Transcriptionally active polymerase chain reaction (TAP): high throughput gene expression using genome sequence data.
An approach is described for making transcriptionally active PCR (TAP) fragments that were used directly in in vitro and in vivo expression experiments. TAP fragments encoding reporter genes were amplified in 1 day using typical PCR methodology and were expressed in cultured cells and in mice at levels comparable with a widely used cytomegalovirus promoter-based plasmid expression vector. Following intramuscular injection, a TAP fragment encoding hepatitis B surface antigen (HBsAg) induced anti-HBsAg antibody titers comparable with those induced by supercoiled plasmid encoding the same antigen. Epitope-tagged TAP fragments were generated and transfected into cells for rapid, high throughput immunocytochemical analysis of the tagged gene products. TAP fragments were also transferred directly into expression vectors by in vivo homologous recombination without conventional cloning, affording a high throughput cloning approach that does not require restriction enzyme digestion, ligations, or thymidine adenine complementation cloning. The methodology has been adapted to a robotic work station enabling the high throughput generation of transcriptionally active genes at the rate of more than 400 different genes per day. This technology offers a practical approach to directly utilize genome sequence data to generate functional proteomes.
Liang
Xiaowu
X
Gene Therapy Systems, Inc., San Diego, California 92121, USA.
Teng
Andy
A
Braun
Dawn M
DM
Felgner
Jiin
J
Wang
Yan
Y
Baker
Scott I
SI
Chen
Shizong
S
Zelphati
Olivier
O
Felgner
Philip L
PL
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D015500:Chloramphenicol O-Acetyltransferase; D002522:Chlorocebus aethiops; D006224:Cricetinae; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016678:Genome; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic; D014162:Transfection
D006514:Hepatitis B Surface Antigens; D015500:Chloramphenicol O-Acetyltransferase