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. genesis 47:545–558, 2009. © 2009 Wiley‐Liss, Inc.

[1]  M. Streiff,et al.  The Chemokine Receptor Cxcr3 Is Not Essential for Acute Cardiac Allograft Rejection in Mice and Rats , 2008, American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.

[2]  V. Papaioannou,et al.  Cre activity causes widespread apoptosis and lethal anemia during embryonic development , 2007, Genesis.

[3]  Dagmar Wirth,et al.  Road to precision: recombinase-based targeting technologies for genome engineering. , 2007, Current opinion in biotechnology.

[4]  S. Semprini,et al.  Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques , 2007, Nucleic acids research.

[5]  A. Zlotnik,et al.  The chemokine and chemokine receptor superfamilies and their molecular evolution , 2006, Genome Biology.

[6]  R. Ransohoff,et al.  The many roles of chemokines and chemokine receptors in inflammation. , 2006, The New England journal of medicine.

[7]  D. Busch,et al.  Chemokine receptor Ccr2 deficiency reduces renal disease and prolongs survival in MRL/lpr lupus-prone mice. , 2005, Journal of the American Society of Nephrology : JASN.

[8]  Marc Parmentier,et al.  Dimerization of chemokine receptors and its functional consequences. , 2005, Cytokine & growth factor reviews.

[9]  B. Ledermann,et al.  Strong and ubiquitous expression of transgenes targeted into the β‐actin locus by Cre/lox cassette replacement , 2005, Genesis.

[10]  H. Ruley,et al.  Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells , 2004, BMC biotechnology.

[11]  R. Balling,et al.  Reduced intragraft mRNA expression of matrix metalloproteinases Mmp3, Mmp12, Mmp13 and Adam8, and diminished transplant arteriosclerosis in Ccr5‐deficient mice , 2004, European journal of immunology.

[12]  U. V. von Andrian,et al.  Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells. , 2004, Annual review of immunology.

[13]  D. Nebert,et al.  Balancer-Cre transgenic mouse germ cells direct the incomplete resolution of a tri-loxP-targeted Cyp1a1 allele, producing a conditional knockout allele. , 2003, Biochemical and biophysical research communications.

[14]  I. Buschmann,et al.  Divergent effects of GM‐CSF and TGFβ1 on bone marrow‐ derived macrophage arginase‐1 activity, MCP‐1 expression, and matrix‐metalloproteinase‐12: a potential role during arteriogenesis , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[15]  N. Maeda,et al.  CCR5 deficiency is not protective in the early stages of atherogenesis in apoE knockout mice. , 2003, Atherosclerosis.

[16]  M. Suematsu,et al.  Peyer's patch is the essential site in initiating murine acute and lethal graft-versus-host reaction , 2003, Nature Immunology.

[17]  J. Nap,et al.  The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA. , 2002, Gene.

[18]  A. Berns,et al.  Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[19]  A. Stewart,et al.  Techniques: Recombinogenic engineering--new options for cloning and manipulating DNA. , 2001, Trends in biochemical sciences.

[20]  C. Deng,et al.  Direct removal in the mouse of a floxed neo gene from a three‐loxp conditional knockout allele by two novel approaches , 2001, Genesis.

[21]  S. Vaulont,et al.  Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene. , 2000, Nucleic acids research.

[22]  T. Graf,et al.  Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages , 2000 .

[23]  Andras Nagy,et al.  Cre recombinase: The universal reagent for genome tailoring , 2000, Genesis.

[24]  R. Doms,et al.  CCR5 binds multiple CC-chemokines: MCP-3 acts as a natural antagonist. , 1999, Blood.

[25]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[26]  I. Saito,et al.  Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. , 1998, Gene.

[27]  R. Bravo,et al.  Impaired macrophage function and enhanced T cell-dependent immune response in mice lacking CCR5, the mouse homologue of the major HIV-1 coreceptor. , 1998, Journal of immunology.

[28]  R. Bravo,et al.  Defects in Macrophage Recruitment and Host Defense in Mice Lacking the CCR2 Chemokine Receptor , 1997, The Journal of experimental medicine.

[29]  Robert V Farese,et al.  Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice. , 1997, The Journal of clinical investigation.

[30]  K. Yamamura,et al.  Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters. , 1997, Journal of biochemistry.

[31]  K. Ley,et al.  Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[32]  B. Sauer,et al.  Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome. , 1996, Nucleic acids research.

[33]  Werner Müller,et al.  Bypass of lethality with mosaic mice generated by Cre–loxP-mediated recombination , 1996, Current Biology.

[34]  S. Sugano,et al.  Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. , 1995, Nucleic acids research.

[35]  J. West.  Manipulating the Mouse Embryo . A Laboratory Manual, 2nd Edition. By Brigid Hogan, Rosa Beddington, Frank Costantini and Elizabeth Lacy. Cold Spring Harbor Laboratory Press 1994, 497 pages. Price $95. ISBN 0-87969-384-3.--- Either ISSN or Journal title must be supplied. , 1995 .

[36]  J. Miyazaki,et al.  Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[37]  K. Rajewsky,et al.  Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting , 1993, Cell.

[38]  B. Sauer,et al.  Genomic targeting with purified Cre recombinase. , 1993, Nucleic acids research.

[39]  A. Berns,et al.  Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[40]  A. Bradley,et al.  Genomic DNA microextraction: a method to screen numerous samples. , 1992, Analytical biochemistry.

[41]  A. C. Chang,et al.  Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid , 1978, Journal of bacteriology.

[42]  A. F. Stewart,et al.  Engineering embryonic stem cells with recombinase systems. , 2006, Methods in enzymology.

[43]  J. Shaw,et al.  Chemokine blockers--therapeutics in the making? , 2006, Trends in pharmacological sciences.

[44]  J. Rossant,et al.  Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors. , 2006, Blood.

[45]  C. Branda,et al.  Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. , 2004, Developmental cell.

[46]  N. Carter,et al.  Karyotyping mouse chromosomes by multiplex-FISH (M-FISH) , 2004, Chromosome Research.

[47]  I. Jentsch,et al.  Animal Cytogenetics and Comparative Mapping , 2004 .

[48]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.

[49]  T. Graf,et al.  Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. , 2000, Blood.

[50]  F. Grosveld,et al.  Microinjection of cre recombinase RNA induces site-specific recombination of a transgene in mouse oocytes. , 1998, Nucleic acids research.

[51]  K. Rajewsky,et al.  A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. , 1995, Nucleic acids research.

[52]  G. Church,et al.  Genomic sequencing. , 1993, Methods in molecular biology.