Lab Report #2

Reverse Genetics:
Creation of a Mouse Model for a Human Disease via the
Transfection and Growth of Mouse Embryonic Stem Cells

Michael Wade Jackson

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BICD 101: Eukaryotic Genetics
May 29th 2003

Group 6:
Michael Jackson
Susan Liem

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 Abstract:
The goal of this series of experimental procedures was to create embryonic stem
cells which contained an inserted transgene that would allow for the creation of a mouse
model of a human disease. This model would then be used to analyze and evaluate new
therapeutic approaches to treatment and pathogenesis of the human disease. The primary
educational goal of this series of experiments was to gain knowledge and practice in cell
culture technique which involved: 1) creation, immortalization, and storage of EF cell
lines 2) Transfection of ES cell lines with linear targeting construct 3) ES cell selection
via antibiotic resistance 4) Growth of monoclonal ES cell line 5) Analysis of target
construct insertion via PCR and 6) Injection of ES cells which exhibit homologous
recombination into blastocyst for the creation of a mouse model after F2 generation in ¼
of progeny. The outcome of these procedures was the production of non-homologous
end joining (NHEJ) cells which were amplified in the process of PCR exhibiting the
control band at 2000 Kb. The PCR amplification displayed a lack of a candidate
monoclonal cell line that exhibited homologous recombination.

Introduction:
The field of genetics is one that is in a process of continual expansion as the
progression of biotechnology and laboratory innovation pushes the boundaries of once a
semi-static field into a multitude of arenas blurring the lines between different specialties
in biology. The methods utilized in these experiments are relatively routine today but
were novel in the 1980’s. The scientific approach that is exemplified in this five week
period is the process of reverse genetics. Reverse genetics is the process by which a gene
in mutated and the phenotypic consequence is then determined. This is a direct opposite
to the process of forward genetics in which a phenotypic mutation is observed and the
hunt is on to trace this mutation back to a target gene. The environment in which reverse
genetics was used in this class is in the field of mouse modeling of human disease
through the creation of homologous recombinant mouse cells to produce chimeric
(agouti) mice and then through selfing of progeny produce ¼ homozygous mutants in the
F2 generation. The production of ES cells that contained HRE is based of the past work
of many scientists and utilized many common techniques practices in labs around the
world.
Embryonic Stem Cell (ES cell) technology finds its roots in the early 1980’s when
the determination was made that ES cells are pluripotent, meaning that these ES cells can
become any cell line in the laboratory animal. The 1st isolation of ES cells came in 1981
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in the 3.5 day old embryo of a mouse (blastocysts). The stem cell is harvested from the
developing mouse embryos a short time after fertilization and before significant zygote
development into a viable embryo. The ES cells are maintained in culture via the use of
embryonic fibroblast cells (EF cells) which have been immortalized (halted in mitotic
development) to provide nutrients and growth factors to keep the ES cells from
differentiating. The first introduction of a germ line mutation in ES cells was in 1984.
The vector for introduction of the mutation was a retroviral vector. This vector was then
used to incorporate the mutation into a blastocyst and the production of an agouti mouse
was the result. This mouse was a chimeric mouse that when the progeny of the F1 were
selfed the F2 generation produces ¼ homozygous mutants which have two copies of the
mutant gene creating an organism that can become a model for human disease.
In 1987 the first gene targeting and knock out experiment was carried out in
which the procedure produced a method by which a normal gene could be replace with a
gene of interest using the DNA repair machinery of the host cell. The creation of this
knockout technique now allows for the ability to create mouse models of human disease
with a higher degree of efficiency. The knockout technique once perfected led to the
creation of skid mice (severe immune compromised) which are models for the human
immunodeficiency virus (HIV). The technique used in these labs to create a mouse
model for a human disease is transfection by electroporation of a linear targeting
construct.
The goal of these labs is to produce mouse models of human disease. This
process is made possible by advances in cellular manipulation and culture since 1980 that
have paved the way for the techniques of today. The major issue surrounding the
procedures and techniques used in this educational lab are the irregular sequence of
events that are not in sync with the actual progression in a diagnostic lab environment.
However, the techniques learned in this lab are the current and frequently used techniques
that will give the students of this class the ability to compete in a genetic biology lab and
provide the background to understand the process of gene targeting.

