Instability of the insertional mutation in <it>Cftr</it><sup><it>TgH(neoim)Hgu </it></sup>cystic fibrosis mouse model
<p>Abstract</p> <p>Background</p> <p>A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains gener...
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| Format: | Article |
| Language: | English |
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BMC
2004-04-01
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| Series: | BMC Genetics |
| Online Access: | http://www.biomedcentral.com/1471-2156/5/6 |
| _version_ | 1817971069227630592 |
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| author | Dorin Julia R Stanke Frauke Dorsch Martina Jansen Silke Charizopoulou Nikoletta Hedrich Hans-Jürgen Tümmler Burkhard |
| author_facet | Dorin Julia R Stanke Frauke Dorsch Martina Jansen Silke Charizopoulou Nikoletta Hedrich Hans-Jürgen Tümmler Burkhard |
| author_sort | Dorin Julia R |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original <it>Cftr</it><sup><it>TgH</it>(<it>neoim</it>)<it>Hgu </it></sup>CF mouse model we have generated using strict brother × sister mating two inbred <it>Cftr</it><sup><it>TgH</it>(<it>neoim</it>)<it>Hgu </it></sup>mouse lines (CF/1 and CF/3). Thereafter, the insertional mutation was introgressed from CF/3 into three inbred backgrounds (C57BL/6, BALB/c, DBA/2J) generating congenic animals. In every backcross cycle germline transmission of the insertional mutation was monitored by direct probing the insertion via Southern RFLP. In order to bypass this time consuming procedure we devised an alternative PCR based protocol whereby mouse strains are differentiated at the <it>Cftr </it>locus by <it>Cftr </it>intragenic microsatellite genotypes that are tightly linked to the disrupted locus.</p> <p>Results</p> <p>Using this method we were able to identify animals carrying the insertional mutation based upon the differential haplotypic backgrounds of the three inbred strains and the mutant <it>Cftr</it><sup><it>TgH</it>(<it>neoim</it>)<it>Hgu </it></sup>at the <it>Cftr </it>locus. Moreover, this method facilitated the identification of the precise vector excision from the disrupted <it>Cftr </it>locus in two out of 57 typed animals. This reversion to wild type status took place without any loss of sequence revealing the instability of insertional mutations during the production of congenic animals.</p> <p>Conclusions</p> <p>We present intragenic microsatellite markers as a tool for fast and efficient identification of the introgressed locus of interest in the recipient strain during congenic animal breeding. Moreover, the same genotyping method allowed the identification of a vector excision event, posing questions on the stability of insertional mutations in mice.</p> |
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| id | doaj.art-e912b2f19c4b4e7b8e78559295bcc970 |
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| language | English |
| last_indexed | 2024-04-13T20:42:05Z |
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| spelling | doaj.art-e912b2f19c4b4e7b8e78559295bcc9702022-12-22T02:30:49ZengBMCBMC Genetics1471-21562004-04-0151610.1186/1471-2156-5-6Instability of the insertional mutation in <it>Cftr</it><sup><it>TgH(neoim)Hgu </it></sup>cystic fibrosis mouse modelDorin Julia RStanke FraukeDorsch MartinaJansen SilkeCharizopoulou NikolettaHedrich Hans-JürgenTümmler Burkhard<p>Abstract</p> <p>Background</p> <p>A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original <it>Cftr</it><sup><it>TgH</it>(<it>neoim</it>)<it>Hgu </it></sup>CF mouse model we have generated using strict brother × sister mating two inbred <it>Cftr</it><sup><it>TgH</it>(<it>neoim</it>)<it>Hgu </it></sup>mouse lines (CF/1 and CF/3). Thereafter, the insertional mutation was introgressed from CF/3 into three inbred backgrounds (C57BL/6, BALB/c, DBA/2J) generating congenic animals. In every backcross cycle germline transmission of the insertional mutation was monitored by direct probing the insertion via Southern RFLP. In order to bypass this time consuming procedure we devised an alternative PCR based protocol whereby mouse strains are differentiated at the <it>Cftr </it>locus by <it>Cftr </it>intragenic microsatellite genotypes that are tightly linked to the disrupted locus.</p> <p>Results</p> <p>Using this method we were able to identify animals carrying the insertional mutation based upon the differential haplotypic backgrounds of the three inbred strains and the mutant <it>Cftr</it><sup><it>TgH</it>(<it>neoim</it>)<it>Hgu </it></sup>at the <it>Cftr </it>locus. Moreover, this method facilitated the identification of the precise vector excision from the disrupted <it>Cftr </it>locus in two out of 57 typed animals. This reversion to wild type status took place without any loss of sequence revealing the instability of insertional mutations during the production of congenic animals.</p> <p>Conclusions</p> <p>We present intragenic microsatellite markers as a tool for fast and efficient identification of the introgressed locus of interest in the recipient strain during congenic animal breeding. Moreover, the same genotyping method allowed the identification of a vector excision event, posing questions on the stability of insertional mutations in mice.</p>http://www.biomedcentral.com/1471-2156/5/6 |
| spellingShingle | Dorin Julia R Stanke Frauke Dorsch Martina Jansen Silke Charizopoulou Nikoletta Hedrich Hans-Jürgen Tümmler Burkhard Instability of the insertional mutation in <it>Cftr</it><sup><it>TgH(neoim)Hgu </it></sup>cystic fibrosis mouse model BMC Genetics |
| title | Instability of the insertional mutation in <it>Cftr</it><sup><it>TgH(neoim)Hgu </it></sup>cystic fibrosis mouse model |
| title_full | Instability of the insertional mutation in <it>Cftr</it><sup><it>TgH(neoim)Hgu </it></sup>cystic fibrosis mouse model |
| title_fullStr | Instability of the insertional mutation in <it>Cftr</it><sup><it>TgH(neoim)Hgu </it></sup>cystic fibrosis mouse model |
| title_full_unstemmed | Instability of the insertional mutation in <it>Cftr</it><sup><it>TgH(neoim)Hgu </it></sup>cystic fibrosis mouse model |
| title_short | Instability of the insertional mutation in <it>Cftr</it><sup><it>TgH(neoim)Hgu </it></sup>cystic fibrosis mouse model |
| title_sort | instability of the insertional mutation in it cftr it sup it tgh neoim hgu it sup cystic fibrosis mouse model |
| url | http://www.biomedcentral.com/1471-2156/5/6 |
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