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Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model.

ABSTRACT: BACKGROUND: 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 CftrTgH(neoim)Hgu CF mouse model we have generated using strict brother x sister mating two inbred CftrTgH(neoim)Hgu 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 Cftr locus by Cftr intragenic microsatellite genotypes that are tightly linked to the disrupted locus. RESULTS: 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 CftrTgH(neoim)Hgu at the Cftr locus. Moreover, this method facilitated the identification of the precise vector excision from the disrupted Cftr 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. CONCLUSIONS: 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.

SUBMITTER: Charizopoulou N

PROVIDER: S-EPMC419339 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model.

Charizopoulou Nikoletta N Jansen Silke S Dorsch Martina M Stanke Frauke F Dorin Julia R JR Hedrich Hans-Jürgen HJ Tümmler Burkhard B

BMC genetics 20040421

<h4>Background</h4>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 CftrTgH(neoim)Hgu CF mouse model we have generated using strict brother x sister mating two inbred CftrTgH(neoim)Hgu mouse lines (CF/1 and CF/3). Thereafter, the in ...[more]

PMID: 15102331

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Project description:BackgroundFrom the original CftrTgH(neoim)Hgu mutant mouse model with a divergent genetic background (129P2, C57BL/6, MF1) we have generated two inbred CftrTgH(neoim)Hgu mutant strains named CF/1-CftrTgH(neoim)Hgu and CF/3-CftrTgH(neoim)Hgu, which are fertile and show normal growth and lifespan. Initial genome wide scan analysis with microsatellite markers indicated that the two inbred strains differed on the genetic level. In order to further investigate whether these genetic differences have an impact on the disease phenotype of cystic fibrosis we characterised the phenotype of the two inbred strains.ResultsReduced amounts, compared to wild type control animals, of correctly spliced Cftr mRNA were detected in the nasal epithelia, lungs and the intestine of both inbred CftrTgH(neoim)Hgu strains, with higher residual amount observed for CF/1-CftrTgH(neoim)Hgu than CF/3-CftrTgH(neoim)Hgu for every investigated tissue. Accordingly the amounts of wild type Cftr protein in the intestine were 9% for CF/1-CftrTgH(neoim)Hgu and 4% for CF/3-CftrTgH(neoim)Hgu. Unlike the apparent strain and/or tissue specific regulation of Cftr mRNA splicing, short circuit current measurements in the respiratory and intestinal epithelium revealed that both strains have ameliorated the basic defect of cystic fibrosis with a presentation of a normal electrophysiology in both tissues.ConclusionUnlike the outbred CftrTgH(neoim)Hgu insertional mouse model, which displayed the electrophysiological defect in the gastrointestinal and respiratory tracts characteristic of cystic fibrosis, both inbred CftrTgH(neoim)Hgu strains have ameliorated the electrophysiological defect. On the basis of these findings both CF/1-CftrTgH(neoim)Hgu and CF/3-CftrTgH(neoim)Hgu offer an excellent model whereby determination of the minimal levels of protein required for the restoration of the basic defect of cystic fibrosis can be studied, along with the modulating factors which may affect this outcome.

Project description:BackgroundCystic fibrosis (CF) is caused by mutations in the CFTR gene that impair the function of CFTR, a cAMP-regulated anion channel. In the small intestine loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have small intestinal bacterial overgrowth, an altered innate immune response, and impaired intestinal transit. We investigated whether lubiprostone, which can activate the CLC2 Cl- channel, would improve the intestinal phenotype in CF mice.MethodsCftr(tm1UNC) (CF) and wildtype (WT) littermate mice on the C57BL/6J background were used. Lubiprostone (10 μg/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in periodic acid-Schiff base, Alcian blue stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit in fasted mice were assessed using gavaged rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray and qRT-PCR.ResultsCrypt width in control CF mice was 700% that of WT mice (P < 0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P = 0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P = 0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P = 0.005). Lubiprostone increased gastric emptying at 20 min postgavage in both WT (P < 0.001) and CF mice (P < 0.001). Lubiprostone enhanced small intestinal transit in WT mice (P = 0.024) but not in CF mice (P = 0.377). Among other innate immune markers, expression of mast cell genes was elevated 4-to 40-fold in the CF intestine as compared to WT, and lubiprostone treatment of CF mice decreased expression to WT control levels.ConclusionsThese results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion.

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Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model.

Publications

Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model.

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