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Plasmids/Protocols
This page identifies some important issues to consider when thinking about generating a targeted mutation in the mouse. It is less a comprehensive guide that a series of tips.
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For additional details:
Guenet, J. L. Animal Models of Human Genetic Disease, in M.A. Vega (Ed.) Gene Targeting. CRC Press.
Plagge, A., G. Kelsey, N. D. Allen. Directed mutagenesis in embryonic stem cells, in I.J. Jackson & C.M. Abbott (Eds.) Mouse Genetics and Transgenics; a Practical Approach. New York: Oxford University Press. |
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Researchers are urged to consider these issues carefully before consulting the specific protocols for generating targeted mutations in murine embryonic stem (ES) cells.
Tips for conceptualizing mouse knockouts
The knockout mouse is a powerful tool for assessing gene function and subtle gene interactions in genetic diseases which have been identified at the molecular level and where the homologous mouse gene has been cloned.
To make a null mutation, one must know the locations of the functional exons to ensure that the target construct includes the entire gene. And if it it turns out that the target construct includes extraneous sequences which effect the mice's functions, then one must remove the extraneous sequences to properly isolate the target gene's effect.
A gene targeting construct should be between 5-10kb to increase the frequency of replacements without sacrificing the construct's manipulitability.
To ensure sequence identity, make the homology arms from the same mouse strain that you will be inserting into.
Since the goal of producing a knouckout mouse is to clearly deliniate the function of the gene knocked out, full homologous gene replacement (rather than insertion) is required.
Since insertion frequency is low and only a portion of replacements are homologous, use baterial gene markers known to resist particular antibiotics to identify homologus replacements. And if you have more than 1 transfection step, you must use more than 1 markers. But multiple exposures to drugs may mean that the ES cells can't generate a mouse. Two seems to be a reasonable maximum for transfection steps.
General protocol for generating targeted mutations in murine embryonic stem (ES) cells
Overview: These protocols were compiled by Renee Hackenmiller when she was a graduate student in our lab. They represent our working methods, and may vary somewhat from other published protocols. Our targetings have been successful using CD1, R1 and RW ES cells. Currently, we use low-passage RW cells obtained from Genome Systems, Inc. In this document, we often refer to ES cell media as "RW media," accordingly.
In general, ES cells are maintained on a layer of non-mitotic (mitomycin C-treated) mouse embryonic fibroblast (MEF) feeder cells. This is CRITICAL for ES cells that will be later injected into blastocysts. For genotyping purposes, ES cells may later be adapted to grow on gelatin-coated plates in the absence of MEFs.
IMPORTANT: It is critical to use the appropriate fetal bovine serum for ES cells that will be used to generate mice. See notes in the "Recipes and Related Protocols" section below. Also, be sure to plate MitoC-treated MEFS one day before plating ES cells onto them. This gives the MEFs a chance to plate and spread out appropriately.
1. Treating plates with gelatin
| A. |
Place 400ml H2O and 0.4g of gelatin (Sigma G-1890) in a 500ml bottle. |
| B. |
Autoclave for 30 minutes to sterilize and to get the gelatin into solution. |
| C. |
Cover the bottom of the wells with this sterile/autoclaved 0.1% gelatin. Approximate amounts:
10 cm- 5 mls/well
6 well- 1.5 mls/well
24 well- 0.5 mls/well
96 well- 200 ul/well |
| D. |
Let the plate sit in the hood for at least 10-30 minutes. |
| E. |
Aspirate off the gelatin before adding cells and media. |
2. Treat MOUSE EMBRYONIC FIBROBLASTS (MEFs) with Mitomycin C
(methods for obtaining MEFs can be found in "Teratocarcinomas and embryonic stem cells - a practical approach" by E.J. Robertson IRL Press, 1987)
| A. |
Aspirate media off of day 14 MEFs that are confluent on a 10cm plate. |
| B. |
Combine 6ml of MEF media with 10 ug/ml mitomycin C. Filter the mixture. |
| C. |
Add the treated MEF media to the day 14 MEFs. Incubate for 2-3 hours at 37º. |
| D. |
Aspirate off the media, and wash 3 times with 5 mls of PBS to get rid of all the MitoC. |
| E. |
Trypsinize plates with 3mls trypsin for 5 minutes. |
| F. |
Add 5ml DMEM + FBS to plates to inactivate trypsin. |
| G. |
Spin down cells and aliquot to freeze.