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 Results:

Table of Reagents:

Name Contents Purpose

EF media (250 ml) 1. DMEM-092 (222.5 ml) 1. Provides the essential
2. 10% FCS (25 ml) nutrients to keep cells
3. Penn/Strep(2.5 ml) happy and provide
4. 50 µM β-mercaptoethanol energy for growth.
(0.85 µL) 2. DMEM-092 is
TOTAL VOLUME = 250ml Dulbecco’s Modified
Eagle’s Media which
contains nutrients for
cell lines.
3. FCS will neutralize
the effects of Trypsin
4. β-mercaptoethanol
prevents oxide
formation
5. Penn/Strep provides
protection against
media bacterial
infection

ES media (250 ml) 1.DMEM-044 (202.5 ml) 1. Provides the proper

2.15% FCS (37.5 ml) environment for ES
3.1x glutamine (2.5 ml) cell growth via
4.1x non-essential amino acids (2.5 ml) nutrients.
5.1x sodium pyruvate (2.5 ml) 2. DMEM-044 specific
6.1x Penn/Strep (2.5 ml) media for ES cells
7.100 µM β-mercaptoethanol provide nutrients
(1.7 µl) 3. FCS- neutralize
8. LIF (Leukemia Inhibitory Factor) (1 µl) Trypsin
TOTAL VOLUME = 250 ml 4. glutamine is a
nutrient
5. non-essential AA is
also a nutrient
6. sodium pyruvate is
nutrient
7. Penn/Strep provides
resistance to bacteria
8. β-mercaptoethanol
prevents oxide
formation which will

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 damage cells
9. LIF is a ingredient
that prevents the
differentiation of the
ES cells
ES media + G418 Same as ES media above + G418 Contains G418 which is a
antibiotic which will kill off
ES cells that do not
recombine with target
construct
PBS Phosphate Buffered Saline Wash solution that is at the
same concentration and
osmotic potential as cells so
cells will not burst when
washed
Trypsin Will cause the detachment of
anchors on cell surface and
cause the resuspension and
breakup cell masses into
single cells to prevent
differentiation
Freeze Media 1. 10 % DMSO DMSO will make the cell
2. 80 % DMEM membrane porous as not to
3. 10 % FCS break and become brittle
during cryogenic freeze.
DMEM and FCS provide
necessary nutrients and
neutralize Trypsin in cell
solution
Linear Target Is the linearized plasmid
Construct construct that will be
transfected into cells by
electroporation and activate
DNA repair mechanisms of
ES cells
Xba1 Involved in the linearization
of the target construct
Digest Enzyme Will digest circular plasmid
Linearize Plasmid into a linear fragment with
blunt ends that will activate
DNA repair machinery of the
ES cells once transfected
6x Load Buffer 1. Glycerol Used in Agarose gel
2. Dye electrophoresis to cause the
weighting down of sample
into well and includes dye
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 front to view gel progress
diH2O Used in almost all solutions
to bring concentration to
appropriate levels
1Kb ladder Used as a analysis tool to
GibcoBRI discern linear insert size,
DNA presence, and PCR
amplification product on
three separate gels
1x TAE Creates and appropriate
electrical conductive
environment for gel
electrophoresis by
surrounding and
encompassing agarose gel
and tray
3M Na Acetate Used to precipitate targeting
construct once linearized in
solution
Ethanol (100%) Used to precipitate target
construct and DNA by out
competing hydrophobic
interactions
Ethanol (70%) Used to wash DNA pellet
after 100% ethanol and then
disposed and allowed to air
evaporate
Lysis Buffer Lysis solution Used to lyse open cells to get
Proteinase K (5µl) pK to DNA which will have
been transfected by
electroporation for PCR
amplification and gel
analysis. pK is used to rid
the solution of all proteins to
aide in DNA purification.
SDS Is mixed with DNA sample
and must be precipitated out
to remove from DNA in
process of purification.
Mostly a detergent
NaCl Used to precipitate DNA by
being a strong Salt which is
imperative in DNA
precipitation for purification
PCR Buffer Mix 1. Taq Used in conjunction with
2. 10x Taq Buffer DNA sample for the proper
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 3. Primers amplification of a targeted
4. 10 mM dNTP sequence for analysis. The
5. H2O buffer contains necessary
molecules for the process of
disassociation, association,
and annealing of DNA
strands to create PCR
products

1. Generate Embryonic Fibroblast (EF) Feeder Layer Cells:

a. Purpose:
i. The EF cells are the essential source of nutrients and support for
the Embryonic Stem Cells (ES). They prevent the differentiation
of the ES cells by providing growth factors that keep the ES cells
in their pluripotent state.
ii. IMPORTANT: EF cells are immortalized cells which mean they
will no longer replicate effectively as to outgrow the ES cells
supplying a base feeder layer for ES cells. The EF cells are
immortalized by mutation by (UV/ IR/ or Gamma) irradiation.
This creates mitotically inactive EF feeder cells.
b. Protocol
i. Take 6cm plate of MEF cells (Note: Growth not yet a feeder layer)
ii. Transfer to new 10cm plate
1. Transfer existing EF media from 6cm to 10cm plate via
pipette (approx 3ml)
2. Wash EF cells in 3ml PBS
a. Dispose PBS
3. Add 1ml Trypsin
a. Incubate at 37 degree C for 5 min
4. Add 6ml EF media
a. Transfer 6cm volume to 10cm plate
i. Approx volume 10ml in 10cm
5. In three days EF cells will be exposed to 30,000 rads of IR
to immortalize cell line
a. Cell life span 2 weeks then death
6. Fourth day after process portion of immortalized MEF
frozen to supply EF cells during process for ES cell growth
and selection
7. Freeze excess EF cells in Corning 430661 2.0ml cryogenic
vial. EF cells viable approx 2 weeks then need new EF
cells.
a. Take excess and freeze with freeze buffer to prevent
cell lysis. Add 1ml of 1x freeze buffer to EF cells
that have been spun down and supernate removed
and then put in -70 C freezer but cool slowly to
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 avoid cell damage. (DMSO in freeze buffer to
cause cell membrane pore formation to prevent cell
membrane lysis which would cause death.

c. Results:
i. MEF cells prepared and irradiated according to protocol. Sterile
technique followed and no contamination observed in the EF cells.
The immortalized cells will be used with ES cells as a feeder layer.

2. Plate Embryonic Stem (ES) Cells on Feeder Plate:

a. Purpose:
i. Once EF cells have been immortalized the ES cells can be added to
the EF plate to allow growth of ES cells and removal of dead ES
cells in culture dish. Move ES cells from a culture dish without an
EF layer to a culture dish with EF layer to start process of growth
of ES cells.
ii. To prevent the differentiation of ES cells by the process of
breaking up clumps and attachment to the culture plate by
trypsinization of cells.
b. Protocol
i. Transfer ES cells from 6cm to 10cm EF (feeder) plate
1. Dispose of existing ES media on 6cm ES plate
2. Wash cells with 3ml PBS
a. Dispose of PBS
3. Add 1 ml Trypsin
a. Incubate at 37 degree C for 5 min
b. Observe if Trypsin effective under microscope
4. Add 3 ml ES media
a. Neutralize Trypsin
b. Make cells happy
5. Resuspend with P1000
a. 10-20x
6. Count Cells with Haemocytometer
a. Add with p20 15 µl of cells

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 b. Count cells in 5 x 5 grid through microscope
c. After count will move 500,000 cells to 10cm plate
7. Put 500,000 cells into 10 cm EF (feeder) plate
a. Through cell calculation add 465 µl to feeder plate
b. Then add 10 ml of ES media to plate
8. Change ES media (Warm to 37 degree C before change)
a. 1 day later change to 15ml ES media (fresh)
i. dispose 10 ml ES via vacuum
b. 4 days later change to 15 ml ES media (fresh)
i. dispose 15 ml ES via vacuum
c. Results:
i. Calculation of Cell Count and Volume Computation

ii. Addition of ES cells to Feeder layer is a success and sterile

technique appears to have been maintained. The media will be
changed on a rotational basis between lab partners. Cell growth
will be checked by Haemocytometer to get a sufficient amount for
Transfection.

