Development of high-titer retroviral producer cell lines by using Cre-mediated recombination.
Retroviral gene transfer is widely used in experimental and human gene therapy applications. We have devised a novel method of generating high-titer retroviral producer cell lines based on the P1 bacteriophage recombinase system Cre-loxP. Incorporation of loxP sites flanking a Neo(r)-SVTK cassette in the proviral DNA allows excision of these selectable markers through expression of Cre recombinase after production of a high-titer producer cell line. The resultant producer line contains a single loxP site flanked by the viral long terminal repeats. Retransfection of this line with the Cre expression vector and a plasmid containing a gene of interest flanked by loxP sites allows insertional recombination of the gene into the favorable preexisting site in the genome and the generation of a new line with a titer equivalent to that of the parental producer cell line. The efficiency of the process is sufficient to allow the generation of multiple new producer lines without the addition of antibiotic resistance genes. We have successfully generated retroviral vectors carrying different genes by using this approach and discuss the potential applications of this method in gene therapy.
Vanin
E F
EF
Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital Research Foundation, Parkville, Victoria, Australia.
Cerruti
L
L
Tran
N
N
Grosveld
G
G
Cunningham
J M
JM
Jane
S M
SM
D000818:Animals; D017101:Bacteriophage P1 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004251:DNA Transposable Elements; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000096:biosynthesis / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics / Q000502:physiology*; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins; D014779:Virus Replication
D004251:DNA Transposable Elements; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases