[Construction of recombinant vectors expressing antisense RNA to CCR5 and expression in eukaryotic cells].
BACKGROUND
To construct recombinant vector expressing antisense RNA to CCR5 in eukaryotic cells and obtain recombinant pseudovirus, which will be used to block HIV-1 infection.


METHODS
The DNA fragment targeted against the initional part of CCR  5 mRNA translation was amplified by using RT-PCR from peripheral blood mononuclear cells (PBMCs) and cloned into retroviral vector pLXSN, then transfected into packaging cell (PA317) with lipofectAMINE. After 2-3 weeks selecting with G418, the pseudovirion in the survival cell's supernatant was detected with RT-PCR (FQ),then was used to infect NIH/3T3 cell.


RESULTS
The psuedovirion packed from expression vector of sense/antisense RNA to CCR5 had infected NIH/3T3 cell successfully. The vector had incorporated into its genome and transcripted into RNA.


CONCLUSIONS
The gene fragment of antisense RNA to CCR5 could be obtained from PBMCs and transfected into eukaryotic cell with retroviral vector. The results made a great foundation for studying its inhibiting effect on HIV-1 infection.
Xing
Huichun
H
Department of Infectious Disease, Peking University First Hospital, Beijing 100034, China.
Xu
Xiaoyuan
X
Wang
Qinhuan
Q
Yu
Min
M
Gong
Weibo
W
Shao
Yiming
Y
D016475:3T3 Cells; D000818:Animals; D005057:Eukaryotic Cells / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D010957:Plasmids / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics*; D019713:Receptors, CCR5 / Q000235:genetics*; D014162:Transfection
D016372:RNA, Antisense; D019713:Receptors, CCR5