Final Report Summary - CATABOLIC SIALIDASES (Understanding the roles of mammalian sialidases in glycolipid catabolism)
Sialidases (neuraminidases) constitute a family of hydrolyses responsible for the removal of sialic acids from sialylated glycolipids, glycoproteins and oligosaccharides. Four mammalian sialidases have been cloned; a lysosomal form (Neu1), a cytosolic form (Neu2), a plasma membrane-associated form (Neu3) and a lysosomal/mitochondrial form (Neu4). Our goal has been to clarify biological functions of mammalian sialidase Neu1 in glycolipid degradation. We proposed to investigate the intralysosomal mechanism by which glycolipids are degraded by sialidase Neu1 and Neu4 as well as HexA using deficient mouse models. Therefore, the main objectives of our proposal are: (1) to generate Neu1-/- single knock-out mouse model (2) to generate HexA-/-Neu1-/- double knock-out mouse model (3) to generate Hexa-/-Neu1-/-Neu4-/- triple knockout mouse models.
In order to generate Neu1-/- single gene knockout mouse model, embryonic stem (ES) cell were transfected with previously constructed Neu1 gene targetting vector (TV-Neu1-1) by electroporation. Although one positive clone among 110 screened colonies was obtained using different sets of 5’ and 3’ primer pairs outside of gene targeting region, S.blot analysis failed to confirm the correct gene targeting event in mouse Neu1 gene locus in that clone. Therefore, new Neu1 gene targeting vector (TV-Neu1-2) was constructed using similar strategy to achive our goal. New targeting vector were electroporated into ES cells to knockout mouse Neu1 sialidase gene by homologous recombination in ES cells. After double selection with geneticine and ganyclovir, this time we obtained 6 positive clones among 163 picked colonies and two of them were confirmed by S. Blot analysis. Positive clones were expanted and microinjected into blastocytes. Chimeric animals were received in September 2013. We observed chimerism between 10-90% as predicted. Chimeric females and males were crossed with C57B6 females and males. So far more than 592 pups were genotyped but no germline transmission were detected. Male chimerics although they are quide old are still in breeding with females and we keep genotyping their offsprings.
In an parallel afford, we used our previously generated mice NeoIn-/- ( here named as Neu1-/-) with ~10% of normal sialidase Neu1 whick is likely to be suitable alternative model to test the role of sialidase Neu1 in metabolic bypass in HexA-/- mice (Seyrantepe et al, 2010, Larbi et al, 2013). Existing hypomorph mice strain Neu1-/- were breeded with HexA-/- mice model while generation of Neu1-/- full knockout mice in progress. Double knockout (HexA-/-Neu1-/-) and triple knockout (HexA-/-Neu1-/-Neu4-/-) mice strain were generated and brain ganglisoides were analyzed by thin layer chromatography. Altered ganglioside pattern were observed in double knock-out HexA-/-Neu1-/- as well as triple knockout (HexA-/-Neu1-/-Neu4-/- mice compared to single knock-out (HexA-/- , Neu4-/-, Neu1-/-) mice. We also performed biochemical (mass spectrometry), imminohistochemical and molecular biological analysis in brain tissue from those animals. Our manuscript showing our interesting results was accepted for publication in Mol Genet Met Reports (September 2015 issue)
In order to generate Neu1-/- single gene knockout mouse model, embryonic stem (ES) cell were transfected with previously constructed Neu1 gene targetting vector (TV-Neu1-1) by electroporation. Although one positive clone among 110 screened colonies was obtained using different sets of 5’ and 3’ primer pairs outside of gene targeting region, S.blot analysis failed to confirm the correct gene targeting event in mouse Neu1 gene locus in that clone. Therefore, new Neu1 gene targeting vector (TV-Neu1-2) was constructed using similar strategy to achive our goal. New targeting vector were electroporated into ES cells to knockout mouse Neu1 sialidase gene by homologous recombination in ES cells. After double selection with geneticine and ganyclovir, this time we obtained 6 positive clones among 163 picked colonies and two of them were confirmed by S. Blot analysis. Positive clones were expanted and microinjected into blastocytes. Chimeric animals were received in September 2013. We observed chimerism between 10-90% as predicted. Chimeric females and males were crossed with C57B6 females and males. So far more than 592 pups were genotyped but no germline transmission were detected. Male chimerics although they are quide old are still in breeding with females and we keep genotyping their offsprings.
In an parallel afford, we used our previously generated mice NeoIn-/- ( here named as Neu1-/-) with ~10% of normal sialidase Neu1 whick is likely to be suitable alternative model to test the role of sialidase Neu1 in metabolic bypass in HexA-/- mice (Seyrantepe et al, 2010, Larbi et al, 2013). Existing hypomorph mice strain Neu1-/- were breeded with HexA-/- mice model while generation of Neu1-/- full knockout mice in progress. Double knockout (HexA-/-Neu1-/-) and triple knockout (HexA-/-Neu1-/-Neu4-/-) mice strain were generated and brain ganglisoides were analyzed by thin layer chromatography. Altered ganglioside pattern were observed in double knock-out HexA-/-Neu1-/- as well as triple knockout (HexA-/-Neu1-/-Neu4-/- mice compared to single knock-out (HexA-/- , Neu4-/-, Neu1-/-) mice. We also performed biochemical (mass spectrometry), imminohistochemical and molecular biological analysis in brain tissue from those animals. Our manuscript showing our interesting results was accepted for publication in Mol Genet Met Reports (September 2015 issue)