Notes:
--Two plates will freeze into 3 vials. 1 vial is good for one 10cm, one 6 well, one 24 well, or one 96 well plate.
--Freeze media is 20% FBS, 10% DMSO.
--MEFs tend to lose steam after passage 3. Accordingly, when you thaw a p.1 vial of day 14 MEFs, split that 1:5, and then split that 1:4. This procedure will produce 20 10cm plates of MEFs, which yields 30 vials of MitoC MEFs. |
3. Thawing ES Cells
| A. |
Remove low-passage ES cells from liquid nitrogen, and thaw quickly in 37º H20 bath. |
| B. |
Transfer cells to a 15ml conical with 5mls of prewarmed ES cell media. |
| C. |
Spin down cells for 5 minutes. |
| D. |
Aspirate off supernatant. Resuspend cells gently (don't use tip on pipette) in appropriate volume of media for culture dish, and then transfer to well containing MitoC treated MEFS. Typically, we freeze ES cells from one well of a 6-well plate (80% confluent) per vial, and replate into one well of a 6-well plate. |
Thaw low passage ES cells which have been frozen in RW Media onto MEFs that have been MitoC treated and plated onto gelatin-treated plates 8-24 hours before plating ES cells.
4. Electroporation with gene targeting construct
| A. |
Pre-warm machine - we use a Bio-Rad Gene Pulser II. |
| B. |
Trypsinize ES cells - we usually electroporate 2 X 10 7 cells (1 confluent 10 cm dish). Resuspend in DMEM + FBS to inactivate trypsin. Spin down. Count cells. Wash several times in DMEM with no additives. |
| C. |
DNA: Precipitate 30ug of linearized DNA. Wash with 70% ethanol (in hood) and resuspend in 30 UL of sterile TE. |
| D. |
Add DNA to cells resuspended in 0.8 ml DMEM no additives in cuvette. Let sit for 5 minutes before electroporation. Electroporate at 250 volts, 500 uF. Time constant should be about 10.0. Do a "no cell" control first to check time constant. Also do a "no DNA" control. As quickly as possible, transfer the zapped ES cells into pre-warmed RW medium to recover. |
| E. |
Put 1/4 of "no DNA" onto a 10cm plate with MitoC MEFs. Split cells with DNA into 4, plating entire electroporation, but on 4 separate 10cm plates |
5. Selecting transformed ES cell colonies
Start selection 24-48 hours after Electroporation.
No DNA plate - select with 200ug/ml G418.
One DNA plate - select with 200 ug/ml G418. This allows a comparison of the number of singly-resistant to doubly-resistant colonies. Ideally, this ratio should be 10:1, but can vary.
Remaining DNA plates (3), select with 200 ug/ml G418 and 1uM Gancylcovir.
6. Changing Media
Change the media everyday so long as it turns yellow. Subsequently, change the media every other day.
7. Picking Clones
On day 10-12, select colonies for screening from the double selection plates.
| A. |
Thaw MitoC MEFs onto gelatin plates one day prior to picking clones. |
| B. |
Wash the double selection plate 2-3 times gently with PBS. Add 5-6ml of warm DMEM no additives to the plate. |
| C. |
Using a p200 set at approx. 50ul, use a sterile pipette tip to pick a single colony into a round bottom (U Bottom, Falcon, 3077) 96 well that has 25ul of trypsin in it. |
| D. |
After a row of 12 clones has been picked, look under scope to insure that each well has a clone. Add 150ul of RW media to inactivate trypsin. Break up the clones by pipetting up and down with either a p200 or the multi channel pipetteman. |
| E. |
After breaking up clones, look under scope to make sure they are broken up. Transfer the entire well contents (approx. 200ul) to a flat bottom 96 well plate (Falcon, 3072) that was gelatin-treated and plated with MitoC MEFs 24 hours prior. Be sure to give each well a number. |
| F. |
To avoid mixing clones, cross off row in U-bottom 96 well after picking clones into it. |
| G. |
The next day, change the media on the clones. Change pipette tips between each well to avoid contamination. Use pre-warmed RW media. Approximately 100ul of media are required. |
NOTE: Many researchers now conduct PCR-based genotype screen on ES colonies at this stage (96-well). We usually expand the cells as described below, partly from habit, and partly because it allows you to group colonies by the rate at which they grow.
