consider. To transfect cell lines, a confluency of 55-80%
should be maintained. Too many cells, too close to each other prevent efficient uptake of
foreign DNA. Less density will cause the culture to grow poorly and the uptake of DNA
will be lesser and inefficient. It is necessary to thaw a new batch of cells every now and
then from a frozen, uncontaminated stock. Many cell characteristics change with time and
the cells respond differently over a period of time after repeated passages. Hence the
number of passages should be kept low.
Reporter gene assays are ideal for determining the success of transfection.
Transfection efficiencies can be validated with plasmids that contain reporter genes. In
case of siRNA or shRNA, the target protein levels are determined by western blotting or
RT-PCR.
Apart from transfecting the gene of interest in a cell line to study gene function,
one can study the function of a gene by silencing the gene using methods such as RNA
interference. RNA interference is an antisense based approach that can be used to
knockdown a particular gene.

23

3.2.2 METHODS
HEK 293 cells grown in T25 flasks to 50-80% confluence were transduced using
rLV encoding P2X4-GFP fusion construct (see 3.1.2 Methods for the details of rLV
production). 3-5 days post-transduction of the cells, protein expression was observed
using a fluorescent microscope. The transduction efficiency was ~80 %. A stable
expression of the gene was achieved by using puromycin-resistance included in the
pLVX rLV vector. Two days post infections puromycin (0.5µg/ml) was added to the cells
and continued growing. Only the cells that have incorporated the gene of interest and
carrying the marker gene survived while the non-transduced did not. The cells were
further grown in media containing puromycin.

3.2.3 RESULT
• rLV-mediated transduction of HEK 293 cells resulting in stable expression of P2X4Rs
The stably transfected HEK 293 cell line expressing the P2X4-GFP protein was
generated in order to use for validation of the knockdown with the rLV shRNA
expression system. A mammalian HEK 293 cell line infected with lenti-X vector carrying
the P2X4 gene along with GFP resulted in a stable expression of the P2X4Rs with the use
of a selection marker gene, in this case, puromycin. The expression of P2X4-GFP was
long-lasting showing no change in fluorescence intensity upon observation for more than
1.5 months. The images shown in Fig. 5 were taken 47 days post initial transductions.
Interestingly, there was also no change in the number of fluorescing cells (80%) over the

24

course of cell growth and re-cultivation. Moreover, the staining pattern of GFP
fluorescence (Fig. 5) showed distinct membrane localization for P2X4R that repeated our
earlier transient HEK 293 and neuronal transductions (see section 3.1.3).

25

Figure 5: rLV mediated stable P2X4 transduction in HEK293 cells.
A, C, E: Phase contrast images of the stably transfected P2X4Rs in HEK293 cells and
B, D, F: FITC images of the stably transfected P2X4Rs in HEK293 cells taken at 10X
magnification
50 µm

50 µm

50 µm

50 µm

50 µm

50 µm

26

To determine whether the observed fluorescence was derived from the presence
of a functional expression of P2X4Rs, we performed patch-clamp electrophysiological
recordings of these cells expressing P2X4Rs. ATP (10 µM) induced currents in these
cells that were characteristic for those of rat P2X4Rs (Fig.6). Similar currents were
observed from HEK 293 cells transiently transfected with rat P2X4Rs. As a negative
control, our laboratory has previously shown that there are no functional endogenous
P2X4Rs in HEK293 cells as evidenced by a lack of ATP induced currents in these cells.
These findings suggested that the rLV technique can be used to generate a cell
line demonstrating a long-lasting functional expression of P2X4Rs in HEK 293 cells.
However, there should be caution taken. For example, we found that there was a loss in
cell viability upon longer cultivation of the HEK 293 cell line stably expressing P2X4Rs.
This could be explained by extremely high level of P2X4Rs which could permit a Ca
2+

influx thus compromising the cell health. On the other hand, the decrease in cell viability
could be related to the rLV itself. Our laboratory is continuing to optimize the use of rLV
techniques in generation of stable cell lines.

27

3.2.4 DISCUSSION
A HEK 293 cell line stably expressing the P2X4R was generated as the next step
towards studying the function and behavioral aspects of P2X4Rs. We sought to use this
cell line to validate the shRNAs for silencing of P2X4Rs.
Our results demonstrate that rLV mediated delivery of P2X4Rs can be
successfully used to generate a cell line that stably expresses P2X4Rs. One advantage of
the rLV transduction is that it has significantly higher transduction efficiency as
compared to the lipid based transfections. Therefore, no sorting of the cells is required for
enrichment of the cell line. Furthermore, the expression of P2X4Rs is long-lasting and
will serve as a good model to perform functional assays.

Figure 6: Patch clamp recordings of ATP currents across P2X4Rs expressed in a
transiently and stably expressed 293HEK cell line.
A; ATP currents in HEK 293 cells stably expressing P2X4Rs
B; ATP currents in HEK 293 cells transiently expressing P2X4Rs

28

3.3 Develop rLV-shRNA techniques for knocking down the P2X4Rs in microglial cells

3.3.1 BACKGROUND INFORMATION
• Mechanism of RNA Interference
RNA interference (RNAi) is a cellular process in which a double stranded RNA
sequence specifically results in the inhibition of gene expression. The numerous other
cellular proteins involved in the efficient process of posttranscriptional gene silencing are
highly conserved in eukaryotes. Andrew Fire and Craig Mello were honored by the 2006
Nobel Prize in Medicine or Physiology for this achievement (C.C. Mello 2007). Craig
and Fire observed inhibition in gene expression when they introduced a double stranded
RNA into C.elegans (Fire et.al 1998). The initiation of the RNAi pathway is carried out
by the enzyme Dicer which is found in many eukaryotes. The RNA – induced silencing
complex is activated by the presence of a short double-stranded RNA molecule in the
cytoplasm of the cell. Dicer cleaves long double-stranded RNA (dsRNA) molecules into
short fragments of ~20 nucleotides. The guide strand or the antisense strand then binds to
the RNA-induced silencing complex (RISC) (Kurreck et.al 2009). The guide strand base
pairs with a complementary sequence of the messenger RNA molecule and undergoes
cleavage by Argonaute (Liu et.al 2004), which is the catalytic component of the RISC
complex. Once target mRNA is cleaved, it lacks the elements like the poly A tail on the
3’ end and 5’ end cap and the cleaved mRNA undergoes rapid degradation by RNases
(Song et.al 2004).

29

Immediately after the siRNA enters the cell, degradation of the target RNA begins,
however a decrease in the amount of protein is seen at a later stage. Usually a significant
decrease in the amount of protein is observed in the cell culture within 48 h of
transfection of a siRNA. However some proteins are stable for a longer period and have a
slower rate of turnover. It is important to consider that siRNA does not cause a complete
shutdown of a gene and hence RNAi is a knockdown technology rather than a knockout
technology (Kurreck et.al 2009). A schematic representation demonstrating the
mechanism of RNAi is given below (Fig.7).

30

Figure 7: Mechanism of RNA interference in mammalian cells.
The enzyme Dicer cleaves the long double -stranded RNA molecules into short fragments
of 20 nucleotides. The guide strand or the antisense strand binds to the RNA induced
silencing complex.(RISC). The guide strand base pairs with the complementary sequence
of the messenger RNA and undergoes cleavage.

31

• Delivery of siRNA

siRNA can be packed into liposomes surrounded by PEG , cationic lipids or can be
coupled with an antibody, aptamer, peptide or cholesterol (Kurreck et.al 2009).
Replication deficient viruses are used to transfer the gene of interest. The central coding
region is replaced with a shRNA expression cassette. The different types of viral vectors
include the retrovirus, adeno-associated virus and the adenovirus.

• Limitations of siRNA
o Off-Target effect
As few as eleven nucleotides are required for the antisense strand of the siRNA to
complement the mRNA and this can result in the down-regulation of an off-target
mRNA. The viability of the cells can be affected by off-targeting (Fedorov et.al
2006). A specific design of the siRNA can reduce the off-target effects. Also
modified nucleotides can be included in the sense strand to completely inactivate it.
An example of such a modification would be the single 2’ O-methyl substitution on
the ribose of the second nucleotide which helped reduce off-targeting effects (Jackson
et.al 2006).

32

o Interferon response
dsRNA greater than 30 nucleotides in length activate an interferon response. In
case of siRNA which are smaller in length, the interferon response is activated by
protein kinase R (Kurreck et.al 2009).

