Instability of the insertional mutation in Cftr(TgH(neoim)Hgu )cystic fibrosis mouse model

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 Cftr(TgH(neoim)Hgu )CF mouse model we have generated using strict brother × sister mating two inbred Cftr(TgH(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 Cftr(TgH(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

CFTR mutations of patients with cystic fibrosis from Republic of Moldova

Departamentul de pediatrie, clinica de pneumologie, Universitatea de Stat de Medicină și Farmacie „Nicolae Testemiţanu”, Chișinău, Republica Moldova, Universitäts-Kinderklinik, Johann-Wolfgang-Goethe Universität, Frankfurt am Main, Germania, Service de génétique médicale, CHU de Bordeaux, Groupe Hospitalier Pellegrin, Bordeaux, Franţa, Klinische Forschergruppe‚ Molekulare Pathologie der Mukoviszidose, Klinik für Pädiatrische Pneumologie, Allergologie und Neonatologie, Medizinische Hochschule Hannover, Germania, ViennaLab Diagnostics GmbH, Wien, AustriaIntroducere. Spectrul de mutaţii ce cauzează fibroză chistică (FC) în Republica Moldova, deocamdată, nu a fost determinat. Material și metode. Analiza mutaţiei genei CFTR a fost efectuată pe mostre de ADN, colectate de la 61 de pacienţi cu FC, examinaţi în Departamentul de pediatrie, clinica de pneumologie, Universitatea de Stat de Medicină și Farmacie „Nicolae Testemiţanu”, în perioada anilor 2006-2011.Rezultate. Mutaţiile cu o frecvenţă mai mare de 3% au fost următoarele: F508del (57%), 2789+5 G-A (4%), 2184insA (4%) și G542X (3%). Concluzii. Distribuţia mutaţiei genei CFTR în Moldova este similară cu cea din România.Introduction. The spectrum of cystic fibrosis (CF) – cau-sing mutations in Republic of Moldova has yet not been determined. Material and methods. CFTR gene mutation analysis was performed on DNA samples from 61 patients with CF seen 2006-2011 at the Department of pediatrics, Division of pneumology, Nicolae Testemitanu State Medical and Pharmaceutical University. Results. Mutations with a frequency of more than 3% were F508del (57%), 2789+5 G-A (4%), 2184insA (4 %) and G542X (3%). Conclusions. The distribution of CFTR gene mutations in Moldova is similar to that in Romania.https://stiinta.usmf.md/sites/default/files/2018-09/MJHS%20nr.%203%20_2015.pd

Spontaneous rescue from cystic fibrosis in a mouse model

BACKGROUND: From the original Cftr(TgH(neoim)Hgu )mutant mouse model with a divergent genetic background (129P2, C57BL/6, MF1) we have generated two inbred Cftr(TgH(neoim)Hgu )mutant strains named CF/1-Cftr(TgH(neoim)Hgu )and CF/3-Cftr(TgH(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. RESULTS: Reduced 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 Cftr(TgH(neoim)Hgu )strains, with higher residual amount observed for CF/1-Cftr(TgH(neoim)Hgu )than CF/3-Cftr(TgH(neoim)Hgu )for every investigated tissue. Accordingly the amounts of wild type Cftr protein in the intestine were 9% for CF/1-Cftr(TgH(neoim)Hgu )and 4% for CF/3-Cftr(TgH(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. CONCLUSION: Unlike the outbred Cftr(TgH(neoim)Hgu )insertional mouse model, which displayed the electrophysiological defect in the gastrointestinal and respiratory tracts characteristic of cystic fibrosis, both inbred Cftr(TgH(neoim)Hgu )strains have ameliorated the electrophysiological defect. On the basis of these findings both CF/1-Cftr(TgH(neoim)Hgu )and CF/3-Cftr(TgH(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

Very mild disease phenotype of congenic CftrTgH(neoim)Hgu cystic fibrosis mice

Background: A major boost to 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)Hgumouse model with a divergent genetic background (129/Sv, C57BL/6, HsdOla:MF1) two inbred mutant mouse strains CF/1-CftrTgH(neoim)Hguand CF/3-CftrTgH(neoim)Hguhad been generated using strict brother × sister mating. CF/1-CftrTgH(neoim)Hguand CF/3-CftrTgH(neoim)Hgumice were fertile and showed normal growth and lifespan. In this work the CftrTgH(neoim)Hguinsertional mutation was backcrossed from CF/3-CftrTgH(neoim)Hguonto the inbred backgrounds C57BL/6J and DBA/2J generating congenic animals in order to clarify the differential impact of the Cftr mutation and the genetic background on the disease phenotype of the cystic fibrosis mutant mice. Clinical and electrophysiological features of the two congenic strains were compared with those of CF/1-CftrTgH(neoim)Hguand CF/3-CftrTgH(neoim)Hguand wild type controls. Results: Under the standardized housing conditions of the animal facility, the four mouse strains CF/1-CftrTgH(neoim)Hgu, CF/3-CftrTgH(neoim)Hgu, D2.129P2(CF/3)-CftrTgH(neoim)Hguand B6.129P2(CF/3)-CftrTgH(neoim)Hguexhibited normal life expectancy. Growth of congenic cystic fibrosis mice was comparable with that of wild type controls. All mice but D2.129P2(CF/3)-CftrTgH(neoim)Hgufemales were fertile. Short circuit current measurements revealed characteristic response profiles of the HsdOla:MF1, DBA/2J and C57BL/6J backgrounds in nose, ileum and colon. All cystic fibrosis mouse lines showed the disease-typical hyperresponsiveness to amiloride in the respiratory epithelium. The mean chloride secretory responses to carbachol or forskolin were 15-100% of those of the cognate wild type control animals. Conclusion: The amelioration of the clinical features and of the basic defect that had emerged during the generation of CF/3-CftrTgH(neoim)Hgumice was retained in the congenic mice indicating that the Cftr linkage group or other loci shared between the inbred strains contain(s) the major modifier(s) of attenuation of cystic fibrosis symptoms

Determination of nutrient salts by automatic methods both in seawater and brackish water: the phosphate blank

9 páginas, 2 tablas, 2 figurasThe main inconvenience in determining nutrients in seawater by automatic methods is simply solved: the preparation of a suitable blank which corrects the effect of the refractive index change on the recorded signal. Two procedures are proposed, one physical (a simple equation to estimate the effect) and the other chemical (removal of the dissolved phosphorus with ferric hydroxide).Support for this work came from CICYT (MAR88-0245 project) and Conselleria de Pesca de la Xunta de GaliciaPeer reviewe