NULL MUTATIONS OF VERTEBRATE NONMUSCLE MYOSIN HEAVY CHAINS

非肌球蛋白是一种普遍存在于所有真核生物中的蛋白质

Nonmuscle myosin is an ubiquitous protein present in all eukaryotic cells. It is involved in the motility of cells and in cellular cytokinesis and capping. This laboratory has isolated two different cDNAs from human and chicken tissues encoding nonmuscle myosin heavy chains (NMMHCs). These two isoforms are called NMMHC-A and -B and are located on two different human chromosomes. In order to understand the in vivo function of the two isoforms, we are trying to perform experiments to eliminate the genes for NMMHC(s) using homologous recombination in embryonic stem cells and in transgenic mice. We generated a construct of 14.5 kb containing a 6.7 kb NMMHC-B fragment cloned from a mouse genomic lambda phage library. A neomycin gene sequence (2 kb) was introduced into exon 2 to interrupt the transcription of the gene and provide neomycin resistance after homologous recombination. Also, the herpes simplex virus thymidine kinase (TK) gene was introduced outside the homologous region at the end of our construct. The TK gene will be integrated only in cases of random integration, but not if homologous recombination occurs. The construct was checked by restriction maps and partial sequencing. The mutated gene was introduced into mouse embryonic stem cells (ESC) (J- 1) by electroporation and selected in the presence of Geneticin (300 ug/ml) and FIAU (0.2 uM). The selected clones were screened using a genomic Southern blot to distinguish homologous from random recombination events. We are also characterizing fragments of mouse NMMHC-B from the actin binding region cloned from the same mouse genomic lambda phage library and screened with a different probe. We will use this region of the gene to knock out the NMMHC-B isoform and we will also make a new construct containing a lox/cre sequence to try to generate a mouse lacking a particular inserted sequence of mRNA present near the actin binding region of brain NMMHC-B. At the same time, a clone for the NMMHC-A isoform is being characterized in order to make a construct similar to the NMMHC-B one.