Development of an in vitro cell culture assay system for measuring the activation of a neighbouring gene by the retroviral vector.
BACKGROUND
The use of retroviral vectors has shown an actual clinical benefit in a few inherited diseases. However, the occurrence of cases of leukemia after the X-SCID gene therapy trial raised concerns about the safety of insertional mutagenesis inherent to the biology of the retrovirus. Although the retrovirus has long been known to integrate into the host chromosome, and thus have the potential to activate the nearby gene, there has been no convenient method of studying or assaying such a cis-activation phenomenon.


METHODS
In the present study, we report an in vitro assay system in which the effect of retroviral integration on the expression of the neighbouring gene can be studied. In this system, a retroviral vector and the neighbouring reporter gene were constructed in a single plasmid as if it had integrated into the chromosome.


RESULTS
Using this assay, we found that the full-length long terminal repeat (LTR) could indeed activate the neighbouring gene expression from a distance and the magnitude of its activation was highly increased when this LTR was placed in the vicinity of the transcription start site of the gene, whereas the truncated LTR exerted little influence.


CONCLUSIONS
This assay system might provide a useful tool for selecting the appropriate vector structure, as well as for studying the molecular mechanism underlying the cis-activation by the viral LTR.
ViroMed Co., Ltd, Gwanak-gu, Seoul, Korea.
Youngtae
Y
Hong
Seung Shin
SS
Yu
Nam-Kyung
NK
Yoon
Sung June
SJ
Kang
Jun-Tae
JT
Lee
Sujeong
S
Kim
Jong-Mook
JM
Kim
Karim
K
Lee
Ji-Won
JW
Jang
Sunyoung
S
Kim
D001483:Base Sequence; D001681:Biological Assay; D018929:Cell Culture Techniques / Q000379:methods*; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry / Q000378:metabolism; D002999:Clone Cells; D004247:DNA / Q000235:genetics; D018274:Electroporation; D015870:Gene Expression; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D012190:Retroviridae / Q000235:genetics*; D020079:Terminal Repeat Sequences / Q000235:genetics; D015533:Transcriptional Activation
D012333:RNA, Messenger; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase