A new system for analyzing LINE retrotransposition in the chicken DT40 cell line widely used for reverse genetics.
Long interspersed elements (LINEs) are autonomous transposable elements that proliferate via retrotransposition, which involves reverse transcription of LINE RNAs. It is anticipated that LINE retrotransposition requires both LINE-encoded proteins and host-encoded proteins. However, identification of the host factors, their roles, and the steps at which they act on retrotransposition are poorly understood because of the lack of an appropriate genetic system to study LINE retrotransposition in a series of mutant hosts. To construct such a genetic system, we applied the retrotransposition-indicative cassette method to DT40 cells, a chicken cell line for which a variety of isogenic mutants have been established by gene targeting. Because DT40 cells are non-adherent, we utilized a selective soft agarose medium to allow the formation of colonies of cells that had undergone LINE retrotransposition. Colony formation was completely dependent on the activities of the LINE-encoded proteins and on the presence of the essential 3' region of the LINE RNA. Moreover, the selected colonies indeed carried retrotransposed LINE copies in their chromosomes, with integration features similar to those of genomic (native) LINE copies. This method thus allows the authentic selection of LINE-retrotransposed cells and the approximate recapitulation of retrotransposition events that occur in nature. Therefore, the DT40 cell system established here provides a powerful tool for the elucidation of LINE retrotransposition pathways, the host factors involved, and their roles.
Honda
Hiroshi
H
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-21 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.
Ichiyanagi
Kenji
K
Suzuki
Jun
J
Ono
Takao
T
Koyama
Hideki
H
Kajikawa
Masaki
M
Okada
Norihiro
N
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D004274:DNA, Recombinant / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D020084:Long Interspersed Nucleotide Elements; D049409:Luciferases, Firefly / Q000235:genetics; D008957:Models, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D013696:Temperature; D014162:Transfection
D004274:DNA, Recombinant; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly