Host-cell reactivation of reporter genes introduced into cells by adenovirus as a convenient way to measure cellular DNA repair.
In order to conveniently measure cellular DNA repair in immortalized and primary human cells we have combined the features of high cellular infectivity of adenovirus (Ad) with that of host-cell reactivation (HCR) of ultraviolet light (UV)-damaged reporter genes. We show that Ads having either the cat (chloramphenicol acetyltransferase) or seap (secreted alkaline phosphatase) reporter gene under control of a strong constitutive promoter can be used to measure relative levels of DNA repair by HCR. Most importantly, the SEAP assay allows for a convenient, inexpensive, and sensitive colorimetric microtiter assay. Only a few steps are involved and it is possible to process many samples simultaneously in a relatively short time, which is not as easily done with other reporter gene assays. Furthermore, we show that co-infection of UV-damaged SEAP Ad with an Ad carrying a prokaryotic repair gene significantly increased the HCR levels in xeroderma pigmentosum cells. The Ad gene delivery system, and the SEAP assay in particular, should simplify existing HCR assays considerably. By using non-lytic Ad as a vehicle it should be possible to quantitatively introduce normal or dominant negative mutant DNA repair genes into bulk cell populations for DNA repair studies.
Valerie
K
K
Department of Radiation Oncology, Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0058.
Singhal
A
A
D000256:Adenoviridae / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004260:DNA Repair; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics* / Q000528:radiation effects; D006801:Humans; D008722:Methods; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum
D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase