There are the 10 known mammalian isoforms of carbonic anhydrases. Each of these isozymes possesses carbon dioxide-hydrating activity that functions to maintain intracellular pH. Carbonic anhydrase isozyme III (CAIII) is distinguished from the other isoforms by several characteristics--particularly, by a low specific activity that is about 1 per cent that of isozyme II. CAIII is not inhibited by acetozolamide, an excellent inhibitor of CAI and CAII. Also, the amount of CAIII is remarkably high in muscle tissue and adipocytes, constituting about 8 and 25 per cent of the soluble protein content of these tissues. Furthermore, CAIII has been reported to exhibit tyrosine phosphatase activity. Despite numerous publications on the enzyme, the physiological function of CAIII remains unknown. We created a knockout to facilitate investigation of the function of CAIII. The genomic DNA of mouse CAIII was cloned by a colony plaque hybridization method. Using a positive/negative type targeting vector, about 4.1 kb of CAIII genomic DNA, including the exons, 3, 4, and 5, was replaced by the bacterial neomycin (neo) resistance gene driven by the phosphoglycerate kinase 1 promoter. The knockout vector was initially electroporated into the embryonic stem cells. G418 was used to select cells containing the plasmid and gancyclovir was used to select against cells in which the construct had integrated randomly. Resistant colonies were then grown to confluence, replica-plated, and genomic DNA from one set was analyzed by a polymerase chain reaction (PCR) method. Eight correctly targeted clones were selected among the 118 double positive clones. Several embryonic cell lines carrying the mutant CAIII allele were injected into host blastocysts, and nine chimeras were born and tested for transmission of the mutation to their offsprings. Screening through both PCR and Southern blotting showed that the heterozygotic mice (F1) lacked one of the alleles of CAIII. To create mice with the knockout, heterozygotic F1 mutant mice were mated. The homozygous (F2) animals are viable, with the oldest currently aged 9 weeks. Thus far, no phenotype has been observed in these F2 homozygotes. - carbonic anhydrase, knockout, acid-base balance
| Kim, G; Selengut, J; Levine, R L (2000) Carbonic anhydrase III: the phosphatase activity is extrinsic. Arch Biochem Biophys 377:334-40 |