8. 96 well to 24 well split
| A. |
Look at the clones under the scope and from the bottom to determine which need to be split. Mark the wells that need to be split. |
| B. |
Wash the double selection plate 2-3 times gently with PBS. Add 5-6ml of warm DMEM no additives to the plate. |
| C. |
Change the media 2-8 hours before splitting. You can split several clones at once. To avoid mixing things up, it is best to do it in groups of 6-12. |
| D. |
Wash the wells to be split with pre-warmed PBS. |
| E. |
Add 50ul of trypsin-EDTA to each well to be split. |
| F. |
Incubate at 37º for 5 minutes. |
| G. |
Add 150ul of RW media to inactivate the trypsin. |
| H. |
Pipette up and down to break into singe cell suspension. Check under scope to make sure the cells are single cell suspension. |
| I. |
Transfer entire contents of well to a 24 well with 1.5mls of RW media in it that has been marked with the appropriate clone number |
| J. |
Change the media the day after the split. Change pipette tips between each well to avoid contamination. Approximately 1 ml of RW media is needed per well |
9. 24 well to 2x24 well split (DNA plates and freeze plates)
Note: One 24 well -plate will be frozen (with MEFs) to preserve the colonies, the other (without MEFs) will be used to prepare genomic DNA for genotyping.
| A. |
The day before splitting, prepare 24 well plates with MitoC MEFs plated onto gelatin-treated plates. The number of plates will depend on how many colonies you believe will need passage, based on their morphology the previous day. |
| B. |
Look at the clones under the scope and from the bottom to determine which need to be split. You can split several clones at once. It is best to do it in groups of 6-12 to avoid mixing things up. |
| C. |
Mark the wells that need to be split. |
| D. |
Change the media 2-8 hours before splitting. |
| E. |
Wash the wells to be split with pre-warmed PBS. |
| F. |
Add 150 UL of trypsin-EDTA to the each well to be split |
| G. |
Incubate at 37º for 5 minutes. |
| H. |
Add 150 UL of RW media to inactivate the trypsin. |
| I. |
Pipette up and down to break into singe cell suspension. Check under scope to make sure the cells are single cell suspension. |
| J. |
Transfer 1/2 the contents of well (150 UL) to a 24 well with feeders (MEF MitoC) and 1.5mls of RW media in it that has been marked with the appropriate clone number. Mark this plate as the freeze plate. |
| K. |
Transfer the other 1/2 (150ul) to the corresponding location on a 24 well that has been gelatin-treated (no MEFs) and has 1.5ml of RW DNA media in each well and is marked with the appropriate clone number. Mark this plate as the DNA plate. L. Change the media the day after the split. Change pipette tips between each well to avoid contamination. Only 1ml of RW media is needed per well |
10. 24 well plate freeze
Freeze plates when the majority of the wells are 80% confluent. Not all the cells will be in the ideal state, but they should survive the freeze. If a well is extremely far behind the rest of the plate, split it 1:1 onto a another 24 well plate with feeders. Be sure to note your having done this (in case that clone turns out to be positive).
| A. |
Assemble freeze media: 6 ml RW media.
1 ml DMSO
3 ml RW Hyclone FBS |
| B. |
Filter sterilize with 10 ml syringe and syringe filter. |
| C. |
Put on ice. |
| D. |
Aspirate media off the plate to be frozen. Change pipette tips between each well to avoid contamination. |
| E. |
Add 0.5ml of ice cold sterile freeze media. |
| F. |
Wrap parafilm around plate. |
| G. |
Place in styrofoam box. If you freeze more than one plate at a time, two plates can fit into one box. Stack them on top of each other separated by either a blue Styrofoam floating eppendorf rack or folded paper towels. Wrap box with colored tape, and record date on tape. |
| H. |
Store the box in -80 freezer. After genotyping the corresponding "DNA Plate" (see next section), you will want to thaw and expand the appropriate clones. To do this: |
| I. |
Thaw quickly by adding 1 ml of 37º media. |
| J. |
Use sterile transfer pipets to remove cells to 5 ml of warm media. |
| K. |
Wash well once with 1 ml of warm media - add to original tube. |
| L. |
Spin down. Aspirate off media. Resuspend gently in 1 ml of RW media and transfer to a new 24 well with MitoC MEFs. Change the media the next day. |
11. 24 Well DNA Prep
When the DNA plates are confluent, they need to be prepped. It is easiest to prep an entire plate at a time, but if the wells are not all ready to be prepped, they can be prepped separately.
