Stable gene expression from a mammalian artificial chromosome
Stable gene expression from a mammalian artificial chromosome
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DOI：
10.1093/embo-reports/kve187
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发表时间：
2001-10-01
期刊：
影响因子：
7.7
通讯作者：
Cooke, HJ
中科院分区：
文献类型：
--
作者：
Grimes, BR;Schindelhauer, D;Cooke, HJ
我们已经研究了基于PAC的载体作为将基因并入哺乳动物人工染色体（MAC）中的途径的潜力。先前我们证明了含有a-卫星DNA的PAC（PAC 7 c5）在人类细胞中产生有丝分裂稳定的MAC。为了确定功能性HPRT基因是否可以在MAC中组装，将PAC 7 c5与含有140 kb人HPRT基因的第二PAC共转染到HPRT缺陷型HT 1080细胞中。分离含有与α-卫星和HPRT探针杂交的MAC的株系。MACs有效分离，与动粒蛋白结合，并在60天后稳定表达HPRT信息。通过在HAT选择上生长证实了亲本HPRT缺陷的互补表型。这些结果表明，可以进一步开发MAC以将一系列基因组拷贝的基因递送到细胞中，并且可以实现稳定的转基因表达。
We have investigated the potential of PAC-based vectors as a route to the incorporation of a gene in a mammalian artificial chromosome (MAC). Previously we demonstrated that a PAC (PAC7c5) containing a-satellite DNA generated mitotically stable MACs in human cells. To determine whether a functional HPRT gene could be assembled in a MAC, PAC7c5 was cotransfected with a second PAC containing a 140 kb human HPRT gene into HPRT-deficient HT1080 cells. Lines were isolated containing a MAC hybridizing with both alpha -satellite and HPRT probes. The MACs segregated efficiently, associated with kinetochore proteins and stably expressed HPRT message after 60 days without selection. Complementation of the parental HPRT deficiency was confirmed phenotypically by growth on HAT selection. These results suggest that MACs could be further developed for delivering a range of genomic copies of genes into cells and that stable transgene expression can be achieved.