Instability of the insertional mutation in Cftr TgH(neoim)Hgu cystic fibrosis mouse model
© Charizopoulou et al; licensee BioMed Central Ltd. 2004
Received: 18 February 2004
Accepted: 21 April 2004
Published: 21 April 2004
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)HguCF mouse model we have generated using strict brother × sister mating two inbred CftrTgH(neoim)Hgumouse 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.
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)Hguat 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.
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.
Cystic fibrosis (CF) is a common and fatal recessive disease, which is caused by dysfunction of a chloride channel, termed the CF transmembrane conductance regulator (CFTR). Since the isolation of the murine homologue of the human CFTR gene on Chromosome 6  several mouse models have been created. These fall broadly into two different categories; those designed to mimic clinical human mutations such as the F508del [2–4], G551D  and G480C , and those with a disrupted Cft r gene resulting in either no or reduced production of CFTR. Although most mouse models share phenotypic characteristics, particularly, the most CF-like severe pathology is observed in the gastrointestinal tract, important variations in phenotype have been observed which may relate to the specific mutation and the genetic background of the targeted strain. Studies using Cftr knockout mice demonstrated differential severity of airway  and intestinal  disease. Candidate modulators for growth, airway and intestinal disease have been mapped to loci on chromosomes 1, 6, 7, 10 and 13 ; 1, 2, 10 and 17 ; 3 and 5 , respectively.
From the original CftrTgH(neoim)Hgumutant mouse generated using insertional mutagenesis in the Cftr exon 10  we have established two inbred CF strains CF/1- CftrTgH(neoim)Hguand CF/3- CftrTgH(neoim)Hguby strict brother sister mating. We have generated three inbred congenic strains by backcrossing the targeted mutation to three different inbred backgrounds C57BL/6, DBA/2J and BALB/c. To observe germline transmission of the mutation after each backcross and after the first incross to develop homozygous congenic strains, mice were genotyped using Southern Blot Hybridisation to indicate the transmission of the insertional vector pIV3.5H (Figure 1). Since Southern analysis is cumbersome and time consuming, we devised an alternative protocol for genotyping, whereby animals are differentiated at the Cftr locus by intragenic microsatellite genotypes tightly linked with the intron 9 and exon 10 of Cftr chosen for insertion mutagenesis in the CftrTgH(neoim)Hgumouse mutant.
Allele distribution between the strains. Consistent genotyping
Expected Southern and microsatellite genotypes for animals backcrossed to the three inbred backgrounds.
Excision of the pIV3.5H vector
Genotypes of the animals with an excised vector. Mouse A in one chromosome, Mouse B in both chromosomes.
Primer sequences used for the amplification of the long range products.
Primer sequence 5'-3'
CCT TCC ATG TAC CCC TCC TCA CTT
CCC GGC ATA ATC CAA GAA AAT TG
TGT GGG AAA TCC TGT GCT GAA A
CTT CCG GCT CGT ATG TTG TGT T
CAC ACA ACA TAC GAG CCG GAA G
TTT ATT GCC GAT CCC CTC AGA A
CTC GTG CTT TAC GGT ATC GCC
TGC TGT AGT TGG CAA GCT TTG A
Primer sequences used for primer walking spanning the entire region from Cftr exon 9 to Cftr intron 10. Location based on the Genome Database Cftr sequence (AF 162137)
TTT GGG GAA TTA CTG GAG AAA G
AGC TCG CTG ATA GGT TAT CCA
CCC CTC CTC ACT TCC ATT AAA
TTT AAG GCT CAG GGC TAA TTG
TTC CAC AAT TAG CCC TGA GC
TGA AGG AAA TCA TTA CTG AAG CA
TGC TTC AGT AAT GAT TTC CTT CA
TAT GGA TCC CCA CAG CAA GT
CTC AGG GAT TGT CAC GGT TT
GCT TTG ATC TCT GGG AGC AC
GAT CAC AGG AGC CTA GCA TAG A
TTC ACT TTA CAT CCT GGC TTC A
ACT GGG AGA GGA TGC AAA AA
CCC AGT GTG AGA AGA TGC AC
TGC TCC CAG AAA TCT TCA CC
AGT TGT CAG AAG GGA ACC CA
TGG GTT CCC TTC TGA CAA CT
TTA GGT CCC CGT GCT TAC AC
