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Generation of Knock out and knock in by gene targeting:
- Consultation with the targeting vector design
- Gene targeting by electroporation into mouse embryonic stem cells (mESC)
- Identification of homologous recombination events by Southern analysis
- Karyotyping for selection of two clones for blastocyst microinjection to generate chimeras
- Breeding of chimeras to ensure their capability for contribution to germline
- Project completed within 6-10 months from initiation of gene targeting
- Guarantee of germline transmission
The gene targeting service is an all-inclusive service that is initiated with electroporation of the vector of interest into the embryonic stem cells (ESCs). The laboratory commonly uses E14TG2A.4 mouse ESC, a stable cell line derived from 129P2/OlaHsd with confirmed germline transmission capability. The C57BL/6NCrf ES cells, generated and established by TMML using the C57BL/6NCr inbred strain, are also available for gene targeting experiments. Following electroporation, homologous recombination events are identified through genotyping with Southern analysis. Karyotyping will be performed on successfully targeted clones to ensure genetic integrity. Two clones with the normal karyotype will be selected for microinjection into wild-type blastocysts from the appropriate background, to generate high-percentage chimeras, judged by percentage of coat color contribution from the ESC. Selected chimeric males will be placed in breeding to ensure germline transmission. Genotypic analysis on the first-generation progeny is performed with Southern blot analysis. The success of the comprehensive gene targeting projects, performed by TMML, is defined by confirmation of germline transmission.
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