Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells

Abstract

The potential pluripotency of rabbit fetal germ cells has been investigated by using them to make chimeric embryos. Gonial cells, isolated from enzyme‐dispersed male and female transgenic fetal rabbit gonads of 18–22 days gestation, were microinjected in groups of about 10 into 640 nontransgenic rabbit embryos between the two‐cell and expanded blastocyst stages. Injections were made with primary isolations of gonial cells, within 48 hr of their collection. The injected embryos were transferred, with or without non‐injected control embryos, into 49 recipient rabbits. Tissues from 159 resulting fetuses, implantation sites, and a few liveborn young were examined by PCR analysis for the two transgenes used (α‐antitrypsin or luciferase). The overall pregnancy rate (about 80%) was not affected by the stage of development of the embryo injected, nor by co‐transfer of control embryos. The survival rate of injected embryos (18% overall, 23.6% in pregnant recipients) was almost identical to that of 243 control embryos. Chimerism was detectable in tissues produced from 4 of 159 (2.5%) of the injected embryos, all four of which had been injected at the 8 to 16‐cell stage. This low rate of success indicates that, although passage of rabbit gonial cells is not an absolute requirement for pluripotency, further investigation should pay particular attention to improving culture conditions with a view to deriving EG cell lines. © 1996 Wiley‐Liss, Inc.

Effects of Protease Inhibitors and Antioxidants on In Vitro Survival of Porcine Primordial Germ Cells1

“…These observations are in agreement with reports by others [52,53]. Similar characteristics were also detected for PGCs of cattle [18], rabbits [19], and rats [20], but there are species-specific characteristics of PGCs, such as the cytoplasmic vesicles of cattle [18], the dense vesicles of mice and pigs [52,54], and the lipid droplets in rabbits [19]. Interestingly, porcine PGCs from Day 25-30 still had pseudopodia, suggesting that they were still migrating or had just colonized the urogenital ridge.…”

“…Pig [17], cattle [7,18], rabbit [7,19], and rat [7,20] PGCs have been collected and characterized by their morphology and immunohistochemistry. We and others have previously shown that porcine EG cell lines can be isolated [21][22][23], genetically transformed [21], and contribute to chimera development in the pig [21][22][23].…”

Pluripotent Stem Cells for Transgenesis in the Rabbit: A Utopia?

“…Another study found that the aggregation of two embryos within the same E3-stage morula had given rise to about 5% chimeric fetuses [88,89]. Microinjection of 1 to 3 blastomeres into an embryo at the 8/16-cell stage had yielded upwards of 2% of germline chimeras [90], and microinjection of dissociated gonadal cells from an E20-stage male fetus into an 8/16-cell embryo produced 2.5% chimeric fetuses [91]. In contrast, the longer-cultured rbPSCs produced at best only 10% of blastocysts with colonized ICM and 3% of E10-stage fetuses that were very weakly chimeric [65].…”

Generation of rabbit pluripotent stem cell lines

“…Yang and Foote used cells recovered at stages of development roughly equivalent to the recipient embryo by combining two morulae [80]. In 1980, ICM cells injected into Day-4 blastocysts [81] or fetal gonadal cells [82], or blastomeres from 8-to 16-cell stage embryos [83] injected into other 8-to 16-cell stage embryos resulted in chimeras. The generation of chimeras through blastocyst injection in rodents has been used to generate knockout animals, where gene targeted ESCs are used to transmit a ma- nipulated genome through the germ line of the chimeric animal.…”