3. Linearize Target Plasmid:

a. Purpose:
i. To check on the efficiency of the linearizing enzyme on the
circular plasmid to create blunt ends and a linear construct that will
induce the host cell DNA repair machinery and integrate the
transgene either by NHEJ or HRE.
ii. Will run the digested plasmid on an agarose gel with a control
sample to view the level of linear form of the plasmid versus the
super coil and open circle to see the efficiency of the digest.
b. Protocol:
i. Linearize plasmid:
ii. Combine the following ingredients:
1. 10 µl of DNA (plasmid)
2. 4 µl of 10x (xba1)
3. 1 µl enzyme (~20 units/µl) (digest 1µg DNA in 1 hr at 37C)
4. 25 µl of H2O
5. Total 40 µl in digest tube
iii. Digest overnight at 37 C and remove from bath and store at 4 C
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 iv. Analyze digest on agarose gel as follows:
1. Take 40 µl digest (100ng/µl)
a. Take 2 µl from digest
b. Take 2 µl 6x load buffer
c. Take 8 µl diH2O
d. TOTAL 12 µl
2. Load 12µl sample into agarose gel and note well #
a. Run agarose gel 1 hr at 100 V
b. Visualize with UV and take photo
c. Run with 1 Kb GibcoBRL ladder (5 µl)
v. Linear DNA purification:
vi. Precipitate DNA
1. Combine 38 µl digest with 4µl 3M Na Acetate
2. Add 2.5x 100% ethanol (105 µl)
3. Spin in centrifuge at RT for 10 min at 14 K
4. Dispose of supernate
vii. DNA pellet
1. Wash 70% ethanol (COLD) (1 ml)
2. Spin again (1 min) (14K) (RT)
3. Dispose Ethanol and Air Dry
4. Once dry dissolve in 40 µl diH2O
c. Results:
i. Gel Photograph:

ii. Group 6 sample in Lane 6/ Ladder lane 12/ Control Lane 13

iii. The gel analysis displays the plasmid digestion was a successful
procedure and the eleven groups all contain the majority of a linear

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 form of the plasmid. The control by comparison is undigested and
displays a high level of the super coil form of the plasmid which
travels farther down an agarose gel irrespective to the actual size of
the sample.
iv. The purified target construct would have been used for the
transfection of the ES cells however the ES cell transfection
process occurred before this procedure so the targeting construct
was previously linearized and purified for the transfection.

4. Prepare Cells for Electroporation:

a. Purpose:
i. Cells must be placed in a media which is conducive to the
electroporation (i.e. a solution that minimizes excess salt). The
low level of salt will allow for directed current flow through
electroporation apparatus.
ii. The cells must be resuspended and then broken up into single cells
to increase the exposed cell membrane surface areas to be
perforated by the electrical current.
iii. A cell count will take place so that the frequency of uptake of the
targeting linear plasmid will be evident in subsequent analysis.
b. Protocol:
i. View ES cells under microscope and draw picture
ii. Wash ES cells with 5ml PBS
1. Dispose of PBS wash
iii. Add 1ml trypsin
1. Incubate 37 C for 5 min
iv. Add 5ml ES media
1. Neutralize trypsin
2. Resuspend 20x
v. Count Cells in Haemocytometer
1. Need 10 million cells for electroporation
vi. Place 10 million cells in 15ml Falcon tube
1. Spin cells in centrifuge 5 min at 1000 rpm (4 C)
2. Dispose media
3. Resuspend cells in 5 ml PBS
a. This is necessary to remove all electrolytes to not
interfere with electroporation
nd
4. Spin 2 time 5 min 1000 rpm (4 C)
5. Dispose PBS
6. Resuspend into 0.5ml PBS (electroporation vol)
a. Add 10 µg linearized targeting construct
b. Mix on ice 10 min
7. Time for electroporation in cuvet made for electroporation

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 c. Results:
i. The sample was prepped for electroporation and then the next step
is the electroporation of the sample. The 10 million cell volume is
calculated from the Haemocytometer results.
ii. The prep results in a cell sample of approximately 10 million cells
which are in a very low salt (low electrolyte) concentration media
as to not interfere with the electroporation.
iii. Cell Count Calculations

5. Electroporate prepared cells with purified linear plasmid:

a. Purpose:
i. The goal of the electroporation is to introduce a linear target
construct with the desired gene of interest to knockout the naturally
occurring gene. The electroporation utilizes a electric current to
open pores in cell membranes and then the flow of particles
towards the positive pole as the DNA has a negative charge to
guide linear plasmid into cell. This targeting construct with its
blunt ends will activate the host cells DNA repair machinery and
then cause the insertion of the targeting construct in either a NHEJ
or HRE event.
b. Protocol:
i. Optimal Electroporation settings
1. 50 µF, 500 Volts
ii. Take the cuvet with the prepared 10 million cells and place it in the
electroporation machine and Electroporate.

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 iii. You will receive a time constant setting from the electroporation
and this value will depict the success of the procedure. Record time
constant value.
iv. Incubate the electroporation sample on ice for 5 min
v. Plate cells back onto a 10cm plate with feeder (EF) layer
1. Add 10 ml ES media without G418 to allow cells to
recover from the stress of electroporation.
vi. Create ES media with G418 as follows:
1. ES media (148.2 ml)
2. Add G418 (0.3mg/ml) = 1.8 ml
3. TOTAL VOLUME = 150 ml
vii. ES Media Change Schedule
1. 1 day out change ES media to ES G418 20 ml
2. 3 days out change ES G418 to ES G418 15 ml
3. 6 days out change ES G418 to ES G418 10ml
4. 9 days out check color of media and see if change needed
c. Results:
i. The cells are left in G418 free media for 24 hrs to allow for
recovery and production of neomycin resistance. Then the media
is changed to the ES with G418 to create a hostile environment to
the cells which did not integrate the target construct and will
therefore be killed off. Both the NHEJ and HRE cells will survive
the addition of G418 as both cell types have gained the ability to
resist G418.
ii. The G418 cells will form monoclonal colonies and these colonies
will present an opportunity to search for both HRE and NHEJ
monoclonal cell lineages.
iii. There are four possible outcomes to the electroporation
1. Cell death
2. Cell survive electroporation do not integrate target
construct
3. Cells survive but integrate construct in a non-homologous
fashion (NHEJ)
4. Cells survive and recombine in a homologous fashion
(HRE)

6. Prepare EF cells on 96 well plates:

a. Purpose:
i. The nature of this procedure is to create a EF cell 96 well flat
bottom plate that will be the future home of the viable monoclonal
ES cell colonies for the growth and selection of possible HRE cell
lines for blastocyst injection and the growth of F2 generation mice
that are homozygous for the mutation.
ii. The plate will be prepped from EF cell stocks which are growing
on culture dish in incubator.
b. Protocol:
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 i. EF cells irradiated at IR 30,000 rads
ii. Wash with PBS (5ml)
1. Dispose of PBS
iii. Add trypsin (1ml)
1. Incubate 5 min at 37 C
iv. Add EF media (3ml)
1. Resuspend 10-20x
v. Count Cells with Haemocytometer
1. Desire 10,000 cells per well
a. Create stock solution that is 100,000 cells /ml
vi. Fill 24 wells per each person in group so a group of two fills 48
wells out of 96 well plate.
vii. Use a Corning 3596 flat bottom 96 well plate.

c. Results:
i. The EF 96 well flat bottom plate is created through this procedure
and it will be used to hold the colonies that survived the G418
selection after the transfection.
ii. The plate will be incubated until it is used with EF media on top of
the cells to prepare the feeder layer for the ES cells.
iii. The plate rows utilized by group 6 are as follows:
1. Susan Liem Row A 1-12 / B 1-12
2. Michael Jackson Row C 1-12 / D 1-12
iv. These selected 48 wells will be used to house 48 monoclonal
colonies of ES cells.
v. Haemocytometer Calculation and Cell Count Data:

