RESUMO
The gene plp on the X chromosome encodes the isoforms proteolipid protein (PLP) and DM(20), two dominant integral membrane proteins of central nervous system (CNS) myelin. DM(20) results from the activation of the cryptic splice site in exon III of the PLP gene. We inserted a sense-orientated loxP flanked neomycin-gene into intron III of the plp sequence, using homologous recombination in embryonic stem cells and generated the homozygous neoS mouse line. Unlike the previously described complete PLP/DM(20) ablation (plp(-/-)), which has been obtained by introducing a neo-gene in antisense-orientation in the same position of intron III, the plp expression surprisingly revealed reduced mRNA levels. The PLP isoform was reduced to 50%, but DM(20) expression was unaffected. This protein pattern resembles the expression profile of the PLP isoforms in the natural occurring rumpshaker mutant. Electron microscopic examination revealed a normal compaction of CNS-myelin and maintenance of axon integrity. PLP expression levels of the wt control were recovered by Cre excision of the neo-selection gene after intercrossing neoS mice and oligodendrocyte-specific Cre-mice. These data strongly hint at different functions of intron III in PLP/DM(20)-specific splicing and mRNA stability. Furthermore evidence is provided for functionally affected translation products of the PLP gene in the rumpshaker mutant, whereas no PLP-isoform occur in plp(-/-) mice generated by introducing a selectable marker into intron III in antisense orientation.
Assuntos
Axônios/metabolismo , Sistema Nervoso Central/anormalidades , Camundongos Mutantes Neurológicos/genética , Proteína Proteolipídica de Mielina/genética , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso , Processamento Alternativo/genética , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/ultraestrutura , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes/métodos , Íntrons/genética , Camundongos , Camundongos Knockout/anormalidades , Camundongos Knockout/genética , Camundongos Knockout/crescimento & desenvolvimento , Camundongos Mutantes Neurológicos/anormalidades , Camundongos Mutantes Neurológicos/crescimento & desenvolvimento , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Neomicina , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição Gênica/genéticaRESUMO
Double (plp-/-mag-/-) and triple (plp-/-mbp-/-mag-/-) null-allelic mouse lines deficient in proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin basic protein (MBP) were generated and characterized genetically, biochemically, and morphologically including their behavioral capacities. The plp-/-mag-/- mutant develops a rapidly progressing axon degeneration in CNS with severe cognitive and motor coordinative deficits but has a normal longevity. CNS axons of the plp-/-mbp-/-mag-/- mouse are hypomyelinated and ensheathed by "pseudomyelin" with disturbed protein and complex lipid composition. The shiverer trait in the plp-/-mbp-/-mag-/- similar to the plp-/-mbp-/- mutant is significantly ameliorated, and its lifespan is considerably prolonged. The longevity of these dysmyelinosis mouse mutants recommends them as suitable models for the long-term evaluation of stem cell therapeutic strategies.
