NULL MUTATION OF A NEURON-SPECIFIC EXON OF NONMUSCLE MYO

Heart, Lung, And Blood Institute

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Abstract

To investigate the physiological function of inserted isoforms of non-muscle myosin heavy chain IIB (nMHC-IIB), knockout mice were generated by the deletion of a specific exon encoding the 10 amino acid insert (B1) or 21 amino acid insert (B2). Previous experience with B1 insert knockout mice indicated that the presence of Neo-cassette in the targeting construct could result in hypomorphism (i.e. a marked decrease) in the general expression of total nMHC-IIB. The floxed Neo-cassette, therefore, was introduced in our new targeting constructs, which enables us to remove the Neo-cassette by crossing with transgenic mice expressing the Cre-recombinase. Both the Neo-cassette containing B1 and B2 knockout mice showed a significant reduction in the total amount of nMHC-IIB expression (similar to the previous B1 knockout mice). About 8% of the hypomorphic B1 knockout mice suffered with severe hydrocephalus between 2 to 3 weeks after birth. No obvious abnormality in general health was found in hypomorphic B2 knockout mice thus far. To further explore the B2 insert function, a more severely hypomorphic B2 knockout model is currently being generated by crossing the present hypomorphic B2 knockout mouse with IIB heterozygous mouse. While nMHC-IIB is expressed in most tissues, the inserted isoforms are only expressed in the nervous system. mRNA expression of the B1 insert appears in the prenatal mouse brain and increases during postnatal development. The B2 inserted mRNA, however, begins expression about one week after birth and reaches its highest level about 3 weeks after birth. To further analyze the regional expression patterns of the B2 insert, a polyclonal peptide antibody was generated specific to the inserted amino acid sequence. Immunohistochemical staining of the adult mouse brain with B2 insert-specific antibody revealed that the B2 insert was mostly enriched in cerebellar Purkinje cells. The cerebellar deep neurons, pontine nuclei and inferior olive also showed positive staining, which indicates that the B2 insert is mainly confined to the cerebro-cerebellar loop which is believed to be important in regulating fine tuning of locomotion. Therefore, functional analysis such as motor coordination and balance will be carried out in B2 knockout mice. In addition, some neurons in the cerebral cortex, especially in layer V, also stained with B2 antibody. Null mutation of the inserted isoforms could, therefore, result in developmental or functional defects in the brain. However, hypomorphism in nMHC-IIB expression could also generate a gene dosage effect. Therefore, the Neo-cassette in these hypomorphic targeted constructs is being removed by crossing these mice with the cre-founder. Whether removal of the Neo-cassette can rescue the hypomorphism is currently under investigation. To mimic a possible human genetic mutational disease of nMHC-IIB, similar to the recently identified mutation in the nMHC-IIA gene, a point mutation knockin mouse model is also under development. Three positive ES cell clones carrying one copy of the mutated allele were separately injected into normal blastocysts to generate chimeras. We are currently assessing germline transmission of these mice.