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1

Rapid and simple screening of transgenic mice: novel extraction-free, filter-based PCR genotyping from blood samples

100%

Suematsu N., Isohashi F.

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2006

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vol. 53

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issue 3

613-613

EN

We evaluated the effectiveness of using Flinders Technology Associates (FTA) filter paper for the polymerase chain reaction (PCR) genotyping of transgenic mice. Tail prick blood sample dried on an FTA filter disc was processed for genomic PCR. It is easy and rapid to prepare DNA templates because the protocol is extraction-free and only requires minimal handling of wash briefly bloodstained FTA filter discs. Progeny of a transgene-positive founder mated with wild-type mice was screened for the presence of the transgene by the filter-based PCR using transgene-specific primers. The resulting amplicons with expected sizes of 3134 bp, 1152 bp, 877 bp and 688 bp were robust and reproducible, allowing a distinction between transgenic (n=44) and wild-type (n=47) mice showing no signal. The filter-based PCR screening took only half a day. The present study confirmed the validity and usefulness of the novel rapid extraction-free genotyping method.

2

Molecular cloning and functional expression of human cytosolic acetyl-CoA hydrolase

100%

Suematsu N., Isohashi F.

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2006

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vol. 53

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issue 3

553-561

EN

A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).

3

Mouse cytosolic acetyl-CoA hydrolase, a novel candidate for a key enzyme involved in fat metabolism: cDNA cloning, sequencing and functional expression.

81%

Suematsu N., Okamoto K., Isohashi F.

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2002

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vol. 49

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issue 4

937-945

EN

A cytosolic acetyl-CoA hydrolase (CACH) cDNA has been isolated from mouse liver cDNA library and sequenced. Recombinant expression of the cDNA in insect cells resulted in overproduction of active acetyl-CoA hydrolyzing enzyme protein. The mouse CACH cDNA encoded a 556-amino-acid sequence that was 93.5% identical to rat CACH, suggesting a conserved role for this enzyme in the mammalian liver. Database searching shows no homology to other known proteins, but reveals homological cDNA sequences showing two single-nucleotide polymorphisms (SNPs) in the CACH coding region. The discovery of mouse CACH cDNA is an important step towards genetic studies on the functional analysis of this enzyme by gene-knockout and transgenic approaches.

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3 Acta Biochimica Polonica

2 2006

1 2002

3 Isohashi F.

3 Suematsu N.

1 Okamoto K.

Rapid and simple screening of transgenic mice: novel extraction-free, filter-based PCR genotyping from blood samples

Molecular cloning and functional expression of human cytosolic acetyl-CoA hydrolase

Mouse cytosolic acetyl-CoA hydrolase, a novel candidate for a key enzyme involved in fat metabolism: cDNA cloning, sequencing and functional expression.