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- Title
1053. Functional Restoration of a Genetic Defect in Multipotent Germline Stem Cells by Human Artificial Chromosome Containing a Genomic Insert.
- Authors
Kazuki, Yasuhiro; Hoshiya, Hidetoshi; Kai, Yoshiteru; Abe, Satoshi; Takiguchi, Masato; Osaki, Mitsuhiko; Kanatsu- Shinohara, Mito; Kajitani, Naoyo; Shirayoshi, Yasuaki; Hiratuka, Masaharu; Ogura, Astuo; Shinohara, Takashi; Oshimura, Mitsuo
- Abstract
Although homologous recombination has been used for gene correction in ES cells, genetic defects with an unknown mutation site or a genomic large deletion could not be restored by homologous recombination. Human artificial chromosomes (HACs) may solve these problems. HACs have several advantages that require for gene therapy vectors, including an efficient transfer to stem cells, a stable episomal maintenance and the capacity for large gene inserts. Although own embryonic stem (nt ES) cells can be made by the nuclear transfer technique, there are many ethical problems. On the other hand, mGS (multipotent germline stem) cells have ES cell-like multipotency and could be generated from neonatal mouse testis. Thus, mGS cells have greater advantages than ntES cells, because individual mGS cells can be obtained without ethical problem. Here we establish a paradigm for treatment of a genetic disorder by combining mGS cells with HAC vector containing a defected gene.As a model, we used the p53 deficient mGS cells. Human p53 genome was cloned into the HAC vector using a HAC-mediated genomic insert knock in (HMGK) system. Briefly, using p53 cDNA probe, we screened RPCI-6 PAC library that had a loxP-site for cloning into the HAC, and the PAC containing p53 genome were inserted into the HAC vector in CHO cells by the Cre-loxP system. The p53-HAC was then introduced into the mouse p53-/- mGS cells using microcell-mediated chromosome transfer (MMCT). Fluorescence in situ hybridization (FISH) analyses showed that the p53-HAC was present independently in the p53-/-mGS cells. Western blot analyses in the p53-/-mGS(p53HAC) after X-ray irradiation showed that the transferred p53 trans-activated the p53-related gene, endogenous mouse p21. Further, radiosensitivity in the p53-/-mGS(p53HAC) had a significantly higher after irradiation as compared to parental p53-/-mGS cells. These data suggest that the HAC vector containing a p53 genomic insert mediates the production of functional human p53 protein and the HAC was able to rescue the p53-/-phenotypes in the p53-/-mGS cells. Next, the p53-/-mGS(p53HAC) cells were injected into nude mice to assess the capacity of differentiation. The transplanted p53-/- mGS(p53HAC) cells gave rise to typical teratomas and some differentiated cell types were detected.Thus, we demonstrated that HAC vector could be transferable to mGS cells and HAC vector containing desired genome corrected a genetic defect in mGS cells by MMCT approach. In future, a combination of mGS cells and HAC-mediated gene delivery may provide a useful treatment for genetic defects, when efficient methods for differentiation in stem cells including mGS cells are developed.Molecular Therapy (2006) 13, S403–S404; doi: 10.1016/j.ymthe.2006.08.1149
- Subjects
GENETIC disorders; GERM cells; STEM cells; ARTIFICIAL chromosomes; GENETIC recombination; GENETIC vectors
- Publication
Molecular Therapy, 2006, Vol 13, pS403
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.1149