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

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Suematsu N., Isohashi F.

2006

vol. 53

issue 3

613-613

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.

Differences in glutathione S-transferase pi expression in transgenic mice with symptoms of neurodegeneration

86%

Kaźmierczak B., Kuźma-Kozakiewicz M., Usarek E., Barańczyk-Kuźma A.

2011

vol. 58

issue 4

621-626

Glutathione S-transferase pi (GST pi) is an enzyme involved in cell protection against toxic electrophiles and products of oxidative stress. GST pi expression was studied in transgenic mice hybrids (B6-C3H) with symptoms of neurodegeneration harboring SOD1G93A (SOD1/+), Dync1h1 (Cra1/+) and double (Cra1/SOD1) mutations, at presymptomatic and symptomatic stages (age 70, 140, 365 days) using RT-PCR and Western blotting. The main changes in GST pi expression were observed in mice with the SODG93A mutation. In SOD1/+ and Cra1/SOD1 transgenics, with the exception of cerebellum, the changes in GST pi-mRNA accompanied those in GST pi protein. In brain cortex of both groups the expression was unchanged at the presymptomatic (age 70 days) but was lower at the symptomatic stage (age 140 days) and at both stages in hippocampus and spinal cord of SOD1/+ but not of Cra1/SOD1 mice compared to age-matched wild-type controls. In cerebellum of the presymptomatic and the symptomatic SOD1/+ mice and presymptomatic Cra1/SOD1 mice, the GST pi-mRNA was drastically elevated but the protein level remained unchanged. In Cra1/+ transgenics there were no changes in GST pi expression in any CNS region both on the mRNA and on the protein level. It can be concluded that the SOD1G93A but not the Dync1h1 mutation significantly decreases detoxification efficiency of GST pi in CNS, however the Dync1h1 mutation reduces the effects caused by the SOD1G93A mutation. Despite similarities in neurological symptoms, the differences in GST pi expression between SOD1/+ and Cra1/+ transgenics indicate a distinct pathogenic entity of these two conditions.

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2 Acta Biochimica Polonica

1 2011

1 2006

1 Barańczyk-Kuźma A.

1 Isohashi F.

1 Kaźmierczak B.

1 Kuźma-Kozakiewicz M.

1 Suematsu N.

1 Usarek E.

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

Differences in glutathione S-transferase pi expression in transgenic mice with symptoms of neurodegeneration