• Therapeutic Applications
In order to study the function of a gene, RNAi is validated in cell cultures and then
used in vivo (Kurreck et.al 2009). By causing a significant decrease in the expression of
the target gene, the physiological role of the gene can be studied.
High-throughput screening methods using microarrays have been used with siRNA
(Janitz et.al 2006)
siRNA has a number of applications in eye diseases, viral infections and cancer. Sirna
therapeutics initiated the first siRNA molecule directed towards the VEGF receptor 1.
This was used in the treatment of age-related macular degeneration (Kurreck et.al 2009;
Haussecker et.al 2008). siRNA has applications in treating most viruses including HIV-1,
HBV, HCV,SARS-coronavirus, polio virus, coxsackie virus have been investigated.
(Kurreck et.al 2009;Haussecker et.al 2008). Many studies have been published to show
that the tumor growth was inhibited in mice treated with siRNA against an oncogene, the
siRNA designed against CD31 inhibited growth of tumors in mouse models (Kurreck
et.al 2009;Santel et.al 2006).

33

• Knockdown studies with Lenti-X vector
The Lenti-X shRNA Expression System from Clontech (Mountain View,CA) was
used to introduce shRNA into target cells for the suppression of genes by the RNA
interference. The pLVx-shRNA vector contains the shRNA silencing cassette, this along
with the Lenti-X Packaging Mix and the Lentiphos HT transfection agents are added to
the HEK 293 T packaging cell lines. The resulting virus is then used to infect target cells
and initiate the silencing process. A small hairpin RNA works via this RNA interference
to silence gene expression. A lentivirus can be used as a vector to deliver the shRNA into
the cells. It uses the U6 promoter so that the shRNA is always expressed. The shRNA
hairpin structure then gets cleaved by the cellular machinery into siRNA which then
attaches to the RNA –induced silencing complex (RISC). The complex binds to the
matching mRNA and cleaves it. The transcription of shRNA is carried out by the RNA
polymerase III.
The design and preparation of rLV vectors expressing siRNA for down-regulation of
specific target genes is described in detail in this section and in the Methods section
(3.3.2). The Human Immunodeficiency Virus type 1 (HIV) has been used as a vector
which is capable of transducing dividing and non dividing cells. These vectors can
integrate in the host genome and result in the long term expression of the gene of interest.
The HIV genome contains nine open reading frames which encode for 15 different
proteins which are involved in the infectious cycle along with structural and regulatory
proteins .There are some cis-acting elements which play an important role in the different

34

stages of the viral life cycle (Singer et.al 2008;Zufferey et.al 1999). To avoid an HIV
infection, the trans-acting factors are separated from the cis-acting factors which are vital
in viral particle production, infection and integration. The widely used third generation of
rLV vectors consists of four plasmids. The transfer vector is the one which carries the
transgene to be delivered and the cis-acting sequences which are important for RNA
production and packaging. The packaging system involves three other plasmids that
provide the trans-acting factors eg pMDL, pRev, and pEnv provide the required trans-
acting factors viz Gag-Pol, Rev and an envelope protein respectively. Gag-Pol codes for
integrase, reverse transcriptase and structural proteins. Integrase and reverse transcriptase
are required for infection whereas the structural proteins are required for production of
the viral particle. Rev interacts with the Rev-responsive element (RRE) which is present
in the transfer vector. This is important in the nuclear export of the viral genomic RNA.
The vesicular stomatitis virus protein G (VSV-G) is a commonly used envelope protein
which helps transduce a wide variety of cell types including the primary cells, stem cells
and early embryos. The transfer vector also contains the woodchuck hepatitis regulatory
element (WPRE) that enhances gene expression (Zufferey et al. 1999) and a central
polypurine tract (cPPt) to increase efficiency of nuclear import. Replication deficient rLV
particles are formed by deletion of the 3’ long terminal repeats. The proviral 5’LTR is
copied from the 3’ LTR during reverse transcription and hence the deletion is transferred
to the 5’LTR. The deleted 5’LTR does not allow the process of transcription (Miyoshi
et.al 1998;Brummelkamp et.al 2002). This prevents further viral replication.

35

• Design of rLV vectors expressing shRNA
siRNAs can be expressed as short hairpin RNAs (shRNA) when pol III promoters are
cloned into plasmids (Brummelkamp et.al 2002;Miyagishi et.al 2002). The commonly
used pol III promoters are H1 and U6. They have a compact size of less than 400bp and
all sequences for promoter function lie upstream of the transcriptional starting site
(Singer and Verma 2008;Miyagishi and Taira 2002). These promoters efficiently express
short RNAs and show ubiquitous expression. They express shRNAs which contain a 21-
23 nucleotide sense sequence that is similar to the target mRNA which has to be
downregulated, followed by a 9 base pair loop sequence, followed by a 21-23 nucleotide
sequence which is antisense to the target sequence. A continuous stretch of 5-Ts is what
gives a pol III signal. The loop is digested by Dicer enzyme and the siRNA eventually
causes degradation of the mRNA target. Usually the silencing vector contains a green
fluorescent protein and an antibiotic resistant gene. The rLV vectors carry a GFP as a
marker and the human H1 –driven silencing cassette into a restriction site in the 3’ LTR
(Singer et.al 2008;Tiscornia et.al 2003). When integration takes place, the 5’LTR in the
provirus gets copied from the 3’ LTR and the H1-driven shRNA gets cloned into the
3’LTR. This results in duplication of the silencing cassette. This helps in increasing the
silencing power of the vector. The multiplicity of infection which is required to silence a
target gene will depend on the levels of expression of the target mRNA, the siRNA and
the cell type transducibility involved.

36

Development and validation of an efficient rLV silencing vector involves some
integral steps like selection of siRNA target sequences, designing the shRNAs, cloning of
the shRNA including validating the effect on the target gene, cloning and testing the rLV
silencing vector. The constraints in the RNAi pathway and accessibility of the target
sequence in the target mRNA can be significant hurdles in the effectiveness of a
particular siRNA. The design of shRNA has some specifications. The target sequence
should be ideally 21-23 bases long however lengths up to 28 bases have been reported
(Singer et.al 2008;Tiscornia et.al 2003;Paddison et.al 2002). Very long targets should be
avoided as the longer double-stranded RNA molecules trigger a protein kinase response
(Clemens et.al 1997). GC content is between 40% -55%. The U6 promoter requires a G
in its first base but shRNAs with the H1 promoter can start with any base. shRNAs can be
directed to the 5’ UTR ,ORF or to the 3’ UTR of the target mRNA. The loop has a
particular 9 -bp sequence (TTC AAG AGA) (Brummelkamp et.al 2002).
Generally several shRNA sequences should be selected and validated for the target
gene. After the shRNAs are cloned in a plasmid which contains the silencing cassette and
are tested, the validated ones are then transferred to the rLV vector.
Screening can be best achieved by cotransfecting a shRNA –RLV plasmid and a
vector which expresses a tag generally myc and FLAG cDNA of the target into a typical
cell line usually 293T. This is followed by western blot analysis. There are other ways of
validating the effectiveness of shRNAs. After successful transfection of the plasmids into
the cell line of interest, reverse-transcriptase-polymerase chain reaction (RT-PCR) can be

37

carried out. Northern blots can also be carried out. Analysis of the target protein by
western blots against the endogenous target is another alternative. Once potential shRNA
candidates are identified they should be cloned into viral vectors. The high titer
preparations should be tested by transduction into a stably transfected cell line expressing
the target protein or a cell line which expresses the endogenous target protein. The
validation of a functional rLV silencing vector is very crucial and can be done by using a
specificity control which includes the use of a rLV vector lacking a silencing cassette or
carrying the cassette against a different target. This type of validation is vital as any of
the shRNAs can cause nonspecific down-regulation of gene expression. Homogenously
transduced cell populations can be examined with fluorescence activated cell sorting
(FACS) for GFP- positive cells or by using a marker which is an antibiotic resistant gene
(Singer and Verma 2008).
A shRNA was designed to knockdown the expression of endogenous P2X4 in
microglial cells. The rLV vector was used for delivery of the shRNA oligonucleotides. A
lentivirus encoding P2X4R-GFP fusion protein and shRNA oligonucleotides targeting
P2X4Rs was designed in the following manner.
The Lenti-X shRNA Expression System from Clontech was used to introduce
shRNA into target cells for the suppression of genes by the RNA interference. The pLVx-
shRNA vector contains the shRNA silencing cassette, this along with the Lenti-X
Packaging Mix and the Lentiphos HT transfection agents are added to the HEK 293 T

