Controlled transgene dosage and PAC-mediated transgenesis in mice using a chromosomal vector.
Previously, we designed a chromosomal vector (CV) and reported germline transmission of the vector by mice and regulated expression of the human tissue factor (F3) gene present on the CV. Further characterization and development of the CV are presented here. Mice could be bred with one to four copies of the CV per cell, and it is shown that F3 expression is proportional to the CV copy number. The insertion of large sequences into the CV was investigated by the insertion of a PAC, carrying 62.5 kb of human genomic DNA containing the CSN2 and STATH genes, into the CV by means of Cre/loxP recombination (CV(PAC)). Retrofitting the PAC with a cytomegalovirus (CMV)-5'HPRT/loxP cassette in Escherichia coli allowed efficient selection of CVs with PAC insert. Mitotic loss rates of the CV(PAC) were similar to the original CV. Furthermore, germline transmission efficiency and mitotic stability of the CV(PAC) in mice were not compromised. The human CSN2 and STATH genes were not expressed in the transchromosomal mice. In contrast, F3, already present on the CV, was expressed in CV(PAC)(+) F(1) mice similar to in CV(+) mice, suggesting that the insertion of large sequences does not interfere with transcription of genes present on the CV.
Voet
Thierry
T
Department of Human Genetics, Flanders Interuniversity Institute for Biotechnology, Herestraat 49, University of Leuven, Louvain B-3000, Belgium.
Schoenmakers
Erik
E
Carpentier
Sebastien
S
Labaere
Charlotte
C
Marynen
Peter
P
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D027042:Chromosomes, Artificial, P1 Bacteriophage / Q000235:genetics*; D003587:Cytomegalovirus; D017931:DNA Primers; D016521:Electrophoresis, Gel, Pulsed-Field; D004926:Escherichia coli; D018628:Gene Dosage; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D008540:Meiosis / Q000235:genetics; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*
D017931:DNA Primers; D013925:Thromboplastin