Inhibitory and enhancing effects of insertion of central polypurine tract sequence on gene expression with vectors derived from human immunodeficiency virus type 1.
Reportedly, in human immunodeficiency virus type 1 (HIV) vectors, insertion of central polypurine tract (cPPT) increased expression of transgenes for a short period. To test this for a stable condition, we constructed a series of vectors carrying a Neo(r) gene as a stable marker driven by a synthetic thymidine kinase (hTK) promoter. Transduction efficiency was increased in about 2-fold and decreased in about 8-fold by insertion of the reported 178bp and our 282bp cPPTs, respectively. PCR analyses revealed that insertion of 282bp cPPT, but not 178bp cPPT, impaired integration, although it did not deteriorate nuclear transport much. Furthermore, we found that insertion of 282bp cPPT between hTK promoter and an upstream LTR sequence reduced reporter gene activity in about 5-fold. This inhibitory effect of 282bp cPPT may partly account for the observed decrease in transduction efficiency. We suggest that actual effect of cPPT insertion should be examined in each HIV vector.
Sakuma
Ryuta
R
Division of Infectious Diseases, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Kobayashi
Noriko
N
Ae
Keisuke
K
Kitamura
Yoshihiro
Y
D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005821:Genetic Techniques; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics*; D016655:HIV Core Protein p24 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011120:Polyomavirus / Q000235:genetics; D011401:Promoter Regions, Genetic; D011687:Purines / Q000737:chemistry*; D013937:Thymidine Kinase / Q000235:genetics; D013997:Time Factors; D014162:Transfection; D019076:Transgenes
D016655:HIV Core Protein p24; D011687:Purines; D008156:Luciferases; D013937:Thymidine Kinase