38

packaging cell lines. The resulting virus can then be used to infect target cells and initiate
the silencing process.
The shRNA contains a defined target sequence for any particular gene and is
expressed from a cloned oligonucleotide template which renders stable RNA interference
in the target cells (Brummelkamp et al.2002; Paddison et al 2002). The RNA hairpin of
the shRNA triggers endogenous RNA interference which generally responds to double-
stranded DNAs. The shRNA forms a stable stem loop on Pol III transcription and is
further processed to form a double stranded small interfering RNA (siRNA) that guides
the RNA –induced silencing complex in the degradation process of target mRNA. The
shRNA target site contains two complementary oligonucleotides. The oligonucleotide
sequences should contain restriction sites which allow annealing into the pLVX –shRNA
vectors. A 5’-BamHI on the upper strand and a 5’-EcoR1 on the lower strand enables the
shRNA sequence to anneal into the vector. For a Pol III transcription start site a guanine
residue should be present upstream of the 5’end of the shRNA sense strand. A 19 base
target sense and antisense sequence is separated by a typical sequence which 7-9
nucleotides. This typical sequence is the hair loop sequence. Usually a diagnostic
restriction site is essential immediately after the terminator sequence.

A schematic representation of the shRNA designed to knockdown the P2X4Rs is
given below (Fig.8).

39

Figure 8: Design of Small hairpin RNA (shRNAs) generated from a cloned oligonucleotide
DNA template to knockdown rat P2X4Rs.
The target sequence is derived from the coding region of the rat P2X4 gene. The shRNA –coding
oligonucleotides containing the sense and antisense target sequence are cloned downstream of a
Pol III promoter in an expression vector designed for gene silencing in mammalian cells. A
hairpin shaped loop is situated between the sense and the antisense sequences on each
complementary strand. The transcribed shRNA acts like a ds siRNA molecule and carries out
gene silencing.

40

3.3.2 METHODS
The following steps were followed in order to use the Lenti-X shRNA System to
efficiently silence the P2X4 gene.

• Plasmid DNA Propagation and Purification
In order to have a renewable source of plasmid DNA, the plasmid vector obtained
from Clontech (Mountain View, CA) was transformed into the XL 10-Gold
Ultracompetent cells, Cat No 200315. The QIA Prep Spin Miniprep Kit (Cat No 27108)
was used for plasmid propagation.

• Selection of Target Sequences and Designing shRNA Oligonucleotides
The mRNA sequence of the rat P2X4 gene obtained from NCBI (NM_031594.1) was
fed into the online Clontech RNAi designer tool and shRNA oligonucleotides were
designed against three target sequences (S1, S2, S3).

41

Target sequence S1 GGTCCTTCCTGTTCGAGTA
Top Strand (66bp)

5'gatccGGGTCCTTCCTGTTCGAGTATTCAAGAGA
CAACCCTGAGTATTTGTGGTTTTTTACGCGTg----
-3'
Bottom strand (66 bp)
5'aattcACGCGTAAAAAACCACAAATACTCAGGG
TTGTCTCTTGAATACTCGAACAGGAAGGACCCg
-----3'

Target sequence S2 CCACAAATACTCAGGGTTG
Top Strand (66bp)
5'gatccGCCACAAATACTCAGGGTTGTTCAAGAG
ACAACCCTGAGTATTTGTGGTTTTTTACGCGTg-
----3'
Bottom Strand (66bp)
5'aattcACGCGTAAAAAACCACAAATACTCAGGG
TTGTCTCTTGAACAACCCTGAGTATTTGTGGCg-
----3'

Target sequence S3 CTCAGATGGGCTTCAGATA
Top Strand (66bp)
5'gatccGCTCAGATGGGCTTCAGATATTCAAGAG
ATATCTGAAGCCCATCTGAGTTTTTTACGCGTg-
----3'
Bottom Strand (66bp)
5'aattcACGCGTAAAAAACTCAGATGGGCTTCAG
ATATCTCTTGAATATCTGAAGCCCATCTGAGCg-
----3'

Table 1: The target sequences of the rat P2X4R and shRNA oligonucleotide design

42

• Cloning of the shRNA Oligonucleotide in the pLVX-shRNA vector
The synthesis and cloning of the oligonucleotides for the S1 target sequence into
pLL3.7 vector was performed at Ernest Gallo Clinic and Research Center/UCSF. The
oligonucleotides for S2 and S3 sequences were synthesized by IDT (Integrated DNA
Technologies, San Diego, CA) and subcloned into the pLVX-shRNA1 vector (Clontech)
in our laboratory. For this, the pLVX-shRNA vector was digested with the BamHI and
EcoR1, the resulting DNA was purified using 1.2 % agarose gel. The purified vector was
resuspended in 10-20 µl TE buffer to achieve a concentration of 48 ng/µl. This was
stored at -20 °C.
The shRNA oligonucleotides were annealed by mixing the upper and the lower
strands in the ratio of 1:1. Each purified oligonucleotide was suspended in TE buffer to a
final concentration of 100 µM. The mixture was subjected to heat and cool cycles and
was then stored on ice. The annealed oligos at a concentration of 0.5 µM were then
mixed with the digested pLVX-shRNA1 vector DNA and the ligation was carried out
using T4 DNA ligase. The resulting pLVX-shRNA1 vector containing the shRNA was
transformed into Supercharge EZ10 Electrocompetent cells. The QIA Prep Spin Miniprep
Kit (Valencia, CA) was used to prepare plasmid DNA minipreps. The desired
recombinant plasmid was identified by using diagnostic restriction site enzymes within
the shRNA oligonucleotide sequences. The USC/Norris DNA Core Facility confirmed
the shRNA insert.

43

• Generation of RLV shRNA
293 T cells were used to package the virus. 3µg at 0.5 µg/ µl of pLVX – shRNA
plasmid DNA was added to the Lenti-X packaging mix provided by Clontech (Mountain
View, CA). The procedure that I used for the production of the lentivirus was described
in detail in Section 3.1.2.

• Infection of target cells with the rLV shRNA
HEK 293 cells which were stably transduced with P2X4-GFP were plated on 35 mm
plates. After incubating at 37 °C for one day, the rLV-shRNA (5 -20 µl) was added to the
plates. Polybrene (2 µg/ml) was added for greater infection efficiency.
A microglial cell line BV-2 expressing endogenous P2X4Rs was also used to validate
the RNA interference provided by the shRNA. The BV-2 cells were plated in 12 well
plates and after 24 hours rLV-shRNA (5 - 20 µl) were added. Infections were carried out
in the presence of polybrene (2 µg/ml).

• Cell lysis and Western blotting analysis
Transduced cells were observed 3 days post infection under a fluorescence
microscope. The cell lysis and western immunoblotting using antibodies against P2X4,
P2X7 and β-actin (Chemicon International, Temekula,CA) proteins was performed as

44

described in Methods section (3.1.2).
3.3.3 RESULTS
• Molecular biology indicates successful generation of shRNAs
The insertion of the shRNA into the pLVX vector was verified by digestion with
restriction enzymes Pst II and EcoRI. The two restriction enzymes generated 1313bp and
516 bp DNA fragments (Fig.9). This confirmed the ligation of the shRNA
oligonucleotides into the pLVX vector. This was verified by DNA sequencing.

• rLV mediated delivery of shRNA to silence P2X4Rs in microglial cells.
First, HEK 293 cells stably expressing P2X4-GFP fusion protein were used to test
shRNAs. We expected to see a decrease in GFP fluorescence after shRNA transductions.
1313 bp

516 bp
Figure 9: Generation of DNA fragments from shRNA1- pLVX vector.
Pst II and EcoRI restriction enzymes were used to validate the inclusion of the shRNA
oligonucleotides into the pLVX vector. Two DNA fragments of size 1313 bp and 516
bp were generated.

45

However, there was no change in the GFP fluorescence with any of the shRNAs tested.
It is possible that the cells develop resistance to rLV infections. This issue requires
further testing and is beyond the scope of this work. Thus, it became necessary to validate
the shRNA using cells endogenously expressing P2X4Rs.
BV-2 mouse microglial cell line expressing endogenous P2X4Rs was selected to
validate the knockdown. Lentivirus encoding shRNA1, shRNA2 and shRNA3 were used
to infect microglial cells. The knockdown of P2X4Rs was validated by western blotting
analysis. ShRNA1 was not effective in knocking down P2X4Rs whereas shRNA2 and
shRNA3 reduced P2X4R expression by more than 50% (Fig.10). Combination of
shRNA2 and shRNA3 inhibited the P2X4R protein expression by 85% (Fig.10). There
was no change in the P2X7R protein levels with any of the shRNAs used suggesting that
these oligos were specifically affecting the expression of P2X4Rs. These data also
suggest that combining two different shRNAs is an effective mean to knockdown
P2X4Rs in BV-2 microglial cells.

46

Figure 10: Protein expression of P2X4R and P2X7 in the microglial transduced with
the shRNA rLV vector.
A) A significant decrease in the protein expression on P2X4Rs on transduction with
the shRNA (2+3) rLV vector is observed. β-actin is used as the internal control.
shRNA 1 was unable to knockdown the expression of P2X4Rs.
B) Densitometry analysis of the P2X4R and P2X7R protein bands normalized on the
β-actin level from 3 separate experiments.

47

3.3.4 DISCUSSION
Validation of shRNAs was attempted in HEK 293 cells stably expressing P2X4Rs
and BV-2 microglial cells. However, HEK 293 cells did not demonstrate inhibition of
P2X4Rs upon shRNA infections. There is a possibility that the cells developed resistance
to repeated rLV infections. To overcome this issue, we have used the microglial BV-2
cell line that endogenously expresses P2X4 and P2X7R subtypes. Currently, the role of
microglia in alcohol-induced behaviors is not well understood. P2X4R knockdown
studies are becoming important in the light of recent findings that support a role for
P2X4Rs in alcohol consumption. Our studies showed that a successful knockdown of
P2X4Rs in microglial cells can be brought about by the rLV mediated delivery of
shRNAs. Importantly, we were able to achieve P2X4R knockdown by combining two
different shRNAs during microglial transductions.

48

CHAPTER 4
CONCLUSION

The goal of this thesis project was to develop and use the rLV technologies for
efficient delivery of P2X4R gene/shRNA in neuronal and microglial cells. The findings
demonstrated the successful use of the rLV mediated gene transfer technique as a tool to
overexpress/knockdown the expression of P2X4Rs in vitro. The results of these studies
outlined below will set the stage for using the rLV technology in vivo and further
investigating the physiological and cellular roles of neuronal and microglial P2X4Rs in
ethanol-induced behaviors.
1. Successful transductions of the P2X4R gene into the neuronal and HEK293 cells
validated the effectiveness of a rLV vector for the delivery of P2X4R gene.
Importantly, the presence of functional P2X4Rs was achieved using rLV-mediated
transductions.
2. The significantly higher transduction efficiencies produced using the rLV gene
delivery developed in this proposal demonstrated its advantage over the lipid-based
transfections for overexpression of P2X4Rs in neurons and HEK293 cells.
3. rLV mediated delivery of P2X4Rs and puromycin selection in HEK293 cells resulted
in a highly enriched cell line that stably expressed P2X4Rs. The P2X4-HEK293 cell
line may serve as a good model to perform functional assays. However, a second
rLV-transduction may not be possible in these cells.

49

4. A successful knockdown of P2X4Rs in microglial cells can be brought about by the
rLV mediated delivery of shRNAs. Importantly, combining two different shRNAs
during microglial transductions resulted in higher degree of inhibition of P2X4R
expression. BV-2 microglial cells endogenously expressing P2X4Rs can serve a
good model for validation of rLV-shRNAs.

50

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Abstract (if available)

Abstract The current study focused on the development of recombinant lentiviral (rLV) mediated gene transfer techniques that would aid in testing the hypothesis that the purinergic P2X4 receptors (P2X4Rs) play an important role in behavioral effects of alcohol. Ethanol inhibits ATP-induced currents in P2X4Rs recombinantly expressed in oocytes and HEK293 cells. Building evidence suggests that P2X4Rs play a role in alcohol consumption. P2X4Rs are widely expressed in neurons and glial cells, however little is known about the contribution of neuronal and/or glial P2X4Rs in alcohol-induced behaviors. In order to study the action of ethanol on P2X4Rs in these cells, it is necessary to develop tools to over-express/knockdown the expression of the receptors. The recombinant rLV gene delivery techniques were developed to express P2X4Rs in neuronal and HEK293 cells (Aim 1)

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Asset Metadata

Creator Inamdar, Sneha (author)

Core Title Development of lentiviral vector mediated gene transfer techniques as tools to overexpress/knockdown P2X4 receptors in vitro

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 07/11/2010

Defense Date 06/05/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag gene transfer,lentiviral techniques,OAI-PMH Harvest,P2X4 receptors

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Alkana, Ronald L. (committee chair), Davies, Daryl L. (committee chair), Okamoto, Curtis Toshio (committee member)

Creator Email sinamdar@usc.edu,snehainamdar@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3186

Unique identifier UC1209632

Identifier etd-Inamdar-3854 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-348629 (legacy record id),usctheses-m3186 (legacy record id)

Legacy Identifier etd-Inamdar-3854.pdf

Dmrecord 348629

Document Type Thesis

Rights Inamdar, Sneha

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu