Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: Comparison of culture media
| Autor: | Vikram Devgan, Polani B. Seshagiri |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Microinjections
Cell Survival Green Fluorescent Proteins Mice Inbred Strains Mice Transgenic Biology Transfection Morula Green fluorescent protein Embryonic and Fetal Development Mice Genetics medicine Animals Inner cell mass Blastocyst reproductive and urinary physiology Gene Expression Regulation Developmental Trophoblast Embryo Embryo culture Cell Biology Embryo Transfer Molecular biology Embryo transfer Culture Media Mice Inbred C57BL Transgenesis Luminescent Proteins medicine.anatomical_structure Animals Newborn embryonic structures Developmental Biology |
| Zdroj: | Molecular Reproduction and Development. 65:269-277 |
| ISSN: | 1098-2795 1040-452X |
| Popis: | To improve efficiency of trans- genesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/ N mouse pronuclear-eggs, microinjected with enhanc- ed 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 � 2.4%) than that in CZB (13.2 � 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 � 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 � 2.6%) and CZBG (83.9 � 3.9%) media than in CZB (31.9 � 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, blas- tocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blasto- cysts 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 devel- opment of biologically viable blastocysts from EGFP- transgene-injected mouse embryos. Mol. Reprod. Dev. 65: 269-277, 2003. 2003 Wiley-Liss, Inc. |
| Databáze: | OpenAIRE |
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