TAT-phiC31 integrase mediates DNA recombination in mammalian cells.
Streptomyces phage integrase phiC31 is capable of mediating site-specific insertions in mammalian genomes. To avoid potential toxicity of long-term expression of phiC31 in host cells, we developed a method employing a cell-permeable TAT-phiC31 integrase. His6-tagged phiC31 proteins with or without an HIV TAT intercellular transducing peptide were generated and purified. Both of them retained integrase activity in vitro. However, TAT-phiC31 but not phiC31 was able to mediate a specific integration between two att sites in the genome of 293-PB [EGFP] report cell line. Transduced TAT-phiC31 was mainly localized in the cytoplasm that is similar to the localization of phiC31 when expressed through cDNA transfection. Adding a nuclear localization signal (NLS) peptide to the C-terminus of TAT-phiC31 facilitated nuclear localization of the integrase with an increased efficiency of recombination in the reporter cell line. These results demonstrated that TAT can mediate a cell membrane entry of phiC31 protein to perform a site-specific integration in mammalian cells. This is a simple and possibly safer method of site-specific recombination for gene delivery.
Zhang
Mao-xiang
MX
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, China.
Li
Zhi-hui
ZH
Fang
Yu-xiang
YX
Zhu
Huan-zhang
HZ
Xue
Jing-lun
JL
Chen
Jin-zhong
JZ
Jia
William
W
D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003627:Data Interpretation, Statistical; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014162:Transfection / Q000379:methods*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D019426:Integrases