| Description |
pCMT-SAhygpA-NP22 (Accession numbrer: AB609714, 10,439bp) is a retrovirus type-promoter trap vector that selects for expressed genes by hygromycin-resistance. This vector was generated by inserting 2-bp nucleotides upstream of the hyg gene in the pCMT-SAhyg-NP21 in order to detect gene trap with a different reading frame. The neo and pu-delta-tk genes are arranged in tail-to-tail orientation, which confers G418-resistance and puromycin-sensitivity. Recombination between inversely-oriented lox2272 flips the orientation of neo-pu-delta-tk, resulting in loss of G418-resistance and acquisition of puromycin-reistance. This feature allows for selection of homozygous mutant cells by G418/puromycin double resistance because two copies of neo-pu-delta-tk cassettes exist in homozygous cells. The U3 region of the 5’ LTR is replaced by hCMV promoter to increase transcription of the viral genome, and the hCMV promoter region is replaced by the U3 region of the 3’ LTR during reverse-transcription and integration into the genome, generating identical LTRs at each end of the vector DNA. FRT sequences within the LTR regions are used to excise internal mutagenic sequences upon Flp expression, generating revertant cells to verify cause-and-effect relationship between mutation and phenotype.
LTR, Moloney Murine Leukemia Virus-long terminal repeat; SA, adenovirus splice acceptor; hyg, hygromcin phosphotransferase; pA1, mouse phosphoglycerate kinase 1 (Pgk1) polyA signal; P, mouse Pgk1 promoter; neo, neomycin phosphotransferase; pu-delta-tk, fusion gene between puromycin N-acetyltransferase and a truncated version of herpes simplex virus type 1 thymidine kinase; pA2, bovine growth hormone polyA signal; hCMV, human cytomegalovirus promoter. |