TAG GTG GAT CCA TAA CCC CA
GGA CAG AGA AGC AGG AGT GG
CCA CTC CTG CTT CTC TGT CC
AAA GAA GAG CGA GCC CCT AC
CCA TAG CCC AAG AGC TTT CA
GTA CCC GGC ATA ATC CAA GA
TTC TTG GAT TAT GCC GGG TA
TTT CCA GTT GGG GGT ACA CT
GGG CTT CAA GGC CTA ATT CT
ATG TGA TCC AGA CTG GCC TA
ATG CAT GGG GTG TGG TAC TT
TCC AAT GAT CTA CCT GTG TCC A
Genetic analysis of complex human diseases such as cystic fibrosis has been successfully supported by the use of various mouse models. In order to dissect the role of the different induced mutations to the murine Cftr gene used from the genetic background, the genomic section carrying the mutation is transferred by repeated backcross cycles to another defined inbred background (introgressing), creating congenic strains. We have generated three congenic CftrTgH(neoim)Hgustrains by crossing the mutant animals to the three inbred backgrounds BALB/c, C57BL/6, DBA/2J. In each generation germline transmission of the disrupted Cftr locus was monitored using Southern Blot Hybridisation . In order to observe germline transmission of the disrupted Cftr locus we have established an alternative 'high-throughput' genotyping protocol using Cftr intragenic microsatellites, which enabled us to identify animals carrying the insertional mutation based upon the different haplotypic backgrounds of the three inbred strains and the mutant CF/3- CftrTgH(neoim)Hguinbred line at the Cftr locus.
The present study is to the best of our knowledge, the first deliberate search for polymorphic intragenic Cftr markers for the establishment of Cftr haplotypic backgrounds of wild type inbred mouse strains. It has been shown that some of the more common polymorphisms in the human CFTR gene have consequences at the functional level. The presence of an allele at a particular locus can determine the proportion of transcripts from which functional CFTR protein can be translated affecting CFTR maturation and the net chloride transport activity of CFTR-expressing cells . Although it remains to be proven whether intragenic changes can account for phenotypic variability in disease expression among mice with different Cftr background carrying the same mutation, it can not be excluded that they may have a potential effect on the severity of the CF phenotype by several mechanisms.
In our study the determination of the Cftr haplotypic backgroung provided a useful tool for the identification of mutant animals. Using this protocol we have successfully verified the genotype of 55 out of 57 animals bred to the three inbred backgrounds, previously genotyped by Southern blot hybridisation using the 1.2H probe.
Mouse A and B littermate genotypes.
The mechanism responsible for this excision repair event must be independent from the mismatch repair (MMR) and nucleotide exchange repair (NER) pathways, since the size of the vector overexceeds the maximum of mismatched nucleotides they can efficiently repair [16–18]. The mechanism involved in the excision of the vector and the subsequent restoration of the mutated Cftr locus to wildtype can not be gene conversion as seen in other organisms [19, 20], because the genetic background is conserved. If the mechanism involved large loop repair by incorporating the vector in a heteroduplex there must be a novel mechanism, which is independent of gene conversion-restoration events.
O type sequence insertion vectors  such as the pIV3.5H, contain an uninterrupted stretch of target- homology with exonic sequence that results in duplication of a large stretch of sequence flanking the heterologous sequence of the plasmid resembling transposable elements, flanked by large direct repeats. Reports  on precise excision events of transposable elements without leaving a footprint involve an alternative mechanism of repair rather than gene conversion which is dependent on length of the repeat flanking the element. It is therefore highly likely that a similar mechanism is responsible for the precise excision of the pIV3.5H insertion vector.
