In order to investigate the function of the KCNE3 subunit, we generated the kcne3 -/-mouse by homologous recombination in -/-mouse embryonic stem (ES) cells.
Figure 6.1A depicts the targeting vector that contained a DNA sequence of the initial intronic sequence and the first 3 exons of the KCNE3 gene, which are part of the 5’untranslated region (UTR), and exon 4, which contains the entire coding region for KCNE3 (Lundquist et al. 2006). Exon 4 was flanked with LoxP sites in order to allow its selective removal by using the Cre/LoxP-recombinase system (see below).
In addition, three selectable genes have been introduced in the targeting construct in order to select for the desired recombination product (Figure 5.1A): the neomycin resistance (neo) gene for positive selection, which allows cells to survive in the presence of the antibiotic neomycin, the diphtheria toxin (dta) gene and the herpes simplex virus type 1 Thymidine Kinase gene (tk). Dta and tk genes are used for “positive-negative”
selection in case of random integration of the targeting vector in the murine genome.
Those cells in which random integration has occurred retain the dta or the tk gene and thus will be specifically eliminated by expression of the dta protein or by the antiviral agent ganciclovir.
As seen in figure 5.1A, both the dta and the tk genes were placed outside the homologous region (5’ of the short arm and 3’ of the long arm of the vector, respectively). In this configuration, if homologous recombination occurs between the targeting construct and the native KCNE3 gene, both the dta and the tk genes will be excluded.
To exclude possible undesired effects due to the insertion of the neomycin cassette into the mouse genome, the neomycin resistance cassette was also flanked by 2 loxP sites to allow its removal by the Cre-recombinase.
ES cell clones that incorporated the recombined allele in the desired fashion were expected to be resistant to neomycin. To further test the specificity of the clones which survived positive selection, we employed a Southern blotting analysis of genomic DNA.
Following BglI enzymatic restriction digestion, genomic DNA was probed with the 5’
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Figure 5.1. Generation of kcne3-/- mice. A, strategy for disruption of the KCNE3 gene. Top, wild type allele showing exons 1-3 (UTR), 4 (CDS, Exon 4 contains the entire coding region) depicted as boxes and unique 12 kb Bgl1 restriction fragment. Middle upper, targeting vector. The neomycin cassette (neo) inserted for positive selection, introduced a new Bgl1 restriction site.
Diphteria toxin (dta) and Thymidine Kinase (tk) gene located outside the homologous region were used for negative selection. The neomycin cassette was flanked by loxP sites (◄). A third loxP site was inserted after exon 4. Middle lower, recombined allele after successful homologous recombination, Bgl1 enzymatic digestion produces a 6 and 8 Kb restriction fragments, detected in Southern blot analysis by the probes indicated as bold bars labeled as 5’ (detected 6 kb Bgl1 restriction fragment) or 3’ (detected 8 kb Bgl1 restriction fragment). Bottom, KO allele after Cre excision. The neocassette and exon 4 (CDS) were removed by cre recombinase leaving instead 1 loxP sites (◄). The 9,5 Kb BglI restriction fragment was detected with either 5’ or 3’ probe (bold bars) by Southern blot (see C). B, Southern blot analysis of embryonic stem cells showing the wild type band (12 kb) and the successfully recombined band detected with probe 3’ (8 kb). The positive clone was afterwards injected into a blastocoele. C, Southern blot analysis of tail DNA from WT (12 kb) and KO (9.5 kb) mice. D, PCR for routine genotyping showing the wild type and kcne3-/- bands .
or 3’ probes. As shown in figure 5.1B, the wild type allele was identified by a sharp band about 12 kb, whilst the recombinant allele band was about 6 kb for the 5’ probe or 8 kb for the 3’ probe.
Positive clones were injected into the blastocoele cavity of C57BL/6 embryos and transferred to a pseudo pregnant foster mother. Offspring showing more than 80%
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chimerism were bred to C57BL/6 females. Successful germ line transmission and hence transfer of the modified ES cell genome to their offspring, was determined by Southern blotting. Subsequently, the selected offspring was crossed with Cre-deleter mice (Schwenk et al. 1995) (in C57BL/6J background) to excise the neomycin selection cassette and the KCNE3 coding region (exon 4).
Downstream 3’ gene
LOX P
Exon 3 UTR …
Figure 5.2. Modified KCNE3 gene region sequence. The modified KCNE3 gene fragment was first amplified by PCR from the kcne3-/- mouse genome and then subjected to sequencing. The nucleotide sequence with the respective color codes are shown, corresponding to the 334 bp covering KCNE3 exon 3 downstream the 3’ end of the KCNE3 gene. As expected, in the kcne3-/- mouse, exon 4 (containing the whole coding region for KCNE3) has been removed, leaving instead a single loxP sequence (the 34 bp loxP sequence is highlighted).
Moreover, also neomycin cassette has been deleted from the recombined allele.
Cre-deleter mice ubiquitously express high levels of cre-recombinase, an enzyme that catalyzes site-specific recombination of floxed DNA segments, meaning that fragments between loxP sites in the same orientation will be deleted.
As a result, KCNE3 gene was permanently modified in the knock-out (KO) allele, leading to a constitutive deletion of kcne3 expression. As shown by Southern blotting analysis performed from homozygous offspring, KO allele was identified by a specific 9,5 kb after BgII digestion (Figure 5.1C). Afterwards, the presence of the KO allele was routinely checked by genotyping PCR (Figure 5.1D).
Further sequencing of the kcne3-/- genomic DNA confirmed that the Cre-recombinase excised both the neocassette and exon 4 (Figure 5.2), leaving instead a unique loxP site in the KO allele.
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Kcne3-/- mice resulting from interbreeding heterozygous animals were obtained at the predicted Mendelian frequency, were viable, superficially indistinguishable from their wild type littermates and showed no obvious phenotypic changes in the cage environment.