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7. Plate Surviving G418 ES cell colonies on 96 well Flat Bottom EF plate
a. Purpose:
i. The goal of this procedure is to isolate monoclonal cell lines that
have grown colonies on the ES G418 plate by segregating
individual colonies into individual wells for further growth and
preservation.
ii. The reduction from 100 colonies to 48 colonies will make the
analysis more manageable for identification of HRE cell lines.
b. Protocol:
i. Get ES cell culture dish from incubator and count colonies in 4
quadrants of the plate. Record colony count in dish.
ii. Dispose of media in dish and replace with 3ml PBS
iii. Locate a 96 well round bottom plate (Corning 3790) which will be
the intermediate home for the ES cell colonies before they are
added to the 96 well flat bottom EF plate prepared in step 6.
iv. Add 30 µl of trypsin to each 48 wells of the round bottom plate.
v. Record the well location of the samples and follow the transfer
from round bottom to EF flat bottom.
vi. Using a p200 set a ~100µl suck up 20 µl trypsin and then carefully
by a technique of harvesting a ES cell colony off the plate add the
colony to the well in the 96 well round bottom plate
vii. Each individual prepares 24 samples for a total of 48 samples and
check for presence of cell colonies.
viii. Go through the entire protocol for each row of twelve to keep the
cells as healthy as possible and to restrict the stress to differentiate.
ix. Select 12 samples and place in respective well on round bottom
plate.
x. Incubate at 37 C for 2 min
xi. Add 120µl of ES media (G418)
xii. Resuspend 20x
xiii. Transfer entire sample (~150µl) into EF plate (feeder).
1. First remove EF media from the well and be careful not to
disturb the bottom of the well.
2. Add the ~150µl volume from the round bottom well to
respective 96 well flat bottom EF plate and then allow to
incubate.
xiv. Repeat colony selection and trypsinization for each of the twelve
samples at a time and then move to EF plate in groups of twelve.
SO THIS PROCEDURE WILL BE REPEATED 4 TIMES TO
MAINTAIN ES CELL HEALTH.
xv. Change ES cell media 1 day after procedure with ~150 µl.
xvi. Change ES cell media 4 days after procedure with ~150 µl.
xvii. Use a 96 round bottom ( Corning 3790)

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xviii. Use a 24 well flat bottom (Corning 3524)

DIAGRAM OF PLATE TRANSITIONS IN PROTOCOL

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 c. Results:
i. The cells were isolated by group individual in groups of twelve
and transferred between dishes from the round bottom plate to the
EF flat bottom plate through careful notation.
1. Round Bottom Rows
a. Susan Liem A 1-12 / B 1-12
b. Michael Jackson D 1-12 / E 1-12
2. Transfer to 96 well Flat Bottom EF plate
a. Susan Liem A 1-12 / B 1-12
b. Michael Jackson C 1-12 / D 1-12
ii. The cell colonies were kept in individual wells because they are all
monoclonal and that means that they all have arisen from same cell
so these specific ES cells should share the same genetic code with
respect to target construct location. Whether the target construct
was adopted by NHEJ or HRE.

8. ES Cell Colony Growth and Transfer of ½ to Round Bottom 96 well for

freezing and then Transfer of ½ into 24 well plate for rapid ES growth for
PCR analysis
a. Purpose:
i. The purpose of this protocol is to divide the cells that have grown
in the 96 well EF plate in half and freeze half in a round bottom
plate and then grow up the other half in 24 well plates for DNA
purification and PCR analysis.
ii. The freezing of half of the sample in a round bottom 96 well plate
is to have frozen samples of the monoclonal cell colonies that can
be thawed to grow into viable blastocyst injectable ES cells.
iii. The other half of the 48 wells will be independently grown in ES
media without EF cells in a 24 well plate to gain sufficient DNA
for purification and PCR analysis via agarose gel electrophoresis.
b. Protocol:
i. Get 96 well flat bottom plate from incubator and look at each well
to determine the presence of cells to determine if transfer is
necessary for each of the wells.
ii. Take the 96 well flat bottom EF plate and prepare samples for
freezing and growth.
iii. Dispose media
iv. Add 100 µl of PBS wash
1. dispose wash
v. Add 30 µl trypsin
1. incubate 37 C for 5 min
vi. Add 100 µl ES media
1. Resuspend 20x
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 vii. 130 µl in each well so half is 65 µl
viii. Take 65 µl and transfer to pre-prepared freeze plate
1. Prepare freeze plate with 2x freeze media (65µl / well)
a. 20% DMSO
b. 20% FCS
c. 60%DMEM
2. Add 65µl 2x freeze media + 65µl ES cell sample = 130 µl
3. Record location corresponding to EF plate for identification
of appropriate cells after PCR analysis
4. Allow to slowly freeze to -70 C
ix. Remaining 65 µl of ES cell culture is now transferred to 24 well
plates with 1 ml of ES media in each well. Total volume = 1065µl
x. Each group member has their own 24 well plates if all 48 wells in
EF plate contain cells. So transfer remaining volume from the EF
plate to 24 well and incubate for 2 days.