This is the first report where an O type vector used in order to generate insertion mutagenesis in the mouse, has been excised. Such events probably remained unnoticed because most of the methods used in order to identify animals which carry the targeted locus base their detection almost exclusively on the presence or absence of the inserted sequence, without taking into consideration the genetic background of the mouse strain adjacent to the insertion, therefore an excision event would not be easily identified. Unlike Southern hybridisation the genotyping protocol that we propose in this study does not indicate the presence of the insertion vector directly based on the presence of its sequence in the disrupted locus, but manages to discriminate insertional mutant animals from the haplotypes associated with the disrupted locus in the Cftr gene. In our study the haplotypes obtained from the three informative intragenic Cftr microsatellites were differential to the haplotypes associated with the insertional mutant mouse, allowing identification of excision events.
Microsatellite markers spanning the mouse genome have been used for the enhancement of congenic breeding, reducing the time to 18–24 months (speed congenics) from an initial 2.5–3 year period [23, 24]. Here we describe the use of Cftr intagenic markers which allowed fast and efficient identification of the differential locus during backcrossing. Moreover, this method provided a useful tool whereby unexpected events such as vector excision from the disrupted Cftr locus have been revealed posing questions for the stability of insertional mutants generated by this strategy. Furthermore, given our observations that different haplotypic backgrounds were found between the inbred strains raises questions on whether alleles at polymorphic loci can affect cftr at the transcript and/or protein level and whether it would be beneficial to study Cftr induced mutations on the respective haplotypic background of the individual strains.
All experiments were approved by the local Institutional Animal Care and Research Advisory Committee as well as by the local government. CftrTgH(neoim)Hgumice were bred under specified pathogen-free conditions in the isolator unit of the Central Laboratory Animal Facility of the Hannover Medical School. Mice were kept in a flexible film isolator. The temperature within the isolator was maintained at 20–24°C with 40–50% relative humidity. Animals were fed an irradiated (50 kGy) standard chow (Altromin 1314) and autoclaved water (134°C for 50 min) ad libitum.
Generation of inbred CftrTgH(neoim)Hgumutant mice
For the establishment of the inbred CF/1-CftrTgH(neoim)Hguand CF/3- CftrTgH(neoim)Hgupopulation, one pair with divergent genetic background (generation F4) of one homozygous male and one homozygous female was obtained from the MRC Human Genetics Unit, Edinburgh. Two separate litters were obtained and two animals of each litter became the starting population for the establishment of the two individual inbred CftrTgH(neoim)Hgulines CF/1- CftrTgH(neoim)Hguand CF/3- CftrTgH(neoim)Hguwhich were generated by brother-sister mating for now more than 26 generations.
Generation of congenic CftrTgH(neoim)Hgumutant mice
CF/3- CftrTgH(neoim)Hgumice served as donors for the development of the three congenic strains C57BL/6, BALB/c and DBA/2J, with selection for CftrTgH(neoim)Hgufor 10 generations. Genotyping of the insertion mutation was conducted by Southern analysis of Xba I/Sal I restricted genomic DNA from spleen .
High molecular weight DNA was isolated from 0.15 g spleen tissue, either fresh or thawed on ice after storage at -20°C based on the protocol by Gross-Bellard et al. .
Southern blot genotyping
Heterozygous and homozygous CftrTgH(neoim)Hguanimals were identified in each backcross generation via Southern Blot Hybridization of Xba I/Sal I genomic digests, using the 1.2H probe located in the Cftr intron 10, after double digestion with Xba I-Sal I (Figure 1). There are no Sal I sites in this region of the Cftr gene, but the targeting vector pIV3.5H carries a unique Sal I site immediately 3' to the neo gene. Animals carrying the mutation were identified by the novel 5 kb Xba I-Sal I fragment hybridizing to 1.2H.
Primer sequences used for the amplification of the intragenic Cftr microsatellites. The forward primer is 5'biotinylated.
