Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double-deficient mice.
The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double-deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double-deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose-binding protein and Cre (MBP-Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks.
Luckow
Bruno
B
Klinikum der Universität München, Campus Innenstadt, Medizinische Poliklinik, Arbeitsgruppe Klinische Biochemie, D-80336 Munich, Germany. bruno.luckow@med.uni-muenchen.de
Hänggli
Amy
A
Maier
Holger
H
Chilla
Silvia
S
Loewe
Robert P
RP
Dehmel
Stefan
S
Schlöndorff
Detlef
D
Loetscher
Pius
P
Zerwes
Hans-Günter
HG
Müller
Matthias
M
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D005822:Genetic Vectors; D005854:Germ Cells; D017404:In Situ Hybridization, Fluorescence; D019426:Integrases / Q000008:administration & dosage* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008845:Microinjections; D054390:Receptors, CCR2 / Q000235:genetics*; D019713:Receptors, CCR5 / Q000235:genetics*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015053:Zygote
C515335:Ccr2 protein, mouse; D017931:DNA Primers; D054390:Receptors, CCR2; D019713:Receptors, CCR5; C045073:Cre recombinase; D019426:Integrases