Diagram of Plate Transfer steps in Protocol #8:

c. Results
i. All 48 wells in EF plate had ES cell growth and therefore all 48
cell lines were harvested and separated into the freeze and growth
batches. The ES cells once grown will then be lysed and the DNA
purified leading to PCR amplification of the target construct
yielding in the identification of the NHEJ and HRE samples.
ii. The 24 well plates do not contain EF layers as that the cells will be
lysed and then analyzed so the desired outcome if rapid growth and
replication in ES media.

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9. Purify DNA Extract & PCR Screen Colonies:
a. Purpose:
i. To identify the NHEJ and HRE samples and if a HRE sample is
present the thawing of the corresponding sample for future
injection into blastocysts to create a chimeric mice leading to F2
progeny that are ¼ homozygous for mutation.
ii. The DNA purification will yield a sample to run on an agarose gel
to determine if it is necessary to run PCR. (i.e. if no DNA no need
to run a PCR reaction)
iii. The sample size will be decreased from the original 24 to 12
samples for DNA purification and then further reduction to a total
of between 5 and 7 samples for PCR amplification. This reduction
is sample size is a modification based on the fact that this is a
teaching lab.
b. Protocol:
i. From 24 well ES cell plate for each individual if enough samples
select 12 wells to harvest for DNA purification. View wells under
microscope to aide in determination of wells. Follow these well
labels through the remainder of the lab to aide in identification and
thaw of the appropriate monoclonal cell colony that may contain
HRE.
ii. Harvest 12 cell samples (samples with most cells)
1. dispose media
2. wash PBS (1 ml) (rinse down side of well)
a. dispose PBS (use p1000)
3. Add 0.5 ml of Lysis buffer + 5 µl pK (proteinase K)
4. Transfer after multiple pipetteing move into label 1.5 ml
ependorf tube.
5. Incubate at 55 C overnight
6. Remove from Water Bath and put in 4 C refrigerator
iii. Purification of DNA
1. Buffer is SDS precipitate at 4 C
a. Cold & Salt aide in precipitation of DNA
b. Add 150µl saturated NaCl invert 10x
c. Incubate on ice 5 min
d. Spin at 14 k for 10 min
e. Transfer supernate to new tube add 1 ml ethanol
(100%) tube contain 1ml ethanol 0.6 ml DNA
f. Let ethanol DNA mix sit on ice 10 min
g. Spin 14k 10 min dispose ethanol
h. Wash 1ml 70% ethanol
i. Air dry
j. Resuspend in 20 µl diH2O
k. Run 2 µl DNA on gel (0.8% agarose, 100V, 30 min)
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 l. Prep sample for gel 2µl DNA + 2µl load buffer +
6µl diH2O = 10 µl sample for agarose gel analysis

iv. PCR amplification and analysis

1. Prep PCR master mix for 11 samples but run only 10.
a. PCR mix (for 11)
i. 2.2 µl Taq
ii. 27.5 µl 10x Taq Buffer
iii. 11 µl primers
iv. 5.5 µl 10mm dNTP
v. 206.8 µl diH20
vi. TOTAL 253µl
vii. 23µl per tube + 2 µl DNA = 25 µl
b. PCR cycle info
i. 94 C 1 min
1. 60 C 1 min
a. 72 C 1 min
ii. 33 cycles rotating through these values

v. PCR product Agarose Gel Electrophoresis

1. Use 8 well comb and modify small tooth comb to make 12
lanes for the gel (1.0% agarose 100V 45 min)
2. Load sample as follows
a. 25µl PCR product
b. 5µl 6x Load dye
c. TOTAL = 30µl
3. Record location of sample in gel and remember to use two
ladders on in each level. (ladder is GibcoBRL 1KB ladder)
4. Add 5µl of ladder
5. Run gel and photograph and analyze.

c. Results:
i. The goal of the DNA purification was to purify the DNA sample to
be amplified by PCR but first the presence of DNA was observed
via agarose gel electrophoresis.
ii. Gel Picture: DNA presence

