Rapid and efficient cloning of proviral flanking fragments by kanamycin resistance gene complementation.
We have developed a technique for the rapid cloning of unknown flanking regions of transgenic DNA. We complemented a truncated kanamycin resistance gene of a bacterial plasmid with a neomycin resistance gene fragment from a gene transfer vector. Optimized transformation conditions allowed us to directly select for kanamycin-resistant bacteria. We cloned numerous proviral flanking fragments from growth factor-independent cell mutants that were obtained after infection with a replication incompetent retroviral vector and identified integrations into the cyclin D2 and several unknown genomic sequences. We anticipate that our method could be adapted to various vector systems that are used to tag and identify genes and to map genomes.
Fehse
B
B
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, an der Universität Hamburg, Martinistrasse 52, 20251 Hamburg, Germany.
Kühlcke
K
K
Langer
A
A
Ostertag
W
W
Lother
H
H
D003001:Cloning, Molecular / Q000379:methods*; D056742:Cyclin D2; D016213:Cyclins / Q000235:genetics; D005816:Genetic Complementation Test; D006412:Hematopoietic Stem Cells / Q000821:virology; D006801:Humans; D007613:Kanamycin Resistance / Q000235:genetics*; D011533:Proviruses / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*
C080336:CCND2 protein, human; D056742:Cyclin D2; D016213:Cyclins