Diploid Blastocyst Injection



Genetically modified mice are used extensively in research for understanding gene function and modeling human disease. In conventional gene-targeting methods, mutant mice are gen­erated by introducing mutations first through fhomologous recom­bination in mouse ES cells, which are then injected into wild-type mouse blastocysts (E3.5). If the targeted ES cell clone contributes to the development of the fetus, the pups produced will be chimeras, exhibiting patches of coat color from the host embryo and patches from the injected ES clone. Chimeric mice must be crossed with wildtype mice to determine if the ES cells can “go germline”, meaning they can contribute to the germline of the chimera and produce offspring arising from an ES cell-derived gamete.


The ESC line most commonly used in our facility is V6.5, which is a hybrid of C57Bl/6 and 129Sv/Jae and agouti in color. The embryos used for injections are B6D2F2 (C57Bl/6xDBA2, F2), which can be black, silver, brown or tan in color. In general, the higher the coat color contribution from the ES cell line, the more likely it is that the mutation will go germline. An approximate percent contribution can be assessed 7-10 days after birth, and sex determined by 3 weeks of age. Chimeric pups are typically weaned at 3-4 weeks of age.

The contribution to the germline by the ES cell lineage in any given chimera is unknown. On average, lower-percentage chimeras (those with less ES cell-derived fur) are less likely to have ES cell-derived gametes. Nevertheless, a high degree of chimerism is not a guarantee of germline transmission. Therefore, it is generally prudent to breed all of the chimeric males (>30% ES coat color contribution). It may be necessary to produce several litters of pups before the first coat-color germline pup is obtained.

Although chimeras can be either males or females, generally the males are the only ones that will go germline, if they are made with XY ES cells. Since the vast majority of ES cell lines – including ourV6.5 – are XY, it is considered a good sign when a majority of the chimeras are males. Strong contributions by ES cells to the germline can cause a female blastocyst to develop into a phenotypically male chimera. Female mice occasionally carry the targeted mutation in the germline, but only if the ES line has lost the Y chromosome, making it XO.

Blastocyst injections include injection of 45-50 blastocysts. A single clone will be injected in a day. It may be possible to inject more embryos – if available – for an increased fee.






External Academic

Injection of ESCs or iPSCs – Charge per Injection Day

Into Diploid Blastocysts to produce chimeras




Additional Costs




$2 cage/day

Additional Injections After Deliverables Reached




Other Costs (Tail biopsies, Pathogen tests, shipping to external labs, etc)






Figure: Standard production of mutant mice from heterozygous ES cells necessitates the generation of chimeric founder animals by introducing targeted male ES cells into diploid blastocysts. Chimeric founders must then be outcrossed to fix the mutation in the male and female germline. These male and female heterozygous offspring are finally intercrossed to produce homozygous mutant progeny.



Figure taken from: Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation.

Eggan K, Rode A, Jentsch I, Samuel C, Hennek T, Tintrup H, Zevnik B, Erwin J, Loring J, Jackson-Grusby L, Speicher MR, Kuehn R, Jaenisch R. Nat Biotechnol. 2002 May;20(5):455-9. doi: 10.1038/nbt0502-455. PMID: 11981557