BICD 101 Lab Report #2

Lab #2: Reverse Genetics A02-92-6779

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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 A02-92-6779

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)

2. 10% FCS (25 ml) 3. Penn/Strep(2.5 ml) 4. 50 µM β-mercaptoethanol (0.85 µL)

TOTAL VOLUME = 250ml

1. Provides the essential nutrients to keep cells happy and provide energy for growth.

2. DMEM-092 is 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)

2. 15% FCS (37.5 ml) 3. 1x glutamine (2.5 ml) 4. 1x non-essential amino acids (2.5 ml) 5. 1x sodium pyruvate (2.5 ml) 6. 1x Penn/Strep (2.5 ml) 7. 100 µM β-mercaptoethanol

(1.7 µl) 8. LIF (Leukemia Inhibitory Factor) (1 µl)

TOTAL VOLUME = 250 ml

1. Provides the proper environment for ES cell growth via nutrients.

2. DMEM-044 specific media for ES cells provide nutrients

3. FCS- neutralize Trypsin

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 2. 80 % DMEM 3. 10 % FCS

DMSO will make the cell membrane porous as not to break and become brittle during cryogenic freeze. DMEM and FCS provide necessary nutrients and neutralize Trypsin in cell solution

Linear Target Construct

Is the linearized plasmid 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 Linearize Plasmid

Will digest circular 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 2. Dye

Used in Agarose gel 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 GibcoBRI

Used as a analysis tool to 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 Proteinase K (5µl) pK

Used to lyse open cells to get 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 2. 10x Taq Buffer

Used in conjunction with DNA sample for the proper


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3. Primers 4. 10 mM dNTP 5. H2O

amplification of a targeted sequence for analysis. The 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

4. Spin 2nd 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.

Documents

BICD 101 Lab Report #2