The fibroblast growth factor 2 is not expressed in these Fgf2 knock-out mice, and they may be useful in studying altered or pathological amounts of the Fgf2 high molecular weight isoforms in cardiac pathophysiology, myocardial ischemia-reperfusion injury, and heart disease.
Thomas Doetschman, University of Arizona
Mice homozygous for the Fgf2hmw-ko (Fgf2hmw or FGF2 HMWKO) allele are viable and fertile with no histological, immunohistochemical, or morphometric abnormalities in myocardial architecture or blood vessel and cardiac capillary density. As the targeted allele disrupts expression of the two Fgf2 high molecular weight (Fgf2 hmw) isoforms, the 20.5 and 21 kDa isoforms are not expressed (as demonstrated in heart, liver, and brain tissues from homozygous mice). The Fgf2 low molecular weight (lmw) isoform (18 kDa) is still expressed in the cytoplasm and nucleus from the targeted allele. Homozygous deficiency of the Fgf2 hmw isoforms is associated with significant improvement in postischemic cardiac/contractile function after ischemia-reperfusion (I-R) injury.
A targeting vector was designed to insert a 14 bp oligonucleotide sequence into the entire first exon of the fibroblast growth factor 2 (Fgf2) locus between the canonical methionine translational ATG start site of the Fgf2 low molecular weight (Fgf2 lmw) core sequence common to all Fgf2 isoforms and the upstream noncanonical CUG/CTG start sites that generate the two Fgf2 high molecular weight (Fgf2 hmw) isoforms. The 14 bp oligonucleotide was designed to introduce stop codons in all three reading frames, preventing production of the Fgf2 hmw isoforms. This construct was electroporated into 129P2/OlaHsd-derived E14TG2A embryonic stem (ES) cells (in which the first exon of Fgf2 had previously been replaced with an Hprt minigene). Targeted ES cells were then selected with 6-thioguanine and injected into recipient blastocysts. Chimeric males were bred with Black-Swiss females to generate the Fgf2hmw-ko colony. Non-sibling heterozygous mice were bred together for approximately 10 generations prior to arrival at The Jackson Laboratory. Upon arrival, mice were bred to C57BL/6J (Stock No. 000664) for at least one generation to establish the colony.
|Allele Name||targeted mutation 3, Thomas Doetschman|
|Allele Type||Targeted (Modified isoform(s))|
|Allele Synonym(s)||FGF2(Lo); Fgf2hmw-ko|
|Gene Symbol and Name||Fgf2, fibroblast growth factor 2|
|Strain of Origin||129P2/OlaHsd|
|Molecular Note||Using a modified Tag & Exchange targeting scheme, the Hprt minigene in the tagged Fgf2 knock-out allele (Zhou et al, Nature Medicine, 4:201, 1998) was "exchanged" by gene targeting with a 14 bp oligonucleotide designed to produce stop codons in all three reading frames and to cause a frameshift in translation products originating from the noncanonical CTG initiation codon. Cells with the exchanged allele were selected for the loss of HPRT function using 6-thioguanine. The loss of high molecular weight protein product expression was confirmed by western blot analysis on heart, liver and brain extracts. The low molecular weight protein product is still expressed.|
|Mutations Made By|| |
Thomas Doetschman, University of Arizona
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