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FBXW7 mutations typically found in human cancers are distinct from alleles and disrupt lung development.

Davis H, Lewis A, Spencer-Dene B, Tateossian H, Stamp G, Behrens A, Tomlinson I - J. Pathol. (2011)

Bottom Line: In most cases, these mutations do not inactivate the protein, but are mono-allelic missense changes at specific arginine resides involved in substrate binding.Fbxw7(-/-) animals died of vascular abnormalities at E10.5.Fbxw7(R482Q) alleles are not functionally equivalent to heterozygous or homozygous alleles, and we propose that they are selected in tumourigenesis because they cause a selective or partial loss of FBXW7 function.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford OX3 7BN, UK.

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Related in: MedlinePlus

Generation of Fbxw7 mouse model. (A) Schematic diagram of targeting scheme to generate Fbxw7 mouse model. The upper panel shows the targeting construct with regions of homology; loxP sites; genomic Fbxw7 exons 9, 10, 11; a neomycin selection cassette; and repeated exons 9, 10, 11 in which a mutation in exon 9 has been introduced by site-directed mutagenesis. The lower panel represents the allele after Cre-mediated recombination. The arrows with corresponding numbers depict the position and direction of the primers used to genotype the R482Q mice. (B) Long-range PCR genotyping of electroporated ES cell clones showing clones with positive 5′ (A10, F12, H3) and 3′ (A10, F12) integration. The band sizes of the 5′ and 3′ PCR products are 5371 and 5533 base pairs, respectively. (C) Southern blot screening showing wild-type and targeted bands (see the Materials and methods section). (D) Genotyping PCR screening of R482Q mice. The left panel shows the PCR product using primer pair 1 and 3 to identify the R482Q and wild-type alleles. The right panel shows the PCR product using primer pair 1 and 6 to identify the R482Q allele. (E) Sequence traces showing heterozygous point mutation in R482Q mice
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fig02: Generation of Fbxw7 mouse model. (A) Schematic diagram of targeting scheme to generate Fbxw7 mouse model. The upper panel shows the targeting construct with regions of homology; loxP sites; genomic Fbxw7 exons 9, 10, 11; a neomycin selection cassette; and repeated exons 9, 10, 11 in which a mutation in exon 9 has been introduced by site-directed mutagenesis. The lower panel represents the allele after Cre-mediated recombination. The arrows with corresponding numbers depict the position and direction of the primers used to genotype the R482Q mice. (B) Long-range PCR genotyping of electroporated ES cell clones showing clones with positive 5′ (A10, F12, H3) and 3′ (A10, F12) integration. The band sizes of the 5′ and 3′ PCR products are 5371 and 5533 base pairs, respectively. (C) Southern blot screening showing wild-type and targeted bands (see the Materials and methods section). (D) Genotyping PCR screening of R482Q mice. The left panel shows the PCR product using primer pair 1 and 3 to identify the R482Q and wild-type alleles. The right panel shows the PCR product using primer pair 1 and 6 to identify the R482Q allele. (E) Sequence traces showing heterozygous point mutation in R482Q mice

Mentions: Figure 2A shows the targeting construct used to generate our Fbxw7-mutant mouse. The targeting construct contained a loxP site upstream of exon 9 and two other loxP sites, flanking a Neo cassette, downstream of exon 11. On the other side of the distal loxP, we inserted a repeated genomic region containing exons 9–11, but with an R482Q allele created by site-directed mutagenesis within exon 9. We anticipated that Cre-mediated recombination would generally remove the genomic sequence between the most proximal and distal loxP sites, removing the NeoR cassette and endogenous exons 9–11 and allowing the transcription of the ‘knock-in’ mutated exon 9; in all the mice subsequently reported in this study, recombination had occurred between these outer loxP sites.


FBXW7 mutations typically found in human cancers are distinct from alleles and disrupt lung development.

Davis H, Lewis A, Spencer-Dene B, Tateossian H, Stamp G, Behrens A, Tomlinson I - J. Pathol. (2011)

Generation of Fbxw7 mouse model. (A) Schematic diagram of targeting scheme to generate Fbxw7 mouse model. The upper panel shows the targeting construct with regions of homology; loxP sites; genomic Fbxw7 exons 9, 10, 11; a neomycin selection cassette; and repeated exons 9, 10, 11 in which a mutation in exon 9 has been introduced by site-directed mutagenesis. The lower panel represents the allele after Cre-mediated recombination. The arrows with corresponding numbers depict the position and direction of the primers used to genotype the R482Q mice. (B) Long-range PCR genotyping of electroporated ES cell clones showing clones with positive 5′ (A10, F12, H3) and 3′ (A10, F12) integration. The band sizes of the 5′ and 3′ PCR products are 5371 and 5533 base pairs, respectively. (C) Southern blot screening showing wild-type and targeted bands (see the Materials and methods section). (D) Genotyping PCR screening of R482Q mice. The left panel shows the PCR product using primer pair 1 and 3 to identify the R482Q and wild-type alleles. The right panel shows the PCR product using primer pair 1 and 6 to identify the R482Q allele. (E) Sequence traces showing heterozygous point mutation in R482Q mice
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757315&req=5

fig02: Generation of Fbxw7 mouse model. (A) Schematic diagram of targeting scheme to generate Fbxw7 mouse model. The upper panel shows the targeting construct with regions of homology; loxP sites; genomic Fbxw7 exons 9, 10, 11; a neomycin selection cassette; and repeated exons 9, 10, 11 in which a mutation in exon 9 has been introduced by site-directed mutagenesis. The lower panel represents the allele after Cre-mediated recombination. The arrows with corresponding numbers depict the position and direction of the primers used to genotype the R482Q mice. (B) Long-range PCR genotyping of electroporated ES cell clones showing clones with positive 5′ (A10, F12, H3) and 3′ (A10, F12) integration. The band sizes of the 5′ and 3′ PCR products are 5371 and 5533 base pairs, respectively. (C) Southern blot screening showing wild-type and targeted bands (see the Materials and methods section). (D) Genotyping PCR screening of R482Q mice. The left panel shows the PCR product using primer pair 1 and 3 to identify the R482Q and wild-type alleles. The right panel shows the PCR product using primer pair 1 and 6 to identify the R482Q allele. (E) Sequence traces showing heterozygous point mutation in R482Q mice
Mentions: Figure 2A shows the targeting construct used to generate our Fbxw7-mutant mouse. The targeting construct contained a loxP site upstream of exon 9 and two other loxP sites, flanking a Neo cassette, downstream of exon 11. On the other side of the distal loxP, we inserted a repeated genomic region containing exons 9–11, but with an R482Q allele created by site-directed mutagenesis within exon 9. We anticipated that Cre-mediated recombination would generally remove the genomic sequence between the most proximal and distal loxP sites, removing the NeoR cassette and endogenous exons 9–11 and allowing the transcription of the ‘knock-in’ mutated exon 9; in all the mice subsequently reported in this study, recombination had occurred between these outer loxP sites.

Bottom Line: In most cases, these mutations do not inactivate the protein, but are mono-allelic missense changes at specific arginine resides involved in substrate binding.Fbxw7(-/-) animals died of vascular abnormalities at E10.5.Fbxw7(R482Q) alleles are not functionally equivalent to heterozygous or homozygous alleles, and we propose that they are selected in tumourigenesis because they cause a selective or partial loss of FBXW7 function.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford OX3 7BN, UK.

Show MeSH
Related in: MedlinePlus