通过整合酶介导的染色体靶向进行人胚胎干细胞工程改造。

hESC engineering by integrase-mediated chromosomal targeting.

作者信息

Liu Ying, Lakshmipathy Uma, Ozgenc Ali, Thyagarajan Bhaskar, Lieu Pauline, Fontes Andrew, Xue Haipeng, Scheyhing Kelly, MacArthur Chad, Chesnut Jonathan D

机构信息

Invitrogen Corporation, Carlsbad, CA, USA.

出版信息

Methods Mol Biol. 2010;584:229-68. doi: 10.1007/978-1-60761-369-5_13.

DOI:10.1007/978-1-60761-369-5_13

PMID:19907981

Abstract

Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency allowing for long-term expression of transgenes. In this chapter, we describe a retargeting system where phiC31 integrase is used to deliver a chromosomal target for a second integrase, R4. The engineered hESC line can be adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture and upon differentiation. The system described here represents a method to rapidly assemble complex plasmid-based assay systems, controllably insert them into the hESC genome, and have them actively express in pluripotent as well as in differentiated lineages there from.

摘要

噬菌体重组酶能够高效靶向人类胚胎干细胞（hESCs）中的特定基因座，从而实现转基因的长期表达。在本章中，我们描述了一种重靶向系统，其中使用phiC31整合酶为第二种整合酶R4提供染色体靶点。经过工程改造的hESC系可采用多位点Gateway技术用于复杂元件组装。重靶向克隆在长期培养和分化过程中显示出转基因的持续表达和适当调控。这里描述的系统代表了一种快速组装基于复杂质粒的检测系统、可控地将它们插入hESC基因组并使其在多能谱系以及由此分化而来的谱系中积极表达的方法。