CRISPR/Cas9-Mediated insertion of loxP sites in the mouse Dock7 gene provides an effective alternative to use of targeted embryonic stem cells

Kathleen A. Bishop, Anne Harrington, Evguenia Kouranova, Edward J. Weinstein, Clifford J. Rosen, Xiaoxia Cui, Lucy Liaw

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

Targeted gene mutation in the mouse is a primary strategy to understand gene function andrelation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resourceof mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novelmouse models have been generated from publically accessible targeted mouse ES cell lines. However,there are limitations, including incorrect targeting or cassette structure, and difficulties with germlinetransmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cellclones, we were successful ~50% of the time in generating germline transmission of a correctly targetedallele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency ofcreating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we wereunsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxPsites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs,and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles,including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles withone of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequentmutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the originaloligonucleotide preparation.

Original languageEnglish
Pages (from-to)2051-2061
Number of pages11
JournalG3: Genes, Genomes, Genetics
Volume6
Issue number7
DOIs
StatePublished - Jul 1 2016

Keywords

  • CRISPR/Cas9
  • Dock7
  • KOMP ES cells

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