ENGINEERING CHO CELLS TO WITHSTAND AMINO ACID STARVATION
A Thesis
Presented to the Faculty of the Graduate School
of Cornell University
In Partial Fulfillment of the Requirements for the Degree of
Master of Engineering
by
Laura Yinran Zheng
August 2008
1
©2008 Laura Yinran Zheng
2
ABSTRACT
Chinese Hamster Ovary (CHO) cells are often times used in the pharmaceutical
and bioprocess industry to produce glycosylated protein therapeutics, and are typically
grown in serum free media to meet quality control standards. This presents the problem
of proteolytic attacks on the final protein product, as the absence of serum also leads to
an absence of serum protease inhibitors. It is possible to inhibit protease activity in CHO
cells using EDTA and ophenantroline, but it is proposed that RNAi can also be used to
inhibit protease activity in CHO cells without permanent alteration of cell genotype.
RNAi was used via introducing small interfering RNAs (siRNAs) produced against the
protease Matrix Metalloproteinase 14 (MMP14) to test the efficacy of silencing the
protease through the use of siRNAs. The most effective siRNAs were then tested via cotransfection with the vector pEGFP-N1 as well as several controls to determine the effect
of silencing the MMP14 on the expression of EGFP. It was then determined that some
siRNAs were effective in silencing the activity of MMP14 which led to an increase in the
activity of EGFP fluorescence. It can be concluded that MMP14 is active in CHO cells
and under normal conditions, will attack the EGFP protein, but once silenced, it cannot
attack EGFP or other proteins and can therefore lead to an increase in their expression.
However, to verify these claims, further testing will need to be performed under
conditions of amino acid starvation. A recombinant plasmid utilizing the sequence of the
most effective siRNA will need to be created and tested under amino acid starvation
conditions to verify the efficacy of RNAi as a silencing mechanism for CHO cells
growing in bioreactors.
3
BIOGRAPHICAL SKETCH
Laura Zheng received her B.S in Biological Engineering Technology at Cornell
University in 2007.
4
ACKNOWLEDGEMENTS
I would like to thank Professor John March, Faping Duan, Emma Risen, and Magdelena
Steinruck
5
TABLE OF CONTENTS
Biographical Sketch
4
Acknowledgements
5
Table of Contants
6
List of Figures
7
List of Tables
8
CHAPTER 1: Introduction
9
CHAPTER 2: Materials and Methods
16
CHAPTER 3: Results
26
CHAPTER 4: Discussion and Future Directions
32
CHAPTER 5: Conclusion
37
REFERENCES
38
6
LIST OF FIGURES
SiRNAs Designed. ........................................................................................................ 19
Layout of DNA/siRNA Co-Transfection. ..................................................................... 23
RT-PCR Results of siRNA Transfection. ..................................................................... 26
RT-PCR Results of DNA/siRNA Co-transfection ........................................................ 28
Average Intensity of EGFP Expression. ....................................................................... 30
Normalized Average Intensity of EGFP Expression. ................................................... 30
Histograns of EGFP Expression. .................................................................................. 31
Layout of DNA/siRNA Co-Transfection. ..................................................................... 32
Schematic of pAARE-MMP14 Vector ......................................................................... 35
Layout of pAARE-MMP14 Testing. ............................................................................ 36
7
LIST OF TABLES
Liquid Volumes of RNA and Water for RT PCR ............................................................. 21
Substances Used for DNA/siRNA Co-transfections......................................................... 22
Volumes of RNA and Water for Second RT-PCR .......................................................... 24
PCR Protocol for RT-PCR ................................................................................................ 25
Average intensities (FITC-A) of EGFP Expression ......................................................... 28
8
CHAPTER 1
Introduction
Chinese Hamster Ovary Cells
Chinese Hamster Ovary (CHO) cells are very important to the pharmaceutical industry,
as these cells are commonly used for the production of glycosylated recombinant
proteins1 such as all therapeutic recombinant antibodies2 and tissue plasminogen
activator (tPA)3 for pharmaceutical purposes4. Current therapeutics on the market
produced from CHO cells include TNKase (Tenecteplase, Genentech), ReFacto(
recombinant Antihemophilic factor, Genetics Institute), Enbre (Etanercept, Immunex),
Herceptin (Trastuzumab, Genentech), Benefix (recombinant Coagulation factor IX,
Genetics Institute), Avone (Interferon β-1a, Biogen), and Rituxan (Rituximab, IDEC
Pharmaceutical and Genentech)5. CHO cells are the pharmaceutical industry’s standard
for the production of complex protein therapeutics because of their capacity for single
cell growth6, which suspension culture in stirred tank bioreactors is now the norm for
recombinant protein production7.
Long term cultures of CHO cells that maintain a high level of productivity are needed for
cost-effective production of recombinant protein products8. However, there are
challenges to maintaining a high level of productivity over time. CHO cells are typically
grown in highly defined serum free media to meet quality requirements, but due to the
absence of serum protease inhibitors, recombinant protein product may be susceptible to
proteolytic attacks that can cause protein heterogeneity, denaturation and loss of function
9
and also decrease recombinant protein yield9.The activity of extracellular proteases can
have a major effect on the production of recombinant proteins in this cell line by reducing
the yield of intact protein10. Amino acid deprivation represents a form of metabolic stress
that presents enormous potential physiological relevance to those in the bioprocess
industry.
Protease Activity
Mammalian cells require a sufficient supply of amino acids for proper protein and
nucleotide biosynthesis11. When mammalian cells are cultured in limiting amino acid
concentrations, the overall rate of protein synthesis falls12. This may be due to several
different reasons but is most predominantly due to the activity of proteases in amino acid
limited conditions. P. Eliot, A Hohmann, and J. Spanos characterized proteases in a
gelatin zymography and Western immunoblotting. They found that the predominant
extracellular protease secreted by CHO-K1 cells was the matrix metalloprotease-9
(MMP-9), also known as gelatinase B. This protease (E.C. 3.4.24.25) is a member of the
matrixin family of zinc-dependent endopeptidases that are responsible for much of the
turnover of matrix components13. Meanwhile, Sandberg et al had mapped and
characterized the proteases that are expressed by a CHO production cell line. What they
found was that the metalloproteinases in the CHO cell line that had homology to human
and mouse protease sequences14. Amino acid sequences of the isolated metalloproteinase
they found had sequence homology with matrix metalloproteinases (MMPs) MMP3,
MMP10, and MMP12 in humans, horses, and rabbits. Their results indicate that an MMP
pro-enzyme of 200 kDa was released from the CHO cells during the production phase
10
and expressed collagenase/gelatinase activity when activated. Due to autoproteolysis, a
number of smaller, less specific MMPs were formed with a 19.4 kDa protein being the
most active15. However, Sandberg et al also tested certain protease inhibitors on CHO
cells such as EDTA, 1,10 ophenantroline, imidazole, aprotinin, 3,4-DCI, Pefabloc Try,
and PMSF, leupeptin, cystatin and E-64; bestatin, pepstatin. What they found was that the
most effective inhibitors were EDTA at 4mM, and 1,10 ophenantroline at 1 mM, which
had inhibition effects of 96 and 98 percent, respectively16. This implies that it is entirely
possible to block protease activity in mammalian cells, and using the right inhibitors, it is
possible to reduce the protease activity by a large percentage17.
Amino Acid Detection Pathways
Mammalian cells have different biochemical pathways for monitoring and responding to
amino acid availability in their surroundings. One of these pathways is designed to detect
amino acid deficiency. Limiting the availability for any single amino acid initiates this
signaling cascade, or the amino acid response (AAR) pathway. The substrate specificity
for this pathway is not well understood, but in general, if an amino acid is “essential” to
the cell, an insufficient amount of that amino acid will trigger pathway activation18. A
number of plasma membrane amino acid transport activities have been demonstrated to
be responsive to amino acid availability19. The two most extensively studied include
neutral amino acid transport System A, mediated by the SNAT2 gene product, and
cationic amino acid transport, mediated by the CAT-1 transporter but there are also many
more transporters that are influenced by amino acid availability20. System A amino acid
transport (SNAT2)21 is a sodium dependent, pH-sensitive transport system that is
11
inhibited by high concentrations of amino acids. The activity is linked to the cell cycle
and cell growth rate as System A transport activity is up-regulated in mammalian cells in
response to amino acid deprivation. System A transport activity is increased by amino
acid limitation. This activation occurs when mammalian cells are deprived of even a
single amino acid that is essential for that particular cell; the amino acid may or may not
be a substrate for the transporter22. Palii et al. determined through on-site mutagenesis
that a 54 nucleotide sequence in the first intron of the SNAT2 gene system functioned as
a transcription inhibitor23. This element, the Amino Acid Response Element (AARE)
regulates transcription in the presence and absence of amino acids24. It is this element that
is of interest to us. AARE is as follows:
Forward: 5-CCTAGGGACGATCGATATTGCATCAGTTTTCTTTCCCATGGG-3
Reverse: 5-CCATGGGAAAGAAAACTGATGCAATATCGATCGTCCCTAGGG-3
RNA interference
RNAi, or ribonucleic acid interference 25 allows for transient inhibition of gene
expression in eukaryotic cells26 but currently has not found applications in the bioprocess
industry. RNAi works through the use of short (21-23 bp) or long (500-800 bp) double
stranded RNA molecules27, which possess perfect complementarity to a target gene and
thus a target gene’s mRNA. This results in the incorporation of the active strand of the
RNAi trigger, which is the antisense, or guide strand. Along with the mRNA, this pairs
into a multiprotein complex known as the RNA-induced silencing complex (RISC)28 The
Argonaute-2 (Ago-2) protein of the RISC complex then cleaves the targeted mRNA at a
defined position, which is subsequently degraded by cellular RNases. This ultimately
leads to efficient and sequence-specific knockdown of a particular gene’s expression29.
12
Because RNAi targets a specific gene for silencing, its transience and specificity make it
an ideal candidate for the use of regulating cell metabolism30. The simplicity of which it
can be achieved is evident. The RNAi complexes can be exogenously introduced to the
target, or alternatively, cells can be induced to intracellularly express the short strands
themselves.
