DEVELOPMENT OF THE EMBRYO
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(141) SUCCESSFUL DEVELOPMENT OF VIABLE BLASTOCYSTS FROM ENHANCED GREEN FLUORESCENT PROTEIN (EGFP) TRANSGENE-MICROINJECTED MOUSE EMBRYOS: COMPARISION OF CULTURE MEDIA.
Seshagiri, Polani1, Devgan, Vikram1, 1 Department of Molecular Reproduction, Bangalore, IN
ABSTRACT- In mammals, transgenic technology is valuable for studying regulation of gene expression. Its limitation, however, is the low efficiency of transgenesis. One of the reasons is a substantial loss of viability of microinjected eggs, 1-8-cell embryos being most vulnerable to mortality. Therefore, it is desirable to culture injected-embryos and select developmentally normal blastocysts for uterine transfer. In this regard, we compared two formulations of mouse embryo culture media viz., M16 & CZB, for supporting development to blastocysts of FVB/N mouse pronuclear eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured for 120 h in M16 (n = 159), CZB (n = 161) and CZB with glucose (CZBG; n = 150) media, 118 (74 ± 2%) developed to blastocysts in M16 medium, while only 23 (14 ± 4%) and 91 (61 ± 4%) developed to blastocysts in CZB and CZBG media, respectively (P <0.0001; n = 10). Under similar conditions, blastocyst development for non-injected embryos was 93 ± 3% (186/201), 32 ± 3% (56/173) and 82 ± 4% (137/168) in M16, CZB and CZBG media, respectively. Blastocyst development of injected- and non-injected-embryos cultured in M16 medium was significantly higher (P <0.03) than those in CZB and CZBG media. Quality of blastocysts (judged by determining mean cell number (MCN) per blastocyst), cultured in M16 medium (80 ± 5), was significantly (P <0.001) higher than those in CZB (34 ± 1) or CZBG media (36 ± 1) for injected embryos; values for non-injected embryos were: 93 ± 2 (M16) 34 ± 1 (CZB) and 49 ± 4 (CZBG). Cell allocation to trophectoderm (TE) and inner cell mass (ICM) i.e., TE:ICM ratio for injected embryos cultured in M16 (3.0 ± 0.4) was significantly lesser (P <0.05) than those in CZB (5.3 ± 0.4) and CZBG (4.7 ± 0.4) media; values for non-injected embryos were: 2.6 ± 0.2 (M16), 3.8 ± 0.3 (CZB) and 5.4 ± 0.3 (CZBG). Moreover, the viability of blastocysts cultured in M16 medium was confirmed by their ability to (i) hatch out of the zona pellucida and attach to substratum, exhibiting trophoblast outgrowth (100%) and (ii) produce live pups (21.4%) following embryo transfer (n = 8). These data indicate that M16 medium is superior to CZB or CZBG media, for supporting development of blastocysts from EGFP transgene-microinjected mouse embryos. (Support: Dept. of Biotechnology, Govt. of India).
KEY WORDS: preimplantation embryo, EGFP-transgene, culture medium, blastocyst