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A mouse model of familial hypertrophic cardiomyopathy (FHC) was generated by the introduction of an Arg 403 --> Gln mutation into the alpha cardiac myosin heavy chain (MHC) gene. Homozygous alpha MHC 403/403 mice died 7 days after birth, and sedentary heterozygous alpha MHC 403/+ mice survived for 1 year. Cardiac histopathology and dysfunction in the alpha MHC 403/+ mice resembled human FHC. Cardiac dysfunction preceded histopathologic changes, and myocyte disarray, hypertrophy, and fibrosis increased with age. Young male alpha MHC 403/+ mice showed more evidence of disease than did their female counterparts. Preliminary results suggested that exercise capacity may have been compromised in the alpha MHC 403/+ mice. This mouse model may help to define the natural history of FHC.

We have developed a simple method for producing embryonic stem (ES) cell lines whereby both alleles have been inactivated by homologous recombination and which requires a single targeting construct. Four different ES cell lines were created that were heterozygous for genes encoding two guanine nucleotide-binding protein subunits, %. and c. , T-cell receptor ca, and 1"-cardiac myosin heavy chain. When these heterozygous cells were grown in high concentrations of G418, many of the surviving cells were homozygous for the targeted allele and contained two copies of the G418 resistance gene. This scheme provides an easy method for obtaining homozygous mutationally altered cells, i.e., double knockouts, and should be generally applicable to other genes and to cell lines other than ES cells. This method should also enable the production of cell lines in which more than one gene have had both alleles disrupted. These mutant cells should provide useful tools for defining the role of particular genes in cell culture.With the discovery that gene sequences in mammalian cells could be targeted by homologous recombination, new methods for constructing mutationally altered cells that lack particular genes have become available. Over the past several years, a number of groups have demonstrated that heterozygous cells in which one of two alleles has been inactivated can be constructed (4,7,8,14,16,18,19).Production of null mutants by using homologous recombination (in which two copies of the gene have been inactivated) has recently been accomplished in embryonic stem (ES) cells (9, 15) and other types of cells (3). Such mutants should allow the investigation of questions more easily addressed in cell lines rather than in whole animals or tissues. The methods used involved sequential targeting by two separate constructs using two different selectable markers. Here, we describe an improved method for obtaining such null mutant cells. Homozygous cells have been known to be spontaneously produced from heterozygous cultured cells by a variety of mechanisms (2, 10, 12, 13). We demonstrate that homozygous cells are routinely produced after initially targeting a single allele and have developed a scheme for the selection of these double knockouts. MATERIALS AND METHODSConstruction of targeting vectors. In each case, the phosphoglycerate kinase (PGK)-thymidine kinase gene was inserted outside the regions of homology. The production of the a12 targeting construct has been described previously (9). The a43 gene clone was isolated as described for the ti2 gene.A HindIII-SalI fragment was subcloned into Bluescript SK (+) (Stratagene). The construct contained the PGK-neo (neomycin resistance) gene interrupting exon 1 at an NcoI site at the translational start ATG. A 5.6-kb fragment of the murine T-cell receptor a (TCR-a) gene containing all four constant-region exons was subcloned from genomic clone * Corresponding author. X*1.2 (6) into Bluescript SK II (+). The fragment extends from an artificial EcoRI site, 5' of the XbaI site, to the XhoI...

We created a mouse that lacks a functional alpha-tropomyosin gene using gene targeting in embryonic stem cells and blastocyst-mediated transgenesis. Homozygous alpha-tropomyosin "knockout" mice die between embryonic day 9.5 and 13.5 and lack alpha-tropomyosin mRNA. Heterozygous alpha-tropomyosin knockout mice have approximately 50% as much cardiac alpha-tropomyosin mRNA as wild-type littermates but similar alpha-tropomyosin protein levels. Cardiac gross morphology, histology, and function (assessed by working heart preparations) of heterozygous alpha-tropomyosin knockout and wild-type mice were indistinguishable. Mechanical performance of skinned papillary muscle strips derived from mutant and wild-type hearts also revealed no differences. We conclude that haploinsufficiency of the alpha-tropomyosin gene produces little or no change in cardiac function or structure, whereas total alpha-tropomyosin deficiency is incompatible with life. These findings imply that in heterozygotes there is a regulatory mechanism that maintains the level of myofibrillar tropomyosin despite the reduction in alpha-tropomyosin mRNA.