Kato M, Yamanouchi K, Ikawa M, Okabe M, Naito K, Tojo H
Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan.
Mol Reprod Dev. 1999 Sep;54(1):43-8. doi: 10.1002/(SICI)1098-2795(199909)54:1<43::AID-MRD6>3.0.CO;2-N.
We have established a reliable method that uses the EGFP (Enhanced Green Fluorescent Protein) gene as a marker for selecting transgenic embryos from preimplantation embryos. Embryos that were subjected to the pronuclear microinjection of the CMV/beta-actin/EGFP fusion gene were cultured in vitro until they developed into the morulae- or blastocyst-stage. The expression of EGFP was easily observed by a fluorescent microscopy. There appeared to be no damage to the in vivo developmental ability of the embryos in response to the EGFP excitation light, which utilized an IB filter for a period of 30 min. Modified PCR analysis using Dpn I and Bal 31 digestion of the embryonic DNA showed that all of the embryos expressing EGFP in all their cells were transgenic, while more than half with mosaic expression of EGFP were not transgenic. Approximately 77% of pups born from the embryos that uniformly expressed the EGFP gene were transgenic, while 21.4% of pups from the embryos with mosaic expression were transgenics. The results showed that the use of EGFP as a marker is very useful and reliable for selecting transgenic embryos, and that it is important to transfer the embryos expressing EGFP in all their cells to obtain truly transgenic animals.
我们建立了一种可靠的方法，该方法使用增强型绿色荧光蛋白（EGFP）基因作为标记，从植入前胚胎中筛选转基因胚胎。对原核显微注射CMV/β-肌动蛋白/EGFP融合基因的胚胎进行体外培养，直至其发育到桑椹胚或囊胚阶段。通过荧光显微镜很容易观察到EGFP的表达。在使用IB滤光片30分钟的情况下，EGFP激发光似乎对胚胎的体内发育能力没有损害。使用Dpn I和Bal 31对胚胎DNA进行消化的改良PCR分析表明，所有细胞都表达EGFP的胚胎都是转基因胚胎，而超过一半EGFP呈嵌合表达的胚胎不是转基因胚胎。从均匀表达EGFP基因的胚胎出生的幼崽中约77%是转基因的，而从EGFP呈嵌合表达的胚胎出生的幼崽中21.4%是转基因的。结果表明，使用EGFP作为标记物对筛选转基因胚胎非常有用且可靠，并且将所有细胞都表达EGFP的胚胎进行移植以获得真正的转基因动物很重要。
