All posts by takahide

Use of PB-Cre4 Mice for Mosaic Gene Deletion

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PLoS One. 2013;8(1):e53501. doi: 10.1371/journal.pone.0053501. Epub 2013 Jan 7.
Birbach A.

Source

Department of Vascular Biology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

Abstract

Transgene expression from short promoters in transgenic animals can lead to unwanted transgene expression patterns, often as a byproduct of random integration of the expression cassette into the host genome. Here I demonstrate that the often used PB-Cre4 line (also referred to as “Probasin-Cre”), although expressing exclusively in the male prostate epithelium when transmitted through male mice, can lead to recombination of loxP-flanked alleles in a large variety of tissues when transmitted through female mice. This aberrant Cre activity due to Cre expression in the oocytes leads to different outcomes for maternally or paternally transmitted loxP-flanked alleles: Maternally inherited loxP-flanked alleles undergo recombination very efficiently, making female PB-Cre4 mice an efficient monoallelic “Cre deleter line”. However, paternally inherited loxP-flanked alleles are inefficiently recombined by maternal PB-Cre4, giving rise to mosaic expression patterns in the offspring. This mosaic recombination is difficult to detect with standard genotyping approaches of many mouse lines and should therefore caution researchers using PB-Cre4 to use additional approaches to exclude the presence of recombined alleles. However, mosaic recombination should also be useful in transgenic “knockout” approaches for mosaic gene deletion experiments.

A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex

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PLoS Negl Trop Dis. 2013 Jan;7(1):e2017. doi: 10.1371/journal.pntd.0002017. Epub 2013 Jan 17.
Boubaker G, Macchiaroli N, Prada L, Cucher MA, Rosenzvit MC, Ziadinov I, Deplazes P, Saarma U, Babba H, Gottstein B, Spiliotis M.

Source

Institute of Parasitology, University of Bern, Bern, Switzerland ; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland ; University of Monastir, Faculty of Pharmacy, Department of Clinical Biology B, Laboratory of Parasitology and Mycology, Monastir, Tunisia.

Abstract

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

Evaluation of a reliable and cost-effective method of DNA isolation for mouse genotyping

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Biotechnol Lett. 2012 Dec 15. [Epub ahead of print]
Sysol JR, Kempf C, Helton MN, Dong Y, Zhu D, Sun H, Garcia JG, Machado RF, Chen J.

Source

Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA, jsysol2@uic.edu.

Abstract

Genotyping is commonly used to define specific gene alterations or the presence of transgenes in mice. This procedure is typically done using DNA isolated from mouse tail tissue. Although there are commercially available kits for tail DNA isolation, they can be time consuming and costly for routine genotyping. In this study, we describe a rapid, “crude” DNA isolation method using mouse tail tissue and compare it to a frequently used, commercially available kit in the genotyping of over 1,000 total mice from 8 genetic lines. Our genotyping results were obtained faster and less expensively but with the same success rate (Crude method: 97.7 %, Kit method: 98.4 %). To our knowledge, this is the first systematic study to compare the reliability of this crude DNA isolation method for mouse genotyping compared to a commercially available kit.

Development of an Efficient Genotyping Method to Detect Obese Mutation in the Mouse Leptin Gene for Use in SPF Barrier Facilities

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J Vet Med Sci. 2012 Dec 10. [Epub ahead of print]
Ayabe H, Ikeda S, Maruyama S, Shioyama S, Kikuchi M, Kawaguchi A, Yamada T, Ikeda T.

Source

Plasma Team, Production Department, CHARLES RIVER LABORATORIES JAPAN, Inc.

Abstract

We have developed a rapid and efficient genotyping method for detection of the mouse leptin obese mutation (Lep(ob)) using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR). In this method, whole blood collected onto gamma-ray sterilized Flinders Technology Associates (FTA) filter paper is used as PCR template without a DNA purification step. Three genotypes (Lep(ob)/Lep(ob), Lep(ob)/+ and +/+) differentiated by single-tube PCR and electrophoresis were perfectly consistent with those determined by PCR-restriction fragment length polymorphism (PCR-RFLP). This method can save material costs and operation time, because it does not require restriction enzyme digestion and could be set up in most specific pathogen-free (SPF) barrier facilities.

 

Save your time and cost

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Dear Researchers,

Did you know that around 30% of researchers and technical staff regard the genotyping   processes as a time-consuming. We know it takes almost one-day operation, even there are 10 samples or 100 samples.

In order to save your time and cost, we always keep our door open to you. Just contact us, if you need!! Do not waste yours.

Regards,

Genotyping Facility

Deconstructing Mus gemischus: advances in understanding ancestry, structure, and variation in the genome of the laboratory mouse

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Mamm Genome. 2013 Feb;24(1-2):1-20. doi: 10.1007/s00335-012-9441-z. Epub 2012 Dec 9.
Didion JP, de Villena FP.

Source

Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, jdidion@email.unc.edu.

Abstract

The laboratory mouse is an artificial construct with a complex relationship to its natural ancestors. In 2002, the mouse became the first mammalian model organism with a reference genome. Importantly, the mouse genome sequence was assembled from data on a single inbred laboratory strain, C57BL/6. Several large-scale genetic variant discovery efforts have been conducted, resulting in a catalog of tens of millions of SNPs and structural variants. High-density genotyping arrays covering a subset of those variants have been used to produce hundreds of millions of genotypes in laboratory stocks and a small number of wild mice. These landmark resources now enable us to determine relationships among laboratory mice, assign local ancestry at fine scale, resolve important controversies, and identify a new set of challenges-most importantly, the troubling scarcity of genetic data on the very natural populations from which the laboratory mouse was derived. Our aim with this review is to provide the reader with an historical context for the mouse as a model organism and to explain how practical decisions made in the past have influenced both the architecture of the laboratory mouse genome and the design and execution of current large-scale resources. We also provide examples on how the accomplishments of the past decade can be used by researchers to streamline the use of mice in their experiments and correctly interpret results. Finally, we propose future steps that will enable the mouse community to extend its successes in the decade to come.

Dear Researchers!!

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Dear Researchrs,

We have been providing the PCR-based genotyping services since 2004 at the Biomedical Research Centre on UBC campus. Services are covered from the disease-model mice genotyping to the pathogen detection in the mice.

The cost is very competitive and the turnaround is five business days.

What you need to bring is just your sample and we extract the genomic DNA to be ready for the genotyping.

Visit our website for the detail at http://www.brc.ubc.ca.

Regards,

Genotyping Facility