So far, RNAi has been used to target genes in different organisms, such as C. elegans,
Drosophila cells, trypanosomes, cultured mammalian cells, and mice. Kemmer and
Neubauer demonstrated the use of RNAi in E. coli bacteria, in which they were able to
reduce the intracellular concentration of RNase. They found that expression of RNase did
not affect cell growth and that they were able to reduce the levels of RNase E 90 minutes
after the introduction of antisense RNAs. They were able to decrease the RNase E levels
down to 35% of the wild type,31 thus proving that significant knockdown of enzyme
activity can be achieved using RNAi.
RNAi has found limited uses in the bioprocess industry, in part due to its transient nature
and its currently inefficient delivery methods. Currently, the industry focuses more on
permanent changes in genotype, especially for altering cell metabolism. However, it is
only in the controlled environment of a bioreactor that high cell densities can be achieved
for most cells. But due to the secretion of proteases into the cell medium, and also due to
release by lysis, proteolytic degradation is a significant problem in bioreactors32.
13
Katsuhiro Mori, et al. were successfully able to use siRNA to convert established
antibody-producing cells to cells producing high antibody-dependent cellular cytotoxicity
(ADCC) antibodies. The conversion was made by constitutive expression of small
interfering RNA (siRNA) against a1,6 fucosyltransferase (FUT8). The team found two
effective siRNAs, which reduce FUT8 mRNA expression to 20% when introduced into
Chinese hamster ovary (CHO)/DG44 cells. This was done via insertion of siRNAs into
plasmids which were later transfected into the CHO/DG44 cells. Selection for Lens
culinaris agglutinin (LCA)-resistant clones after introduction of the FUT8 siRNA
expression plasmids yielded clones producing highly defucosylated (c60%) antibody with
over 100-fold higher ADCC compared to antibody produced by the parental cells (c10%
defucosylated). Moreover, the selected clones remain stable, producing defucosylated
antibody even in serum-free fed-batch culture. Their results demonstrate that constitutive
FUT8 siRNA expression can control the oligosaccharide structure of recombinant
antibody produced by CHO cells to yield antibodies with dramatically enhanced ADCC
production33. This also demonstrates that a plasmid can be used to constitutively express
siRNAs in a cell and therefore alter its phenotype and metabolic activity.
For this experiment, the purpose is to utilize this Amino Acid Response Element (AARE)
to target proteases present in CHO cells in order to increase recombinant protein yield.
We hypothesize that under conditions of amino acid starvation, cells will make more
proteases which will degrade recombinant protein products, and therefore, we will
investigate if blocking these proteases will increase recombinant product yield under
conditions of amino acid starvation, and this is because under some conditions, we feel
14
that it may be best to block protease activity. To do this, we will utilize the Amino Acid
Response Element as reported by Palii et al, to make a recombinant plasmid that allows
the cells themselves to make siRNAs against endogenous proteases. It is hypothesized
that during conditions of amino acid starvation, the Amino Acid Response Element will
become active and stimulate the synthesis of siRNAs in the cells from the recombinant
plasmid targeting the proteases and therefore will knock down their activity and increase
recombinant protein yield.
15
CHAPTER 2
Materials and Methods
Cloning of Amino Acid Response Element (AARE)
Constructs with AARE element were created using the TOPO TA gene expression kit
(Invitrogen, Carlsbad, CA) to insert the AARE into the pGlow with GFP vector. AARE
insert was ordered from IDT (Coralville, IA) in forward and reverse sequences, and
annealed to form a double stranded product by mixing together in equal molar amounts
and then heating to 94ºC before being brought back to room temperature, creating a
double stranded 60 bp insert. Constructs were created by adding 4 µL of annealed AARE
product with 1 µL of provided salt solution and 0.5 µL of TOPO TA vector (both
Invitrogen, Carlsbad, CA). This mixture incubated for 5 minutes at room temperature
before being placed back on ice. Constructs were then inserted into TOP10 E.coli
bacteria (Invitrogen, Carlsbad, CA) using the heat shock method by incubation at 42ºC
for 30 seconds before being returned to ice. 250 µL of SOC media was added and
bacteria were then grown for 1 hour in a 37ºC incubator at 200 rpm, then spread in 50
and 150µL amounts onto LB plates containing 50 µL of ampicillin (concentration of
50mg/mL) and grown at 37ºC overnight. Ampicillin resistant colonies were then screened
by PCR to test for presence of the AARE. Overnight tubes with 3 mL of LB media and 3
µL ampicillin (concentration of 50 µg/mL) were made from colonies that tested positive
in the PCR. Sixteen hours later the plasmids were extracted from the bacteria using the
Qiagen QIAprep Miniprep kit. (Qiagen, Valencia, CA) and sent for sequencing.
Construct was then subsequently dubbed pGlow-AARE.
16
Cell Culture
CHO K1 cells were grown in 40 mL flasks using F12K media (Mediatech, Herndon, VA)
supplemented with 50 mL of Fetal Bovine Serum and 5 ml of Penicillin and
Streptomycin. Cells were incubated at 37ºC, 95% air and 5% CO2. Cells were passaged
on an average of once every seven days when they had reached approximately 90-100%
confluency. Media was removed and cells were washed with 10 mL of 1X PBS. 2 mL of
trypsin was added and cells were incubated at 37ºC for 5 minutes. 4mL of media was
added back and cells were split among several flasks. Media was changed 8 hours
afterward. Cells were grown for 5 passages before experiments began. For amino acid
deprivation experiments, the complete F12K media was removed and fresh complete
media, or media lacking histidine was added, each one was supplemented with FBS and
antibiotics.
