Refined human artificial chromosome vectors for gene therapy and animal transgenesis.
Refined human artificial chromosome vectors for gene therapy and animal transgenesis.
复制标题
用于基因疗法和动物转基因的精致人工染色体载体。
作者：
人类人工染色体（HAC）作为基因治疗载体具有几个优点，包括稳定的附加型维持和携带大基因插入物的能力。我们以前开发的HAC载体从正常的人类染色体使用染色体工程技术。然而，内源性基因仍然存在于这些HAC中，限制了它们的治疗应用。在这项研究中，我们在同源重组熟练的鸡DT 40细胞中改进了不含人21号染色体内源基因的HAC载体。HAC的物理特征在于使用转化相关重组（TAR）克隆策略，然后对TAR-细菌人工染色体克隆进行测序。HAC中无内源基因残留。我们证明，任何所需的基因可以克隆到HAC使用Cre-loxP系统在中国仓鼠卵巢细胞，或同源重组系统在DT 40细胞。HAC可以通过微细胞介导的染色体转移有效地转移到其他类型的细胞，包括小鼠ES细胞。转基因的HAC在体内外均能稳定表达。此外，更昔洛韦在体外和体内选择性地杀死了含有携带自杀基因的HAC的肿瘤细胞，单纯疱疹病毒胸苷激酶（HSV-TK）。因此，这种新的HAC载体不仅可用于基因和细胞治疗，而且可用于动物转基因。
Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.
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影响因子：
12.4
作者：
Hoshiya, Hidetoshi;Kazuki, Yasuhiro;Oshimura, Mitsuo
通讯作者：
Oshimura, Mitsuo
影响因子：
14.9
作者：
Kouprina, N;Ebersole, T;Larionov, V
通讯作者：
Larionov, V
影响因子：
14.9
作者：
Ebersole, T;Okamoto, Y;Larionov, V
通讯作者：
Larionov, V
DOI：
10.1073/pnas.93.14.7125
发表时间：
1996-07-09
影响因子：
11.1
作者：
Heller, R;Brown, KE;Brown, WRA
通讯作者：
Brown, WRA
影响因子：
14.8
作者：
Kouprina, Natalay;Larionov, Vladimir
通讯作者：
Larionov, Vladimir