Mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny.

Murine embryonic stem cells (mESCs) inoculated at passage P13 with the mycoplasma species M. hominis, M. fermentans and M. orale and cultured over 20 passages showed reduced growth rate and viability (P < 0.0001) compared to control mESCs. Spectral karyotypic analysis of mycoplasma-infected mESCs showed a number of non-clonal chromosomal aberrations which increased with the duration of infection. The differentiation status of the infected mESCs was most affected at passage P13+6 where the infection was strongest and 46.3% of the mESCs expressed both POU5F1 and SSEA-1 markers whereas 84.8% of control mESCs expressed both markers. The percentage of germline chimeras from mycoplasma-infected mESCs was examined after blastocyst injection and embryo transfer to suitable recipients at different passages and, compared to the respective control group, was most affected at passage P13+5 (50% vs. 90%; P < 0.07). Further reductions were obtained at the same passage in the percentage of litters born (50% vs. 100%; P < 0.07) and in the percentage of pups born (22% vs. 45%; P < 0.001). Thirty three chimeras (39.8%) obtained from blastocyst injection with mycoplasma-infected mESCs showed reduced body weight (P < 0.0001), nasal discharge, osteoarthropathia, and cachexia. Flow cytometric analysis of plasma from chimeras produced with mycoplasma-infected mESCs revealed statistically significant differences in the proportions of T-cells and increased levels of IgG1 (P < 0.001), IgG2a (P < 0.05) and IgM (P < 0.05), anti-DNA antibodies (P < 0.05) and rheumatoid factor (P < 0.01). The present data indicate that mycoplasma contamination of mESCs affects various cell parameters, germline transmission, and postnatal development of the resulting chimeras.

Emergency prevention of extinction of a transgenic allele in a less-fertile transgenic mouse line by crossing with an inbred or outbred mouse strain coupled with assisted reproductive technologies.

Certain transgenic mouse lines are difficult to breed or archive and, consequently, their transgenes become lost. We examined a C57BL/6 mouse line (B6-tg), transgenic for green fluorescent protein (GFP) with low fertility, and its crosses with the more prolific inbred C3HeB/FeJ (C3) and outbred Swiss (SW) strains in order to assess the possibility of emergency prevention of extinction of a transgenic allele by using assisted reproductive technologies (ART). Out-crossing was performed by natural mating or in vitro fertilisation (IVF) with heterozygous mice. Most of the crossing combinations resulted in improved archiving and rederivation efficiencies of the transgenic allele. Natural crossing increased both mean litter size by two to three pups and the superovulatory rate from 69% for B6-tg to 70-90% for females from the out-crosses. Each plug-positive B6-tg female yielded an average of 4.6 two-cell embryos, whereas females from the out-crosses produced three- to fivefold that amount. After thawing, 13% of B6-tg embryos and 6-12% of out-crossed embryos developed into transgenic pups after transfer into recipients. After IVF with cryopreserved spermatozoa, cleavage rates were 4% for B6-tg, 22-37% for B6-tg oocytes out-crossed with C3 and SW spermatozoa, 9-49% for gametes from out-crossed mice and 28-44% for back-crosses with B6 oocytes. Transgenic pups were not derived from IVF with B6-tg gametes when either fresh or cryopreserved spermatozoa were used. Rederivation efficiencies were 7% and 4% from out-crosses of B6-tg oocytes with C3 and SW spermatozoa, respectively, 6-22% for gametes from out-crossed mice and 4-10% for the back-crosses. Although out-crossing changes the original genetic background, the strategy of crossing coupled with ART prevents the extinction of an allele of interest, especially where archiving and rederivation of the transgenic line fail.

Transmission of mouse minute virus (MMV) but not mouse hepatitis virus (MHV) following embryo transfer with experimentally exposed in vivo-derived embryos.

The present study investigated the presence and location of fluorescent microspheres having the size of mouse hepatitis virus (MHV) and of mouse minute virus (MMV) in the zona pellucida (ZP) of in vivo-produced murine embryos, the transmission of these viruses by embryos during embryo transfer, and the time of seroconversion of recipients and pups. To this end, fertilized oocytes and morulae were exposed to different concentrations of MMVp for 16 h, while 2-cell embryos and blastocysts were coincubated for 1 h. In addition, morulae were exposed to MHV-A59 for 16 h. One group of embryos was washed, and the remaining embryos remained unwashed before embryo transfer. Serological analyses were performed by means of ELISA to detect antibodies to MHV or MMV in recipients and in progeny on Days 14, 21, 28, 42, and 63 and on Days 42, 63, 84, 112, 133, and 154, respectively, after embryo transfer. Coincubation with a minimum of 10(5)/ml of fluorescent microspheres showed that particles with a diameter of 20 nm but not 100 nm crossed the ZP of murine blastocysts. Washing generally led to a 10-fold to 100-fold reduction of MMVp. Washed MMV-exposed but not MHV-exposed embryos led to the production of antibodies independent of embryonic stage and time of virus exposure. Recipients receiving embryos exposed to a minimum of 10(7) mean tissue culture infective dose (TCID(50))/ml of MHV-A59 and 10(2) TCID(50)/ml of MMVp seroconverted by Day 42 after embryo transfer. The results indicate that MMV but not MHV can be transmitted to recipients even after washing embryos 10 times before embryo transfer.