Media Preparation
Two separate media were prepared for experiments, one with the full range of amino
acids, identical to F12 media, and another lacking histidine but otherwise identical.
Media was prepared in 100x and 1000x stock solutions and filtered according to R.G
Ham’s protocol34 and diluted and mixed with an appropriate amount of Millipore water.
11 Different stock solutions were prepared in 50 mL Falcon Tubes (BD, Franklin Lakes,
NJ) by weighing out the appropriate amount of each compound and mixing with
autoclaved Millipore water. All 11 stock solutions were then added to 2224.7663 mL of
Millipore water. NaCl and NaHCO3 were then added. Media was then balanced to a pH
of 7.23. Media was then sterilized via filtration with Stericup filter cups (Millipore
17
Corporation, Billerica, MA) with 0.22 µm filters and stored in autoclaved glass bottles at
4°C.
Transfection of DNA
The vector pGlow-AARE was transfected into CHO cells to test its activity. Cells were
seeded into 12 well plate (BD, Franklin, NJ) with two 100% confluent 40 mL flask and
supplied with complete F12K (Mediatech, Atlanta, GA) media and grown for 24 hours.
Transfection was performed with 5.9724 µL (1.6 µg) of plasmid DNA (pGlow-AARE5)
per 8 wells out of 12, Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was present
at a ratio of 4 µL per 100 µL of reduced serum F12 medium, according to the
manufacturer’s protocol. 7.075µL of pEGFP-N1 (1.6µg) of plasmid was used in
remaining 4 wells to test transfection efficacy. After 4 hours, cells were rinsed once with
PBS and given fresh F12 K media. 16 hours post-transfection, medium was removed,
cells rinsed once with PBS, incubated for 28 hours in 1 mL/well of either complete F12k
or F12K lacking histidine (F12K-His), each one supplemented with FBS and antibiotics.
After completion of treatment, cells were checked for fluorescence under a microscope.
SiRNA Design
SiRNAs were designed using IDTdna’s online RNAi Design software. The target gene
sequence selected was the Matrix Metalloproteinase 14 (MMP14) of the Chinese
Hamster, Genbank assension number AAG34676. Although available MMP9 and
MMP12 were available online for the mouse, only MMP14 of the Chinese Hamster was
available as a sequenced gene, and thus, MMP14-CHO was selected as the target for the
18
siRNAs. The sequence was uploaded into IDTdna’s RNAi Design program, selecting
Screening DsiRNA 27mer as the duplex class. Three sequences were subsequently
chosen and 10nanomoles of each complex were ordered (IDT, Coralville, IA). SiRNAs
were resuspended as per IDT’s RNAi Resuspension Protocol. 100µL of RNase-Free
Duplex Buffer (IDT, Coralville, IA) were added to form a 100µM solution. Suspension
was then heated on a hot plate in a beaker of water to 94ºC for 2 minutes, and then
removed from heat and cooled to room temperature. Once product was at room
temperature, it was transferred to storage at -20ºC.
Figure 1: siRNAs designed using IDTdna’s RNAi Design software.
Transfection of siRNA
Cells were plated at 30% confluency into two six well plates for a total of 12 wells. Cells
were plated with a volume of 2 mL of F12K media (Mediatech, Atlanta, GA),
supplemented with FBS. 20 µL of Lipofectamine 2000 (Invitrogen, Carlsbad, CA).was
added to 225µL of F12K medium. This was then incubated for 5 minutes at room
temperature. 50µL of siRNA (500pmol siRNA) was added to 200µL of F12K. These two
volumes were then combined and incubated for 20 minutes at room temperature. 500µL
19
of the mixture was added to each well, and gently rocked to mix, and incubated at 37ºC
for 4 hours. Media was then changed after 4 hours, activity was assessed via RT-PCR.
mRNA extraction and RT-PCR
Elution solution was preheated to 80ºC. 300 µL of Lysis/Binding Buffer was added and
pipetted several times along the surface of each plate. 300µL of 64% ethanol solution was
applied. All three samples were applied with the same siRNA treatment to 1 column
(three times), spun for 30 seconds at 10,000 RCF, and the flow through was discarded.
700µL of Wash Solution #1 was added, centrifuged, the flow through was then discarded.
Then 500µL of Wash Solution #2/3, was added and centrigued and the flow thought was
subsequently discarded. This process was repeated with another 500 µL of Wash Solution
#2/3. RNA was eluted with 50µL of preheated Elution Solution, and centrifuged at
10,000 RCF for 30 seconds, and eluted again with 15 µL of Elution Solution. Solution
was then treated with 7.5µL of 10x DNase1 buffer and 1µL of DNase1 enzyme to destroy
residual DNA. The product was then incubated for 30 minutes at 37ºC before 8µL of
DNase1 inactivation reagent was added and incubated for 2 minutes at room temperature.
The tube was then spun down at 10,000 RCF for 1 minute to pellet the inactivation
reagent. Purity was measure for absorbance at 260 and 280 nm against a blank of plain
water using a 1:10 dilution. RT-PCR was done with 300µg of RNA. Four samples were
prepared, a control and one for each siRNA tested.
