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ES Cell Gene Targeting

  • Receiving frozen embryos, thaw, perform embryo transfers: $ 500
  • Receiving blastocysts at ambient temperature: $ 500 for 3 embryo transfers, then $150 for each additional transfer (OAR charges will be separate)

For all services, download the Service Order Form and submit to the GMF Administration via email: hdemanbey@mcb.harvard.edu or to Biolabs room 1062. Arrange with OAR for transfer of animal to GMF; form can be downloaded here.

Obtain mycoplasma and mouse pathogen test data (Charles River Lab provides an economical solution) from ES cell clones to be submitted for blastocyst injection. Submit two vials of frozen ES cells with test result data and Service Order Form to the GMF (see PDF).

We will then inject > 30 blastocysts per ES cell clone, transfer embryos, produce offspring; Wean offspring, transfer to investigator's colony.

Disclaimer:
The GMF does not guarantee germ line transmission of a mutant allele by breeding chimeras. Based upon past experience, however, approximately 80% of service projects yielded germ line transmitting chimeras from either of the injected ES cell clones.


Gene Targeting - Tasks and Approximate Timelines


Tasks Timeline Responsible Parties

 

Targeting Vector Construction


Variable

> 12 weeks


1) Design and clone gene targeting vector
2) Confirm construction by sequencing vector
3) Linearize 50 μg DNA, precipitate in EtOH
4) Schedule ES cell transfection with GMF

 

ES Cell Transfection and Selection



3 weeks
1) Grow batch of 107 ES cells (J1, V6.5, JM8)
2) Electroporate with targeting vector, put under drug-selection
3) Isolate ~ 200 drug-resistant colonies, produce replica plate, freeze master plate
4) Forward replicate plates to investigator for genotyping

 

Genotyping ES Cell Clones



Variable:
2 weeks to n weeks



1) Genotype ES cell clones, confirm targeting at 5' and 3' end.
2) Request for GMF to grow putative targeted clones for confirmation

 

Blastocyst Injection


G0 : Analysis as early as a few days after injection.

F1 - Fn : Analysis up to several years

Analysis of transgene expressing mice

1) G0 - mice during gestation or postnatally
2) Expression and phenotype analysis in F1 to Fn mice

 

Chimera Analysis



Germline transmission:
8 weeks

Homozygous mutants:
15 weeks

1) Breed chimeras at 6 weeks of age
2) Check for germline transmission by genotyping ES cell derived (agouti) offspring
3) Intercross heterozygous mutant mice
4) Analyze homozygous mutants