Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.
We have examined the ability of single-stranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them. We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mp11. The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo. The resulting single-stranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. We were able to obtain recombinant molecules containing wild-type neo genes in all three systems. Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate. We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction. We have also tested the ability of single-stranded DNA to yield transfectants. When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TK+ colonies. From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.
Rauth
S
S
Song
K Y
KY
Ayares
D
D
Wallace
L
L
Moore
P D
PD
Kucherlapati
R
R
D000818:Animals; D002467:Cell Nucleus / Q000502:physiology; D002522:Chlorocebus aethiops; D004277:DNA, Single-Stranded / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004351:Drug Resistance; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009355:Neomycin / Q000494:pharmacology; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D013937:Thymidine Kinase; D009355:Neomycin