20
Sample
Control
MMP14_CHO1
MMP14_CHO4
MMP14_CHO5
µL of RNA
3.76
3.7
10.0
4.1
µL water
6.3
6.3
0
5.8
Table 1: Liquid volumes of RNA and water used for RT PCR
10 µL dilution solution was mixed with 1µL of oligo dT and 1µL of dNTPs and
incubated for 5 minutes at 65ºC. To this mix, 4µL of MgCl2, 2µL 10x buffer, 2µL DTT
and 0.25µL DT and 0.5µL RNase Out, and 0.25 µL of Super Script III Reverse
Transcriptase enzyme were added. This was incubated at 50ºC for 50 minute and then 5
minutes at 85ºC. 0.2µL RNase H was added. Then this was incubated for 20 minutes at
37ºC. Solution was then stored at -20ºC. cDNA PCR was done in a similar manner with a
total of eight samples. Each tube contained 7.5 µL water, 12.7 µL Q-Load 2x Master Mix
(NEB, Ipswich, MA), 2 µL forward and reverse primers mix, and 3 µL cDNA template.
PCR was done for 27 cycles with annealing temperature set for 55ºC and a 1 minute
extension time.
Co-transfection of DNA and siRNAs
Cells were seeded into two 24 well plates (BD, Franklin Lakes, NJ) and grown to 80-90%
confluence in F12K, supplemented with 50mL of FBS and 5mL of Penicillin and
Streptomycin. The day before transfection, media was changed
to F12K supplemented with FBS but no antibiotics. Five tubes were prepared as follows:
First tube
pEGFP-N1
EGFP siRNA
F12K Medium Only
Positive Control
200 ng/well
50 pmol/well
Second tube
Experimental
12.381 µL
3 µL
300 µL
21
pEGFP-N1
MMP14_CHO1
F12K Medium Only
200 ng/well
50 pmol/well
Third tube
pEGFP-N1
MMP14_CHO5
F12K Medium Only
Experimental
200 ng/well
50 pmol/well
Fourth tube
pEGFP-N1
dsRED siRNA
F12K Medium Only
Negative Control
200 ng/well
50 pmol/well
Fifth tube
pEGFP-N1
Default Control
200 ng/well
12.381 µL
3 µL
300 µL
12.381 µL
3 µL
300 uL
12.381 µL
3 µL
300 µL
12.381 µL
F12K Medium Only
Sixth tube
Lipofectamine
F12K Medium Only
Volume to distribute
300 µL
Lipofectamine
120 µL
1500 µL
324 µL
324 µL to all other tubes
Table 2: Concentrations and volumes of DNA, siRNA, Lipofectamine 2000, and media used for
DNA/RNAi cotransfections.
After five minutes of room temperature incubation, 324 µL of diluted Lipofectamine
2000( Invitrogen, Carlsbad, CA) in F12K media was added to each tube, increasing the
total volume to 639.381 µL. After incubating for 20 minutes at room temperature, each
well received 106.654 µL of a mixture of pEGFP-N1, a particular siRNA, Lipofectamine,
and F12K media. The default control had a total volume of 636.381 µL, leading to an
individual volume of 106.064 µL per well. Plate was mixed gently by rocking back and
forth. Media was changed 5 hours after the transfection to F12K supplemented with FBS
and antibiotics. Cells were incubated at 37ºC for 38 hours afterward and fluorescence was
observed.
22
Figure 2: Layout of cells, DNA, and siRNA for the DNA/siRNA co-transfection. Names of siRNAs are in
boldface.
Cell Harvesting
Cells were then harvested by removing the media, washing gently with 200µL PBS per
well, and 100µL of trypsin was used to detach cells from the well. Another 200µL of cold
CaCl2-, MgCl2- PBS (Invitrogen, Carlsbad, CA) was added and cells were gently spun at
1000rpm for 5 minutes at 4ºC. A portion of the supernatant was removed and discarded,
and cells were resuspended in a cold solution of 4% formaldehyde in CaCl2-, MgCl2PBS. Cells were then analyzed using a BD-Biosciences LSRII Flow Cytometer (BD
Biosciences, San Jose, CA) for number and intensity of GFP expressing cells at 530 nm.
mRNA extraction and RT-PCR
RNAqueous-4PCR kit (Ambion, Austin, TX) was used. Elution buffer was preheated to
80ºC. 150 µL of Lysis/Binding buffer was added to two wells of each sample of the plate.
Sample was pipetted up and down to mix and adjacent wells were combined to form a
300 µL sample. 300 µL 64% Ethanol was added and carefully pipetted to mix. Solution
was then applied to an Ambion filter cartridge in a collection tube and centrifuged at
10,000 RCF for 30 seconds. Flow through was discarded. 500 µL of Wash Solution #1
23
was applied and centrifuged again at 10,000 RCF for 30 seconds, and flow through was
discarded. 500 µL of Wash Solution #2/3 was added and centrifuged again at 10,000
RCF for 30 seconds and flow through was discarded. 500 µL of Wash Solution #2/3 was
added again and centrifuged. Filter cartridge was transferred to a new collection tube and
RNA was eluted with 50 µL of 80ºC Elution Solution, centrifuged at 10,000 RCF for 30
seconds. This process was then repeated a second time for 21 µL of Elution Solution.
RNA was then treated with 7.7µL of 10x DNase1 buffer and 1µL of DNase1 enzyme to
destroy residual DNA. The product was then incubated for 30 minutes at 37ºC before
7.97 µL of DNase1 inactivation reagent was added and incubated for 2 minutes at room
temperature. The tube was then spun down at 10,000 RCF for 1 minute to pellet the
inactivation reagent. Purity was measure for absorbance at 260 and 280 nm against a
blank of plain water using a 1:5 dilution. RT-PCR was done with 100 ng of RNA.
cDNA synthesis was performed using the Superscript III 1st Strand Synthesis System
(Invitrogen, Carlsbad, CA)
Sample (siRNA used)
EGFP-1
MMP14_CHO1
MMP14_CHO5
DsRed
Control (no siRNA)
µL RNA
12.0
6.9
7.9
8.6
7.5
µL water
0.0
5.1
4.1
3.4
4.5
Table 3: Volumes of RNA and water used for RT-PCR
To each tube 1 µL of 50 µM oligo(dT), 1 µL 10 mM dNTP mix were added. Mixture was
incubated at 65ºC for 5 minutes before being lowered to 4ºC. 2 µL 10X RT buffer, 4 µL
MgCl2, 2 µL DTT, 0.5 µL RNase Out, and 0.25 µL Super Script III Reverse
Transcriptase enzyme was added to each tube, and incubated at 50ºC for 50 minutes and
24
then at 85ºC for 5 minutes before being ramped back to 4ºC. 0.5 µL of RNase H was
added, and reaction was incubated at 37ºC for 20 minutes before cDNA was taken out
and stored at -20ºC. PCR reactions mixtures were made as follows: 4 µL of each cDNA
sample was applied to a PCR tube containing 10 µL Q-Load 2X PCR MasterMix (NEB,
Ispwich, MA), 4 µL nuclease free water (Fermentas, Glen Burnie, MD), and 1 µL of each
forward and reverse primers for MMP14-CHO (IDT, Coralville, IA) to produce 5 PCR
tubes of 20 µL each to test for MMP14 activity. Another 4 µL of each cDNA sample was
applied to a PCR tube containing 10 µL Q-Load 2X PCR MasterMix (NEB, Ispwich,
MA), 4 µL nuclease free water (Fermentas, Glen Burnie, MD), and 1 µL of each forward
and reverse primers for β-Actin (IDT, Coralville, IA) to produce 5 PCR tubes of 20 µL
each as positive controls.
PCR reactions were performed on a Bio-Rad iCycler (Bio-Rad, Hercules, CA). PCR
protocol was as follows:
Cycle
1 (1x)
2 (27x)
3 (1x)
Temperature and Time
95ºC, 4 minutes
95ºC, 30 seconds
55ºC, 30 seconds
72ºC, 60 seconds
72ºC, 7 minutes
4ºC, hold indefinitely
Table 4: PCR Protocol for RT-PCR
3 µL of each β-Actin PCR product and 19 µL of each experimental sample were loaded
onto a 1% agarose gel (0.5g agarose, 50 mL 1X TAE buffer). Gel was run for 15 minutes,
13 seconds at 154 V.
25
CHAPTER 3
Results
SiRNA effect on MMP14
SiRNAs MMP14_CHO1, MMP14_CHO4, and MMP14_CHO5 were transfected into
cells to assess the silencing effect of the siRNAs on the activity of Matrix
Metalloproteinase 14 (MMP14). RT-PCR results showed significant silencing of the
matrix metalloproteinase following transfection of the siRNA MMP14_CHO1 and slight
silencing of the protease following the transfection of the siRNA MMP14_CHO5, while
the siRNA MMP14_CHO4 did not seem to significantly silence the protease. In Figure
Figure 3: RT-PCR results of transfection with siRNA. Β-Actin was used as a positive control.
3, the resulting gel run of the RT-PCR shows dark bands of MMP14 activity in the
control band (no siRNAs tranfected). . What this suggests is that matrix metalloproteinase
14 is active in CHO cells during normal conditions of growth where the nutrient, and
26
particularly, amino acid supply, is sufficient for normal cell growth. The second lane,
showing the resultant band of MMP14 activity in CHO cells transfected with the siRNA
MMP14_CHO1 is significantly lighter than the control band, therefore showing less
MMP14 activity is present when the siRNA is present. The third lane, showing the
resulting band of MMP14 activity in CHO cells transfected with the siRNA
MMP14_CHO4 is darker, showing minimal silencing of the protease when it is present.
The fourth lane showing the resulting band of MMP14 activity in CHO cells transfected
with the siRNA MMP14_CHO5 is slightly lighter than the band of the control lane,
therefore showing that slight silencing of the protease has taken place. Lanes 5 through 8
hold the β-Actin PCR products, run as positive controls to test the efficacy of the PCR
protocol and the gel electrophoresis. This therefore shows that the silencing of matrix
metalloproteinase 14 (MMP14) is indeed effective through the use of siRNAs.
SiRNA Effect on EGFP Activity
The effect of various levels matrix metalloproteinase 14 activity on EGFP production was
assessed via RT-PCR and flow cytommetry.
27
Figure 4: RT-PCR results of transfection with DNA and siRNA. Β-Actin was used as a positive control.
While RT-PCR results were similar to the results found in the first RT-PCR, the flow
cytommetry data was more telling.
Sample
FITC-A 1
FITC-A 1
Average
Standard
Deviation
Normalized
units
EGFP
4,721
3,487
4,104
872.5698
MMP14_CHO1
25,813
27,711
26,965
1338.553
MMP14_CHO5
24,129
21,092
23,016
1574.726
DsRed
15,698
23,700
19,699
5658.268
Control
26,678
14,066
20,372
8918.031
0.2
1.3
1.1
1.0
--
Table 5: Average intensities (FITC-A) of co-transfected cells, as reported by flow cytommetry.
While it was shown through the RT-PCR that the activity of MMP14 was silenced by the
siRNAs, it also showed that the silencing of the protease also had an effect on the activity
of EGFP in the transfected CHO cells. While the number of cells was unusually low, it
was clear from the flow cytommetry data that although slight, the silencing of the
protease did in fact, increase the activity of the EGFP. While in the absence of any
siRNA, the average intensity of the EGFP expression in cells silenced with MMP14
28
siRNAs was slightly higher than those that weren’t silenced by the siRNAs. EGFP
siRNA served as a positive control to test the effects of silencing the EGFP expression,
while DsRed siRNA served as a negative control to test how tranfection of any other
exogeneous material affected the activity of EGFP, while the control tested both the
activity of EGFP in the absence of any siRNA and also served as a test for transfection
efficacy. The average fluorescence of the cells targeted with siRNAs against MMP14 is
higher than those of the control sample and the sample transfected with dsRed, therefore
showing that the transfection of the dsRed siRNA, which was designed for a gene that the
CHO cells do not have, and therefore, do not express, does not have any effect on the
activity of the EGFP. The higher levels of EGFP expression in the cells targeted with the
MMP14_CHO1 and MMP14_CHO5 siRNAs suggests that the protease is indeed active
during normal conditions in the CHO cells, and that its inherent activity decreases the
activity of EGFP under normal circumstances. When the protease is silenced by siRNAs,
the increased expression of EGFP shows that in the absence of any silencing, the protease
attacks EGFP and decreases its expression. In the presence of siRNA silencing, the
protease is unable to attack the EGFP, and therefore, unable to attack other protein
products as well.
29
35,000
Average Intensity
30,000
25,000
20,000
15,000
10,000
5,000
0
Control
MMP14_CHO1 MMP14_CHO5
DsRed
EGFP
Sample
Figure 5: Average intensity of samples from flow cytommetry. Error bars represent one standard deviation
of cells done in duplicate.
1.6
Normalized Units
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
MMP14_CHO1
MMP14_CHO5
DsRed
EGFP
Sample
Figure 6: Average intensity of samples from flow cytommetry in normalized units, normalized to the
control samples as 1.0. Error bars represent one standard deviation of cells done in duplicate.
The cells showed a modest increase in flourescence when co-transfected with both
pEGFP-N1 and the MMP14_CHO5 siRNA, but the increase in fluorescence was not as
great as that of the co-transfection of pEGFP-N1 and the MMP14_CHO1 siRNA.
Therefore, only the MMP14_CHO1 siRNA was chosen for further testing.
30
Figure 7: Histograms of different cell samples from flow cytommetry. Number of cells is displayed on the
x-axis and average intensity is displayed on the y-axis. Each sample showed the same distribution of
intensity, but average intensity was higher for cells that were transfected with both pEGFP-N1 and
MMP14_CHO1 and MMP14_CHO5 siRNA.
31
CHAPTER 4
Discussion and Future Directions
Overview
What the results of our experiment have proven are that the silencing of MMP14 is
possible through RNAi and that decreasing the activity of MMP14 can possibly increase
recombinant protein yield. The next steps are to conduct further tests using the F12 Hismedia and the pGlow-AARE construct.
Co-transfection of DNA and siRNAs
The first experiment would be identical to the DNA/siRNA transfection using pEGFP-N1
and various siRNAs but would be done under conditions of amino acid limiting
conditions using the F12 His- media. 16 hours post-transfection , the existing media
would be carefully removed, the cells would be washed with PBS, and the new F12 Hismedia will be applied and incubated for 20 hours before cells would be harvested.
Figure 8: Layout of cells, DNA, and siRNAs for the DNA/RNAi co-transfection. Names of siRNA are in
boldface.
Activity of MMP14 will be determined using the RT-PCR protocol in the Materials and
Methods section, while activity of EGFP will be assayed using the Cell Harvesting and
32
Flow Cytommetry protocol mentioned in the Materials and Methods section. The results
of that experiment should show that the activity of the MMP14 is higher in amino acid
deprivation, while EGFP activity should increase. The application of the siRNAs would
have a more pronounced effect on the activity of EGFP, since the silencing of the
MMP14 should lead to a greater increase in activity of the EGFP as compared to the
control transfection of pEGFP-N1 only. What this experiment would show is that effect
of applying siRNAs on EGFP production is more pronounced during amino acid limiting
conditions.
Transfection of DNA
We will once again test the pGlow-AARE vector using the DNA transfection protocol as
detailed in the Materials and Methods Section. 16 hours post-transfection, media will be
removed, cells will be washed with PBS, and new F12 His- and F12 His+ media will be
applied. F12 His- will once again serve to test the activity of the AARE under amino acid
deprivation conditions and F12 His+ media will act as a control. Cells will be incubated
for an additional 20 hours after the media change and GFP activity will be assayed
through flow cytommetry for number and intensity of GFP expressing cells. It is expected
that the cells will express more GFP in the cells given the F12 His- media as the amino
acid deprivation will cause the AARE element to become activity and initiate synthesis of
GFP.
33
pAARE-MMP14 Design and Testing
The next and final experiment would first involve the creating of a new plasmid using the
pGlow-AARE construct. Currently, the construct utilizes the AARE element as a
promoter and GFP as a reporter gene. The GFP sequence will be excised from p-GlowAARE construct using restriction enzymes and the siRNA sequence, constructed as an
inverted repeat, will be inserted into the vector instead. The siRNA sequence will be the
identical sequence to the siRNA MMP14_CHO1 forward, and then an inverted repeat
sequence with perfect complementarity so that when the inverted repeat sequence is
transcripted into mRNA, it forms a double stranded piece of siRNA that resembles the
MMP14_CHO1 siRNA. This way, the cells can produce the siRNA themselves and
therefore the cells do not need to be transfected with introduced siRNA. This construct
will be inserted into E. coli bacteria using the heat shock method as stated in the
Materials and Methods section for the Cloning of the AARE Element. This will be
screened using PCR and gene specific primers to determine the presence of the
MMP14_CHO1 sequence. Overnight and freezer stocks will be picked from colonies that
were positive in the PCR. This vector will be named pAARE-MMP14.
34
Figure 9: Schematic of the pGlow-MMP14 vector. It uses the AARE as a transcriptional regulator paired
with the forward and reverse sequences for the MMP14_CHO1 siRNA to produce a double stranded RNA
product upon transcription
Once this plasmid has been collected, it will be co-transfected into CHO cells along with
pEGFP-N1 using Lipofectamine 2000 (Invitrogen, Carlsbad. CA) and a 24 well plate.
The samples tested will include two samples of the new vector co-transfected with
pEGFP-N1, one sample transfected with pEGFP-N1 only as a test for transfection
efficiency, and one sample of CHO cells co-transfected with pEGFP-N1 and the
MMP14_CHO1 siRNA as a comparison to earlier experiments. The protocol for DNA
transfection and DNA/siRNA co-transfection will be used as outlined in the Materials
and Methods section. After 16 hours, media will be removed and cells will be rinsed with
PBS and given either F12 His- or F12 His+. Cells will then remain incubated for an
additional 28 hours before being harvested for testing of activity of EGFP via flow
cytommetry and testing of MMP14 activity via RT-PCR. Testing of pGlow-MMP14
should show an increase in the activity EGFP in cells co-transfected with both pEGFP-
35
N1 and pGlow-MMP14 in relation to the activity of cells transfected with pEGFP-N1
only. Activity of EGFP in the cells co-transfected with pEGFP-N1 and pGlow-MMP14
in His+ media should be comparable to the activity of EGFP in the cells co-transfected
with pEGFP-N1 and the MMP14_CHO1 siRNA. EGFP activity in the cells cotransfected
with pEGFP-N1 and pGlow_MMP14 in F12 His- media will not be as high as the activity
in the previous two, due to the fact that protein synthesis rates fall in limiting amino acid
concentrations35. RT-PCR results should show that the activity of MMP14 decreases in
the presence of both the MMP14_CHO1 siRNA and the pGlow-MMP14 vector, as
compared to the control sample.
Figure 10: Layout of cells for the testing of pGlow-MMP14. Wells highlighted in yellow will receive F12
His- media 16 hours post-transfection.
36
CHAPTER 5
Conclusion
From the results of past experiments done for this project, it is clear that the use of RNAi
to block the activity of proteases is a feasible strategy for use of CHO cells in bioreactors,
as it was able to successfully block protease activity while increasing the activity of other
proteins such as EGFP. However, in order to prove that this concept has long term
potential, further testing needs to be done under both conditions of optimal amino acid
concentrations and amino acid starvation conditions.
37
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P Elliott, A. Hohmann and J. Spanos. “Protease expression in the supernatant of
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14
H. Sandberg et al., “Mapping and Partial Characterization of Proteases Expressed by a
CHO Production Cell Line,” Biotechnology and Bioengineering 95, no. 5 (2006): 961971
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16
H. Sandberg et al., “Mapping and Partial Characterization of Proteases Expressed by a
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38
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Ibid
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21
Stela S. Palii, Hong Chen, and Michael S. Kilberg. “Transcriptional Control of the
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18
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Ibid
Ibid
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Ibid
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27
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29
Ibid
John C. March ,William E. Bentley. “RNAi-based tuning of cell cycling in Drosophila
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30
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31
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32
39
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34
Richard Heal and John McGivan. “Induction of calreticulin expression in response to
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35
40