Entire Plate Prep:
| A. |
DNA Prep does not have to be done in the hood if the entire plate is being prepped. |
| B. |
Wash plate twice with -/- PBS. |
| C. |
Add 0.5ml of DNA Lysis buffer. |
| D. |
Seal the 24 well with an ELISA stick seal, then put lid on. Place the plate in the ziplock bag on top of a moist paper towel. Place at 55º overnight. |
| E. |
Next day, remove digested material (using a new pipette tip for each sample), and place the DNA into an eppendorf labeled with the clone # and the date. Add 500ul of isopropanol. Mix until you see fluffy DNA. |
| F. |
Spin at 14K, 10 min, room temp. Remove supernatant, wash with cold 70% etoh. Remove 70% etoh. Get the majority of the ethanol out off the eppendorf. Do Not Speedy Vac. |
| G. |
Add 50ul of TE. Do not attempt to resuspend at this point. Just add the TE and put at 55º overnight. |
| H. |
8-10 hours later, Digest 10ul for Southern(?). |
Single Well Prep: Essentially the same, except for the following:
| A. |
Do the PBS wash in the hood. |
| B. |
Add 500ul Lysis buffer in hood, let sit for about 5 minutes in hood, then transfer to an eppendorf. Be sure to get most of the cells out of the well. The mix will be a little goopy/snotty, so be careful not to suck it up into the pipetteman. Incubate eppendorf with cells + lysis overnight at 55º. |
Recipes and Related Protocols
Heat Inactivation of Serum
All serum needs to be heat inactivated before use in tissue culture.
A. Thaw serum at 37º. B. Place at 55º for 30 minutes to heat inactivate. C. Serum can be stored for 2-3 months at 4º. Otherwise, aliquot and refreeze.
Gelatin
400ml H20 0.4g of gelatin (Sigma G-1890) in a 500ml bottle Autoclave for 30 minutes to sterilize and get gelatin into solution.
FBS
Note: It is imperative to use the correct serum for any ES cells that may ultimately go into a mouse, as well as MEFs on which the ES cells will grow. Contact HYCLONE and order FBS that has been tested for ES cell work, and is known to have given successful germline transmission.
In order to conserve on the RW FBS and the LIF ESGRO, one can use a 10% FBS media to inactivate trypsin when you will be spinning down the cells. If you are not spinning down the cells, you need to inactivate with the media the cells grow in (normal RW media).
500ml DMEM (any kind) 50ml FBS (any kind of heat inactivated serum-cheap stuff, e.g. GIBCO FBS)
MEF Media (Use for any cell that will go into a mouse)
500ml DMEM (high glucose, no L-glut, no Na Pyruvate) 50ml HYCLONE FBS (heat inactive). 5ml Penn/strep 5ml Non Essential Amino Acids 3ml L-Glutamine Filter sterilize before using. Replace L-glut every 2-3 weeks.
RW Media
410ml DMEM (high glucose, with L-glut, no NA Pyruvate) 75ml HYCLONE FBS 5ml Penn/strep 5ml Non Essential Amino Acids 10ml L-Glutamine 4ul BME -- Dilute 4ul of BME in 5ml of DMEM before adding to mix 50ul LIF ESGRO (GIBCO) -- Dilute 50ul of LIF ESGRO in 5ml DMEM before adding to mix Filter sterilize before using. Replace L-glut every 2-3 weeks.
RW DNA Media (Do NOT use for cells that will go into a mouse)
400ml DMEM (high glucose, with L-glut, no NA Pyruvate) 50ml of Fetal Clone II FBS 50ml of Newborn Calf serum-NBS 5ml Penn/Strep 5ml non essential amino acids 10ml L-glut 4ul BME-- dilute 4ul of BME in 5ml of DMEM before adding to mix 250ul of homegrown lif Filter sterilize before using. Alternative: Replace the 50ml of NBS with an additional 25ml of FCII and an additional 25ml of DMEM.
Lysis Buffer 15 ml of lysis buffer: 150 UL 1M Tris pH 7.5
300 UL 0.5 M EDTA
600 UL 5M NaCl
188 UL 20 mg/ml Proteinase. K (BMB)
0.075 g Sarocosyl
13.762 ml H20 Note: Lysis buffer can be stored for approximately 2 weeks at 4º. After that, Prot K goes bad.
Note: Only thaw the proteinase K once. No repeated freeze/thaw.
Amount of media for well types:
10 cm - 10 mls
6 well - 3 mls
24 well - 1 ml
96 well - 100 UL
*These amounts will be altered when the trypsin needs to be diluted out.
Related Links
Penn's Transgenic and Chimeric Mouse Facility |
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