Primer sequence 5'-3'
BIOTIN-TGC TTG AGC TAT CCA TTC TGA
TAC CCA ATG TTG CCA TCT GA
BIOTIN-TTG GAA GTG AGG ATT GCC TT
TGC CTC AGT CTC ATA TTA TTG C
BIOTIN-TCT CAG CCT GTC TTC CTC TCA
TCC TCC CAA AAC AGC TTC AC
BIOTIN-GAG TTG GAG AGG CTG TTT GG
TGT GCC AGG ACA CTG TGA CT
BIOTIN-TTC AAA TGA CCA AAA TCC CC
TGG CAA ATT TTC AAC AAC AAA
Genotyping of microsatellites
Microsatellite markers were genotyped in 96 well plates purchased from Greiner, Frickenhausen, pre-coated with 50 ng DNA per well in a Hybaid Thermocycler (Hybaid, Teddington) with a heated lid. One of the two primers per microsatellite was 5'-terminal biotinylated. PCR was performed in a total volume of 30 μl, without oil overlay, using InViTaq polymerase (InViTek, Berlin). After PCR an 8 μl aliquot was transferred to a multiwell plate and allowed to dry overnight at 37°C, dissolved in 10 μl loading buffer (0.2% w/v xylenecyanol and bromphenolblue in formamide) and denatured for 5 min at 95°C. The PCR products were separated by direct blotting electrophoresis (GATC 1500, MWG Biotech, Ebersberg, Germany) on a denaturing acrylamide gel (4% acrylamide/N,N'-methylenebisacrylamide 29:1 containing 6 M urea in 0.9 M Tris-0.9 M boric acid-0.02 M EDTA buffer) and simultaneously transferred to a Hybond N+membrane (Amersham). Signals were visualised by blocking the membrane in 1.5%(w/v) of blocking reagent in Buffer 1 (100 mM Tris-HCl, 150 mM NaCl, pH 7.5), followed by incubation in diluted solution of anti-biotin alkaline phosphatase conjugate in Buffer 1. The membrane was further washed three times with 1% Triton X-100 in Buffer 1 and equilibrated for 15 min in assay buffer (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5). The membrane was covered for 5 min with reaction buffer containing 10%(v/v) Sapphire II (Tropix) and 60 μl CDPstar (Tropix)in 50 ml assay buffer, followed by rinsing with a solution containing 1% v/v Sapphire II and 6 μl CDPstar in 50 ml assay buffer. Signals were exposed to Kodak XA-R films and the exposition time varied from 10 min to 45 min. Evaluation of results was performed as described by Mekus et al. .
150 ng of DNA template was each amplified in 12 different premixes using the Failsafe ™ PCR System (EPICENTRE Technologies, WI USA). PCR products were amplified using primers described in Table 3 separated by 1% agarose gel electrophoresis and visualised under UV illumination, the optimal reaction mixture was thereafter chosen for further amplifications (Figure 3).
Primer sequences used for the amplification of the Cftr intron 9-pMC1 vector plasmid sequence (CFneo2) and the neomycin-Cftr intron 9 (CFneo1) products.
Primer sequence 5'-3'
CGT TGG CTA CCC GTG ATA TT
CTT CCA CAA GGC TTC CTG AG
CCT GAT GTT GAT TTT GGG AGA
ATT AAT GCA GCT GGC ACG AC
Excision scanning by primer walking
Based on the Genome Database Cftr sequence (AF162137) 15 overlapping pairs (Table 4) of primers spanning the entire region from exon 9 to intron 10 of the murine Cftr gene were designed, using the Primer 3 oligo design program http://frodo.wi.mit.edu. PCR reactions were performed on DNA with inconsistent Southern and microsatellite insertional mutation genotypes and controls in 96 well plates precoated with 50 ng of DNA template using InViTaq polymerase (InViTek, Berlin). Full length products were separated on 2.5% agarose gels and visualised under UV illumination.
Following PCR amplification the chosen PCR products were sequenced by Qiagen GmbH.
We thank Petra Adomat and Harry Dettmering for excellent animal care and Margit Ritzka for experimental advice. We thank David J. Porteous for thorough discussions during the initial stage of the project. Financial support by the Deutsche Forschungsgemeinschaft to H.-J. H. and B.T. (HE 1058/3-2) is gratefully acknowledged.
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