SV 40-transformed normal and DNA-repair-deficient human fibroblasts can be transfected with high frequency but retain only limited amounts of integrated DNA.
The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0-40 kb in length. The transfection frequency for the selected (G418R) marker was between 2 x 10(-4) and 2 x 10(-3) for all cell lines, comparable to many other mammalian systems. About 50% of the G418R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.
Mayne
L V
LV
Sussex Centre for Medical Research, Sussex University, Falmer, Brighton, U.K.
Jones
T
T
Dean
S W
SW
Harcourt
S A
SA
Lowe
J E
JE
Priestley
A
A
Steingrimsdottir
H
H
Sykes
H
H
Green
M H
MH
Lehmann
A R
AR
D002472:Cell Transformation, Viral; D002874:Chromosome Mapping; D003360:Cosmids; D004260:DNA Repair; D004274:DNA, Recombinant / Q000032:analysis / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D005819:Genetic Markers; D006801:Humans; D013539:Simian virus 40; D014162:Transfection
D004274:DNA, Recombinant; D005819:Genetic Markers