Inactivation of the {beta}-subunit of lysosomal {beta}-hexosaminidase by targeted disruption of the HEXB gene in mice

{beta}-Hexosaminidase is a lysosomal enzyme that catalyzes the hydrolysis of ganglioside G{sub M2} and other substances. Two major catalytically active isozymes are expressed in human tissues: Hex A (a{beta}) and Hex B ({beta}{beta}). Sandhoff disease is a rare autosomal recessive disorder caused by mutations in the HEXB gene, inactivating both isoenzymes. We have characterized the structure of the mouse HEXB gene from the strain 129/Sv. It contains 14 exons in a span of 22 kb; a 1200 by 5{prime}-sequence showed promoter activity when expressed in monkey COS cells. A survey of mRNA levels in mouse tissues showed the highest levels in kidney and epididymis, with brain intermediate, and lung, testis and liver with the lowest levels. We used homologous recombination of a distrupted HEXB gene to inactivate the endogenous gene in pluripotent embryonic stem (ES) cells. The sequence replacement vector contained {approximately}11 kb of the mouse HEXB gene (exons 1-5) and was interrupted in the second exon with the neomycin-resistance (neo{sup r}) gene. The targeting vector was introduced into male CGR8 ES cells by electroporation followed by selection for G418-resistant colonies. Analysis of the genomic DNA of 100 independent colonies by Southern blot allowed us to identify six cell linesmore » with the expected band pattern generated by specific recombination at the targeted site. Cells of three of the cell lines were microinjected into C57BL/6J blastocysts in order to generate chimeric mice. Three chimeric males were obtained with ES cell contributions ranging from 80 to 95% as judged by the proportion of agouti coat color. These chimeras are currently being bred with C57BL/6J mice for the generation of HEXB heterozygous and homozygous mice.« less