Devgan Vikram, Seshagiri Polani B
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
Mol Reprod Dev. 2003 Jul;65(3):269-77. doi: 10.1002/mrd.10306.
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 +/- 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, 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 EGFP-transgene-injected mouse embryos.
为提高转基因效率，我们比较了M16和CZB胚胎培养基对注射增强型绿色荧光蛋白（EGFP）转基因的FVB/N小鼠原核受精卵发育至囊胚的支持能力。当培养注射了EGFP的受精卵（120小时）时，M16培养基中的囊胚发育率（72.5±2.4%）显著高于CZB（13.2±4.3%）或CZBG（培养48小时时添加5.6 mM葡萄糖的CZB）（62.1±3.7%）培养基（P<0.03）。未注射胚胎在M16（92.0±2.6%）和CZBG（83.9±3.9%）培养基中的囊胚发育率高于CZB（31.9±2.8%）培养基（P<0.0001）。然而，所有处理组在72小时时桑葚胚的比例相当。在M16培养基中发育的囊胚比在CZB或CZBG培养基中发育得更好。与此一致的是，由注射胚胎发育而来的每个囊胚的平均细胞数，在M16培养基中（79.6）显著高于CZB（31.3）或CZBG培养基（60.7）（P<0.002）；未注射胚胎的情况类似。对于注射了EGFP的囊胚，M16培养基中滋养外胚层（TE）和内细胞团（ICM）的细胞分配比例，即TE:ICM比值（3.0）低于CZB（4.2）和CZBG（4.4）培养基（P<0.05）；未注射囊胚的情况类似。此外，在M16和CZBG培养基中发育的囊胚能够孵化、附着并表现出滋养层生长；其中18%显示出EGFP表达。重要的是，来自M16培养基的囊胚在胚胎移植后产生了存活的转基因“绿色”幼崽（11%）。综上所述，我们的结果表明，培养48小时时添加葡萄糖（CZBG）对于桑葚胚向囊胚的转变是必需的；从培养开始就含有葡萄糖的M16培养基在支持注射了EGFP转基因的小鼠胚胎发育成具有生物学活性的囊胚方面优于CZB或CZBG。
