[Construction of inducible lentiviral vector containing human Notch1 and EGFP gene and its expression in PC12 cells].
OBJECTIVE
To construct inducible lentiviral vector containing human Notch1 intracellular domain (NICD) gene and enhanced green fluorescent protein (EGFP), and to study its expression in PC12 cells.


METHODS
NICD cDNA was amplified by RT-PCR from human placenta tissue. EGFP gene was amplified by PCR from pEGFP-C1. Both NICD and EGFP were cloned into pcDNA 3.1 (+) plasmid to form pcDNA3.1-Notch1-EGFP. Then the Notch1-EGFP fragment was separated and cloned into pLVX-Tight-puro to form pLVX-Notch1-EGFP. The lentivirus were packaged and harvested, which were used to infect PC12 cells. After antibody selection for 2 weeks, the PC12 cells were induced by doxycycline (Dox). The expression of Notch1-EGFP was detected by fluorescence microscope and flow cytometry.


RESULTS
The recombinant inducible lentiviral vectors (pLVX-Notch1-EGFP) were success fully constructed. The EGFP positive cell percentage was over 90% in transfected PC12 cells after 500 ng/ml Dox induction for 36 h. The expression of Notch1 was posited correlated to the Dox concentration. The expression of Notch1 increased with the duration of Dox induction, which got the peak at 36 h after Dox induction.


CONCLUSIONS
The recombinant inducible lentiviral vectors containing Notch1 and EGFP gene are successfully constructed, which provides an effective and simple method to regulate the expression of Notch1 in PC12 cells.
Stem Cell Research Center, Zhengzhou University, Zhengzhou 450001, USA.
Yong-Min
YM
Liu
Ping
P
Duan
Chun-Tian
CT
Huang
Bo
B
Li
Xue-Fei
XF
Han
Yan
Y
Xu
Wen-Hai
WH
Yan
Ying
Y
Xing
D000818:Animals; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016716:PC12 Cells; D010957:Plasmids; D051381:Rats; D051881:Receptor, Notch1 / Q000235:genetics*; D014162:Transfection
D051881:Receptor, Notch1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins