Faster regeneration associated to high expression of Fam65b and Hdac6 in dysferlin-deficient mouse.

Dysferlin is a sarcolemmal muscle protein associated with the processes of membrane repair, trafficking, and fusion of intracellular vesicles and muscle regeneration. Mutations in the DYSF gene cause clinically distinct forms of muscular dystrophies. The dysferlin-deficient SJL/J mouse model presents a reduction of 85% of the protein but shows mild weakness and discrete histopathological alterations. To study the effect of dysferlin deficiency in the muscle regenerative process, we used a model of electrical injury by electroporation to induce muscle degeneration/regeneration in the SJL/J mouse. The relative expression of the genes Pax7, MyoD, Myf5, and Myog was accompanied by the histopathological evaluation during muscle recovery at different time points after injury. We also investigated the effects of dysferlin deficiency in the expression of genes encoding FAM65B and HDAC6 proteins, recently described as forming a tricomplex with dysferlin at the beginning of myoblast differentiation. We observed an altered time course through the process of degeneration and regeneration in dysferlin-deficient mice, with remarkable regenerative capacity characterized by a faster and effective response in the first days after injury, as compared to the WT mice. Also, dysferlin deficiency seems to significantly alter the gene expression of Fam65b and Hdac6 during regeneration, since higher levels of expression of both genes were observed in dysferlin-deficient mice. These results need further attention to define their relevance in the disease mechanism.

Comparison of DNA extraction using proteinase K and extraction kit: analysis of the quality of the genetic material / Comparação da extração de DNA utilizando proteinase K e kit de extração: análise da qualidade do material genético

ABSTRACT INTRODUCTION: Deoxyribonucleic acid (DNA) is the raw material for genetic studies, and therefore laboratory techniques are developed to obtain it with appropriate concentration and integrity. OBJECTIVE: To compare two methods of DNA extraction regarding sample concentration and integrity. METHODS: DNA was extracted from the tail end (2 mm long) of mice, stored at -20ºC. The proteinase K method (PKM) and the Kappa Express Extract® kit were used for extraction. The concentrations and ratios 260/280 and 260/230 were determined by spectrophotometry. DNA integrity was checked on 2% agarose gel with ethidium bromide. For the final test of the extracted samples, a multiplex polymerase chain reaction (PCR) was performed, with primers for the Large gene. RESULTS: Samples extracted by the PKM presented mean concentration value of 59.4 ± 18.5 ng/µl (260/280 = 1.74 ± 0.04 and 260/230 + 1.85 ± 0.14) and the samples extracted by the commercial kit presented mean concentration value of 178.8 ± 42.0 ng/µl (260/280 = 1.09 ± 0.04 and 260/230 = 0.62 ± 0.66). PCR amplified the Large gene in the DNA extracted, regardless of the methodology used. CONCLUSION: Both methodologies studied can be used, and the PKM is cheap but a time-consuming method, while the commercial kit is more expensive, however DNA extraction is faster.

RESUMO INTRODUÇÃO: O ácido desoxirribonucleico (DNA) é a matéria-prima para os estudos genéticos, por isso são desenvolvidas técnicas laboratoriais para sua obtenção, com concentração e integridades adequadas. OBJETIVO: Comparar dois métodos de extração de DNA em relação à concentração e à integridade da amostra. MÉTODOS: O DNA foi extraído da extremidade das caudas (2 mm de comprimento) de camundongos, as quais foram estocadas a -20ºC. Utilizou-se a metodologia de extração com proteinase K (MPK) e o kit Kappa Express Extract®. As concentrações e as relações 260/280 e 260/230 foram determinadas por espectrofotometria. A integridade do DNA foi verificada em gel de agarose a 2%, com brometo de etídeo. Para o teste final das amostras extraídas, realizou-se reação em cadeia da polimerase (PCR) multiplex, com primers para o gene Large. RESULTADOS: As amostras extraídas pela MPK apresentaram concentração média de 59,4 ± 18,5 ng/µl (260/280 = 1,74 ± 0,04 e 260/230 + 1,85 ± 0,14) e as extraídas pelo kit comercial, concentração média de 178,8 ± 42 ng/µl (260/280 = 1,09 ± 0,04 e 260/230 = 0,62 ± 0,66). A PCR amplificou o gene Large no DNA extraído, independente da metodologia utilizada. CONCLUSÃO: As metodologias estudadas podem ser utilizadas, sendo a MPK uma metodologia barata, porém demorada, enquanto o kit comercial é mais oneroso, contudo a extração do DNA é célere.