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A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice
The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding fr...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Japanese Association for Laboratory Animal Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547999/ https://www.ncbi.nlm.nih.gov/pubmed/25765875 http://dx.doi.org/10.1538/expanim.14-0085 |
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author | WANG, Qingkai WANG, Na ZHANG, Xin HU, Weiguo |
author_facet | WANG, Qingkai WANG, Na ZHANG, Xin HU, Weiguo |
author_sort | WANG, Qingkai |
collection | PubMed |
description | The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds. |
format | Online Article Text |
id | pubmed-4547999 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Japanese Association for Laboratory Animal Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-45479992015-08-26 A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice WANG, Qingkai WANG, Na ZHANG, Xin HU, Weiguo Exp Anim Original The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds. Japanese Association for Laboratory Animal Science 2015-03-10 2015 /pmc/articles/PMC4547999/ /pubmed/25765875 http://dx.doi.org/10.1538/expanim.14-0085 Text en ©2015 Japanese Association for Laboratory Animal Science http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. |
spellingShingle | Original WANG, Qingkai WANG, Na ZHANG, Xin HU, Weiguo A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice |
title | A simple PCR-based method for the rapid genotyping of inherited fifth
complement component (C5)-deficient mice |
title_full | A simple PCR-based method for the rapid genotyping of inherited fifth
complement component (C5)-deficient mice |
title_fullStr | A simple PCR-based method for the rapid genotyping of inherited fifth
complement component (C5)-deficient mice |
title_full_unstemmed | A simple PCR-based method for the rapid genotyping of inherited fifth
complement component (C5)-deficient mice |
title_short | A simple PCR-based method for the rapid genotyping of inherited fifth
complement component (C5)-deficient mice |
title_sort | simple pcr-based method for the rapid genotyping of inherited fifth
complement component (c5)-deficient mice |
topic | Original |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547999/ https://www.ncbi.nlm.nih.gov/pubmed/25765875 http://dx.doi.org/10.1538/expanim.14-0085 |
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