Generation of Fgfr3 conditional knockout mice

Abstract

Fibroblast growth factor receptor 3 (FGFR3), highly conserved in both humans and murine, is one of key tyrosine kinase receptors for FGF. FGFR3 is expressed in different tissues, including cartilage, brain, kidney, and intestine at different development stages. Conventional knockout of Fgfr3 alleles leads to short life span, and overgrowth of bone. In clinic, human FGFR3 mutations are responsible for three different types of chondrodysplasia syndromes including achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). For better understanding of the roles of FGFR3 in different tissues at different stages of development and in pathological conditions, we generated Fgfr3 conditional knockout mice in which loxp sites flank exons 9-10 in the Fgfr3 allele. We also demonstrated that Cre-mediated recombination using Col2a1-Cre, a Cre line expressed in chondrocyte during bone development, results in specific deletion of the gene in tissues containing cartilage. This animal model will be useful to study distinct roles of FGFR3 in different tissues at different ages.

Keywords: Cre-Loxp; FGFR3; conditional knock out; gene targeting.

Figure 1

Generation of the Fgfr3^floxneo allele. (a) Strategy for generating the Fgfr3 targeting vector and Fgfr3^floxneo (targeted) allele. Blue boxes represent exons. The 5' external probes for Southern Blot is indicated by thick lines. The predicted length of Southern fragments are indicated with double arrow lines. Cl, Cla I; No, Not I; Sm, Sma I; Sp, Spe I; Xb, Xba I; Hp, Hpa I; p, primer. (b) Targeted events were identified by Southern analysis of Spe I- digested genomic ES cell DNAs with a 5'flanking probe. (c) The third Loxp in targeted allele was confirmed by PCR.

Figure 2

Generation of Fgfr3^{floxneo/floxneo} mice. (a) Map of Fgfr3^floxneo allele. The position of primers was marked in the map. (b) Genotype of Fgfr3^{floxneo/floxneo} mice was identified by PCR (primer p1, p2, p3 and p4). (c) There is no expression of Fgfr3 in Fgfr3^{floxneo/floxneo} mice (d) Fgfr3^{floxneo/floxneo} mice showed kinky tails, which is also found in Fgfr3 knock out mice. (e) Increased expansion of proliferating and hypertrophic chondrocytes in the growth plate in Fgfr3^{floxneo/floxneo} mice on P15.

Figure 3

Validation of exons 9-10 and neo gene in Fgfr3^floxneo alleles deleted by Cre recombinase. (a) Mice containing Fgfr3^floxneo alleles were crossed with EIIa-Cre transgenic mice resulting in three kinds of deletion between three Loxp sites (b-d). Position of primers was also marked. (b) Primer p1 and p5 amplify fragments of 390 bp from Fgfr3^null allele with deletion exons 9-10 and neo between Loxp1 and 3 (1/3), no amplification for the wild type (WT). (c) Primer p1 and p4 amplify fragments of 408 bp from Fgfr3^neofloxed allele with deletion exons 9-10 between Loxp2 and 3 (2/3), no amplification for WT. (d) Primer p3 and p5 amplified fragments of 320 bp from Fgfr3^flox allele with deletion neo between Loxp1 and 2 (1/2), but only 260bp from WT. n, Fgfr3^null allele; nf, Fgfr3^neofloxed allele; f, Fgfr3^flox allele; w, wild type.

Figure 4

Identification of Fgfr3 null mice and validation of Fgfr3 conditional knockout allele. (a) There was no expression of Fgfr3 in brain RNA of Fgfr3 null mice (homozygous mice with both exons 9-10 and neo deleted in Fgfr3). (b) The length of femur in 3-month-old Fgfr3 null mice was longer than that in wild-type mice. (c) X-ray analysis showed increased length and decreased bone mineral density of femur from Fgfr3 null mice (Arrows). (d) Tissue-specific inactivation of the Fgfr3 conditional allele by a Col2a1-Cre transgene in tissues containing cartilage was revealed by PCR analysis using primer pair p3/p5, which amplifies about 320 bp from the unrecombined allele, and primer pair p1/p5, which amplifies about 390 bp from the recombined allele.