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iii. The gel above displays the samples that were to be amplified using
PCR and this was a check of the presence of DNA in the sample.
If no DNA was present there would be no need to amplify a non-
existent segment of DNA so this allows for the identification of
viable samples.
iv. All the wells that contained samples provided results and depicted
the desired outcome of the DNA gel analysis and the DNA band is
the bright band above the 12 Kb value on the ladder.
v. The results of the PCR amplification was then also run of an
agarose gel with large wells to allow the use of the entire 30µl
sample to guarantee the loading of a complete PCR sample. The
PCR analysis of the construct contained a positive control to show
that the PCR worked so a band at 2000 Kb is the positive control
and displays a NHEJ event whereas a lane with two bands would
depict a HRE if the second band was larger (ie 2500 Kb) to show
the extension of the target site by the inserted gene of interest.
vi. PCR product agarose gel diagram:

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 vii. The gel above shows that the 1st five sample which are those of
Michael Jackson displayed NHEJ but did not show a HRE event.
These events are quite rare and in 500 samples you would expect
approximately 3 to 4 events. The lack of bands presents a failed
PCR reaction which could be caused by a multitude of issues.
Especially the Taq polymerase being denatured due to long
exposure time outside ice.
viii. The goal of these procedures is to identify a HRE sample colony
and then unfreeze the sample and inject those ES cells into a
mouse blastocyst to produce agouti mice (chimeric) P and then to
produce ¼ homozygous mutant by the F2 generation after selfing
the F1 generation.

ix. Possible integration events and PCR analysis

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x. Agouti Mouse

xi. Creation of germ line mutation from injectable blastocyst

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 Discussion:
The goal of these laboratory procedures was to produce a HER embryonic stem
cell and to take these monoclonal cells from a frozen 96 well plate, thaw them, and inject
them into a blastocyst to create chimeric mice. The major concerns surrounding this
procedure with relation to this laboratory environment (teaching lab) is that the
prevention of differentiation of ES cells was not maintained at a high level of efficiency
during the growth on the 96 well flat-bottom EF plate. The color change of the ES media
from a magenta/pink to a yellow displayed the creation of waste by the cells and this
waste if not frequently removed will cause the ES cells to differentiate into epidermal
cells to survive the harsh nutrient starved environment. The issue with the premature
differentiation of the ES cell line is that the injection of an ES cell into a blastocyst and
the subsequent viability rely heavily upon the ES cell remaining in an undifferentiated
state. If the ES cell differentiates the blastocyst may be rejected completely and expelled
or it may kill off the mutated gene providing a useless generation of progeny from a
surrogate mouse mother. This is a major weakness of the procedure as that the media
change interval is not adequate to prevent differentiation and as well the sample size also
presents the possibility that no HRE will occur.
A second concern is the infrequency of HRE events versus the NHEJ events
within the targeting construct and the host cell DNA. The blunt ends provide two
possible methods of integration of the target construct and these two are non-homologous
end joining and homologous recombination. These events create distinct PCR products
for analysis as the construct contains a control to differentiate between a HRE and a no
PCR amplification reaction. The HRE event will contain two bands to exhibit
homologous recombination. It has been suggested that in a sample size of 500
monoclonal cell colonies that you can expect 3 to 4 HRE events and this yields about a
1% occurrence rate for the event. So NHEJ will be the predominant form of
recombination present in the ES cells that survive G418 selection.
The overall results provided by analysis of 10 samples yielding 5 results
maintains the 1% frequency of an HRE event. The use of a positive control to provide
evidence for a successful PCR reaction is a major benefit to analysis. The process of

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PCR amplification is still not providing consistent returns but that is a major lesson to
learn in the field of biological lab work in that the ratio of a successful procedure to a
failure is usually heavily in the favor of error. However, error is not always a bad event
in the process of science as it may bring a new technique to the fore-front such as the use
of spray bottles to transform Arabidopsis thaliana with agrobacteria. A process that once
was thought to require meticulous targeting of the gametes to produce sufficient results.
The technique of reverse genetics is a beneficial and highly utilized process in
biology today. It plays a crucial role in the analysis and treatment of human disease by
creating a mouse model or other animal models by targeting a specific gene for knockout
and then through the process of recombination the development of HRE allows for the
production of germ-line mutants that can model a necessary human disease and allow for
advance and benefit to humanity.

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