Establishment and analyses of mouse models of glycine-encephalopathy by conditional knockout of mouse H protein-gene.

点击翻译按钮获取中文摘要

1.We made the knock-out vector (H protein gene-KO) that carry mouse H protein 5'-genomic DNA, loxP, exon1, loxP, pGK-neo, loxP, mouse H protein 3'-genomic DNA in this sequence.2.We introduced the linearized knock-out vector into mouse ES cells and got 11 cell lines of ES cells that have undergone homologous recombination between the ES genome and knock-out vector.3.Next, we microinjected recombinated ES cells to C57 Black mouse blastocysts and obtained 7 male chimeras, 3 of them have undergone germ-line transmission of the mutated allele.4.We then mated F1 mice with Cre recombinase-expressing transgenic mice. In the next generation, excision of exon1 DNA sequence was confirmed by genome PCR and Southern blot analyses.5.To determine whether the H protein expression is downregulated in the tissues of H protein +/-mice, we performed Western blot analysis using anti-H protein rabbit polyclonal antibody. Western blot analysis indicated that the H protein is decreased to 50% in the brains of +/-mice as compared to +/+ mice.6.We mated males and females of H protein +/-mice and analyzed the genotypes of newborn pups and embryos. PCR analyses have clearly shown that homozygous H protein knockout mice can not be born alive or even develop to E14.7.In the present study, we obtained the H protein +/-mice that have reduced (50%) levels of H protein and showed that H protein is essential for normal development and survival of mouse embryos. By using Cre/lox P system, we will be able to know the unknown functions of H protein in vivo.