Successful Development of Viable Blastocysts From Enhanced Green Fluorescent Protein Transgene-Microinjected Mouse Embryos:Comparison of Culture Media

Devgan, Vikram and Seshagiri, Polani B (2003) Successful Development of Viable Blastocysts From Enhanced Green Fluorescent Protein Transgene-Microinjected Mouse Embryos:Comparison of Culture Media. In: Molecular Reproduction and Development, 65 (3). pp. 269-277.

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Abstract

To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P<0.03) higher in M16 medium (72.5 \pm 2.4%) than that in CZB (13.2 \pm 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 \pm 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 \pm 2.6%) and CZBG (83.9 \pm 3.9%) media than in CZB (31.9 \pm 2.8%) medium (P<0.0001). However, percentages of morulae at 72 hr were comparable in all treatments. Developed blastocysts were better in M16 than in CZB or CZBG media. Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P<0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos. Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P<0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts. Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blastocysts from M16 medium produced live transgenic green pups (11%) following embryo transfer. Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFPtransgene injected mouse embryos.

Item Type:	Journal Article
Publication:	Molecular Reproduction and Development
Publisher:	Wiley-Liss, Inc.
Additional Information:	The copyright belongs to Wiley-Liss, Inc.
Keywords:	green fluorescent protein;culture media;mouse embryo;blastocyst development;transgenesis
Department/Centre:	Division of Biological Sciences > Molecular Reproduction, Development & Genetics
Date Deposited:	16 Dec 2005
Last Modified:	19 Sep 2010 04:21
URI:	http://eprints.iisc.ac.in/id/eprint/4395

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