Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells.
The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3-4 weeks by using Gateway cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.
Nyabi
Omar
O
Department for Molecular Biomedical Research, VIB, Gent, Belgium.
Naessens
Michael
M
Haigh
Katharina
K
Gembarska
Agnieszka
A
Goossens
Steven
S
Maetens
Marion
M
De Clercq
Sarah
S
Drogat
Benjamin
B
Haenebalcke
Lieven
L
Bartunkova
Sonia
S
De Vos
Ilse
I
De Craene
Bram
B
Karimi
Mansour
M
Berx
Geert
G
Nagy
Andras
A
Hilson
Pierre
P
Marine
Jean-Christophe
JC
Haigh
Jody J
JJ
D000483:Alleles; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004171:Diploidy; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D006822:Hybrid Cells; D051379:Mice; D008822:Mice, Transgenic; D011506:Proteins / Q000235:genetics*; D022661:RNA, Untranslated; D011995:Recombination, Genetic; D019076:Transgenes
C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D011506:Proteins; D022661:RNA, Untranslated