Rapid construction of phosphatase and tensin homolog-deleted on chromosome ten gene recombinant adenovirus using the AdEasy system  

作　　者：Yongqiong Wei Lixue Chen Zhaofang Zeng Chongbiao Shen 

机构地区：[1]Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing 400016, China [2]Chongqing Key Laboratory of Neurology, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China

出　　处：《Neural Regeneration Research》2010年第15期1166-1170,共5页中国神经再生研究（英文版）

摘　　要：Recent studies have shown that phosphatase and tensin homolog-deleted on chromosome ten （PTEN） gene plays an important role in ischemic brain damage and synaptic plasticity. The AdEasy system, which has been widely used, greatly simplifies preparation of recombinant adenovirus. Therefore, recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene （Ad-PTEN） was constructed using the AdEasy-1 system and was transfected into HEK293 cells for packaging and amplification. Infection efficiency and expression intensity were observed in primary cultured rat hippocampal neurons infected with Ad-PTEN in vitro. Results revealed a cytopathic effect in green fluorescent protein expression, which increased with prolonged time. After three cycles of amplification, the adenovirus titer was increased to an adequate titer for infecting hippocampal neurons. The entire process typically requires 4-5 weeks for completion. Results suggested that recombinant defective adenovirus vector carrying the PTEN gene was successfully and rapidly constructed using the AdEasy system.Recent studies have shown that phosphatase and tensin homolog-deleted on chromosome ten （PTEN） gene plays an important role in ischemic brain damage and synaptic plasticity. The AdEasy system, which has been widely used, greatly simplifies preparation of recombinant adenovirus. Therefore, recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene （Ad-PTEN） was constructed using the AdEasy-1 system and was transfected into HEK293 cells for packaging and amplification. Infection efficiency and expression intensity were observed in primary cultured rat hippocampal neurons infected with Ad-PTEN in vitro. Results revealed a cytopathic effect in green fluorescent protein expression, which increased with prolonged time. After three cycles of amplification, the adenovirus titer was increased to an adequate titer for infecting hippocampal neurons. The entire process typically requires 4-5 weeks for completion. Results suggested that recombinant defective adenovirus vector carrying the PTEN gene was successfully and rapidly constructed using the AdEasy system.

关 键 词：phosphatase and tensin homolog-deleted on chromosome ten recombinant adenovirus AdEasy system vector construction nerve factors neural regeneration 

分 类 号：Q78[生物学—分子生物学] Q782