Germline transmission rates
PGT achieves high rates of germline transmission for its ES cell lines. Specifics are as follows:
A) V6.5 (129Sv x C57BL6) ES cells
If timing and price are important parameters, and the genetic background is not a major issue, we recommend this hybrid ES line. Our stock of V6.5 ES cells was expanded at the Core from a stock originally derived at the Jaenisch lab at MIT. Germ line transmission occurs in > 80% of all injected clones, except in cases of embryonic lethality of the mutant allele.
In practice this means that often only one, and sometimes two independent ES clones need to be injected for a given project to obtain germline transmission.
B) Our C57BL6 KOMP line EAP6
This line is of particular utility when a pure C57BL6 background is necessary for the project and backcrossing either would take too much time or would not be sufficient to completely remove 129 alleles, particularly in the vicinity of the targeted gene.
The EAP6 C57BL6 ES cells were derived at Penn under contract from the NIH – KOMP consortium. They have been extensively tested and characterized by the Penn Gene Targeting Core and Laboratory in collaboration with the laboratories of Drs. Eric Pierce and Klaus Kaestner. By comparative genomic hybridization the EAP6 line was demonstrated to be free of genetic rearrangements at a 15 KB resolution. The Penn Gene Targeting Core has developed special growth conditions for these ES cell lines. Based on extensive germline tests involving more than 30 independently targeted C57BL6 ES clones the Core routinely achieves rates of germline transmission that are comparable to the rates for 129 ES cells (about 56 % for a single C57BL6 ES clone). For detailed documentation on germline transmission rates of our C57BL6 ES cell lines please see below. In practice, usually 3 independently obtained ES clones need to be injected into blastocysts for germline transmission.
|Traditional published growth conditions for ES cell lines:
Low germ line transmission rates: 3 of 15 tested independently derived ES cell lines go germline
|New growth conditions developed by the Penn Gene Targeting Core and Laboratory:
High germline transmission rates. 9 of 16 independently derived ES cell lines go germline