Production of Knock Out Mice

PRODUCTION OF KNOCKOUT MICE

Engineering of a targeting vector and its subjection to homologous recombination in embryonic stem (ES) cells, when done by well established procedures, will result with ES cells that are available for injection into mouse blastocyts for further germ line transmission. As in the case of Transgenic Mouse production, the injected blastocyst (minimum of 40) will be implanted into foster mice. High rate of germ line transmission of the mutated allele depends on the quality of the ES cells used. Therefore, the investigator must provide evidence that the ES cells have been carefully treated under sterile condition, as well as some indication that the ES cell line used has colonized the germ line. Our core had successfully produced mice in which genes such as casein kinase or Interleukin-16 were knocked out.

As outlined in the Schematic Overview below, embarking on a gene knock out project will require several steps. Begin by cloning the gene (from 129/Sv genomic library). Map it, construct the targeting vector, electroporate the ES cells (derived from 129/Sv strain of mice), drug selection of resistant cells, isolate and screen by Southern blotting or by PCR drug resistant clones. All of these procedures are to be done by the investigator. Following these procedures, the core will inject the selected clones into blastocy sts and implant them into foster mice as well as initiate the breeding of pups. The investigator will resume responsibility again at the stage of breeding chimeric mice and identifying by Southern blot analysis (or PCR) heterozygous as well as knock out mice. The later will rely on a careful, initial planing of the targeting vector, so that it will be possible, by enzyme digestion of genomic DNA, to distinguish between the wild type gene, heterozygous knock out and homozygous knock out. Detailed protocols of preparation of ES cells, electroporation, clonal selection and more are available in the following book: Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994) Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory. ** you may want to replace this with the new edition of the same …

The transgenic core is currently testing freshly derived (in the core) ES cells for their ability to colonize the germline. Once tested, they would be available for investigators on campus. Until then, the core would be able to direct the investigator to other potential sources. G418 resistant mouse fibroblast feeder cells, needed for growing ES cells, and the pGK-neo and pGK-TK DNA constructs used for targeting are available in the core and from a few investigators on campus.

The lengthy process outlined here often yields knock out mice with exciting and intriguing phenotypes, depending on the gene of interest.

GENE TARGETING IN MICE: THE PRODUCTION OF KNOCK OUT MICE
Schematic Presentation

GENERATION OF KNOCK OUT MICE
Overview
PHASE A
Procedure Estimated Time
- Screen a genomic library (129/SV)
- Obtain a genomic clone
     
- Engineer construct to abrogate gene and allow double selection  
   

12 months

       

 

- Electroporation into ES cells
1 day
- Select cells resistant to G418 and gancyclovir
- Select individual clones

2-3 weeks

- Use Southern blots, PCR to identify homologous recombinants
1-5 months
PHASE B
Procedure Estimated Time
- Culture targeted ED cell clones
2 days
- Inject ES cells into C57BL/6 blastocysts and implant into uterus of foster mother
1 day
         
- Pups born
3 weeks post injection
- Identify chimeric offspring by coat color
2 week old pups
- Confirm by Southern blot of tail biopsies
3 week old pups
- Outcross male chimeras
8 week old mice
- Southern blots on tail biopsies to determine germ line transmission
3 week old pups
- Breeding of germ line chimers
6-8 week old pups
         
- Southern blots on tail biopsies to identify hetrozygous and homozygous mice
2-3 week old pups
         
* All of phase B should take approximately 5-6 months *

Boston University School of Medicine/Transgenic Knock Out Core Facility


Scientific Director of Transgenic/Knock out Core, Dr. Katya Ravid
Transgenic Specialist and Manager, Greg Martin

Primary teaching affiliate
of BU School of Medicine