A more efficient method to generate null mutants using Hprt-Cre with floxed alleles

Abstract

Purpose-The generation of non-viable homozygous null mouse embryos from heterozygote null/+ breedings can be highly resource consuming, with only 25% of the embryos in the litter being null mutants. We hypothesized that 1) we could double the number of homozygous null mouse embryos in a litter without reducing litter size using Hypoxanthine-guanine phosphoribosyltransferase-Cre (Hprt-Cre) (which is active in the female germ line at the time of fertilization) and 2) these homozygous null mutants would be identical to mutants generated through traditional null/+ breedings.Methods-To test this hypothesis we used a conditional allele Fgfr2IIIb flox . This allele when recombined is identical to the Fgfr2IIIb null allele. An F1 generation of Fgfr2IIIb rec/+ ; Hprt Cre/+ females was created by mating Fgfr2IIIb +/+ ; Hprt cre′/cre females to a Fgfr2IIIb flox/flox male. The F1 females were then mated to a Fgfr2IIIb flox/flox male. F2 embryos were genotyped and the morphology and histology of the lungs, intestine, limbs and brain was analyzed.Results-The Hprt-Cre mating strategy results in 51% of pups being genotypic homozygous null embryos (85/166) versus 23% for the standard null/+ approach (38/167). These embryos did not express the Fgfr2IIIb transcript and were phenotypically identical to null embryos generated through standard null/+ breedings. Conclusions-TheHprt-Cre mating strategy increases the number of homozygous mutant embryos in a litter without decreasing litter size. Embryos generated through this approach are phenotypically identical to those from standard heterozygous breedings. We recommend this Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. , and cleft palate [9,10] have been described. Generation of homozygous null embryos through null/+ (n/+) heterozygous breedings can be highly resource consuming. When employing the n/+ breedings for one gene, a single homozygous null (n/n) embryo is generated for every 4 embryos. Thus, a litter of 10 will yield 2 or 3 homozygous null mutant embryos. Generating compound homozygous null mutants for 2 or 3 genes with this process becomes even less efficient as the frequency of homozygous null mutants decreases to 1 in 16 embryos and 1 in 64 embryos, respectively. Researchers also face increasing pressure to minimize the number of animals used while maintaining, or even increasing, the quality of basic science research. This pressure is driven in part by both the animal rights movement [11,12] To test the efficiency of the Hprt-Cre strategy, we determined the number of phenotypically null embryos in a litter as compared ...

Utility and limits of Hprt-Cre technology in generating mutant mouse embryos

“…It has been previously reported that homozygous null embryos can be generated with greater efficiency than the standard heterozygous breedings using Hprt-Cre in combination with a conditional allele [1]. This was fully characterized for only one gene, that is, Fgfr2IIIb .…”

“…Finally, we wanted to determine whether the Hprt-Cre breeding strategy could be successfully used to generate either double or triple compound mutants as originally postulated [1]. Double and triple homozygous-heterozygous compound mutants Fgfr2IIIb rec/rec ; Raldh2 rec/+ and Fgfr2IIIb rec/rec ; Raldh2 rec/+ ; Cyp26b1 rec/+ were generated and constituted 50% of the embryos per litter (Table 3).…”

“…We have previously reported that the efficiency of generating homozygous null mutants can be significantly increased by using the Hprt-Cre breeding strategy (Fig. 5) [1]. This approach can generate homozygous null embryos effectively identical to those generated using a standard heterozygous breeding strategy.…”

“…They allow for tissue specific mutations, although preserving normal levels of gene expression in adjacent tissue. Another application of Cre technologies is in increasing the efficiency and production of homozygous mutant embryos [1,2]. Increased efficiency is accomplished by introducing the Cre into the female germ-line under the control of the hypoxanthine-guanine phosphoribosyltransferase ( Hprt , a housekeeping gene) promoter enhancer.…”

“…It reduces the number of matings required to generate a desired number of null embryos by doubling the percentage of mutants per litter. To date, this approach has only been fully characterized with one conditional allele, that is, Fgfr2IIIb [1]. We were interested in duplicating this methodology with several early embryonic conditional alleles.…”