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Maternal transmission of a humanised Igf2r allele results in an Igf2 dependent hypomorphic and non-viable growth phenotype.

Hughes J, Frago S, Bühnemann C, Carter EJ, Hassan AB - PLoS ONE (2013)

Bottom Line: Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality.In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r.This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Tumour Growth Group, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The cation independent mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) functions in the transportation and regulation of insulin-like growth factor 2 (IGF2) and mannose 6-phosphate modified proteins. The relative and specific titration of IGF2 by high affinity binding of IGF2R represents a mechanism that supports the parental conflict theory of genomic imprinting. Imprinting of Igf2 (paternal allele expressed) and Igf2r (maternal allele expressed) arose to regulate the relative supply of both proteins. Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality. In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r. Here we show that the knock-in construct resulted in over-growth when the humanised Igf2r allele was maternally transmitted, a phenotype that was rescued by either paternal transmission of the humanised allele, expression of a wild-type paternal allele or loss of function of Igf2. We also show that expression of IGF2R protein was reduced to less than 50% overall in tissues previously known to be Igf2 growth dependent. This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele. The phenotype following maternal transmission of the humanised allele resulted in overgrowth of the embryo, heart and placenta with partial peri-natal lethality, suggesting that further generation of hypomorphic Igf2r alleles are likely to be at the borderline of maintaining Igf2 dependent viability.

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Transgene targeting and genotyping.(A) Schematic diagram of Igf2r humanisation targeting strategy. Exons are represented as numbered boxes, loxP sites as small triangles, FRT sites as large triangles, human IGF2R mini-gene (Exons 3–48) as open box, mouse 3′ UTR as crossed box. WT, wild type; TV, targeting vector; TA, targeted allele; HA, humanised allele; P, PspOMI; N, NsiI; O1, oligo1; O2, oligo 2; O3, oligo 3; O4, oligo 4. (B) To detect correct homologous recombination at 5′, 5 clones of ES cells were digested with PspOMI and hybridised to probe 5e1 (left panel), 3 clones gave the expected band at 14.9 kb. For the 3′flank, cells were digested with NsiI and hybridised to probe 3e1 (central panel), all 5 gave the expected band at 8.7 kb. To detect single insertion, the clones were digested with NsiI and hybridised to probe Puro and all 5 gave a single band at the correct size of 8.7 kb (right panel). * denotes clone 2 chosen for breeding (A-D09). (C) Genotyping analysis was carried out by PCR on pups bred from clone 2 in (B). (Left panel) The 396 bp fragment (arrow) amplified by oligos 1 and 2 denotes the presence of heterozygous/homozygous humanised alleles. Wild type allele is not detected. (Right panel) The 167 bp fragment (arrow) amplified by oligos 3 and 4 denotes the presence of heterozygous and homozygous wild type alleles. In both PCR reactions, an internal control fragment of 585 bp was included. Marker bands of 15 bp and 7000 bp are shown. NC, negative control.
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pone-0057270-g001: Transgene targeting and genotyping.(A) Schematic diagram of Igf2r humanisation targeting strategy. Exons are represented as numbered boxes, loxP sites as small triangles, FRT sites as large triangles, human IGF2R mini-gene (Exons 3–48) as open box, mouse 3′ UTR as crossed box. WT, wild type; TV, targeting vector; TA, targeted allele; HA, humanised allele; P, PspOMI; N, NsiI; O1, oligo1; O2, oligo 2; O3, oligo 3; O4, oligo 4. (B) To detect correct homologous recombination at 5′, 5 clones of ES cells were digested with PspOMI and hybridised to probe 5e1 (left panel), 3 clones gave the expected band at 14.9 kb. For the 3′flank, cells were digested with NsiI and hybridised to probe 3e1 (central panel), all 5 gave the expected band at 8.7 kb. To detect single insertion, the clones were digested with NsiI and hybridised to probe Puro and all 5 gave a single band at the correct size of 8.7 kb (right panel). * denotes clone 2 chosen for breeding (A-D09). (C) Genotyping analysis was carried out by PCR on pups bred from clone 2 in (B). (Left panel) The 396 bp fragment (arrow) amplified by oligos 1 and 2 denotes the presence of heterozygous/homozygous humanised alleles. Wild type allele is not detected. (Right panel) The 167 bp fragment (arrow) amplified by oligos 3 and 4 denotes the presence of heterozygous and homozygous wild type alleles. In both PCR reactions, an internal control fragment of 585 bp was included. Marker bands of 15 bp and 7000 bp are shown. NC, negative control.

Mentions: To generate a humanised allele of mouse Igf2r, we targeted the human IGF2R exons 3–48 cDNA into the intron 2 region by homologous recombination (Fig. 1). The targeting vector replaced the intron 2 Airn promoter region but retained the 3′ splice site of mouse exon 2, with the 5′ splice site of human exon 3 distal to a flp site flanked human cDNA and mouse derived 3′UTR. C57BL6 ES lines were transformed under positive (puromycin) and negative selection with gancyclovir. Following PCR verification of correct targeting, and Southern blot to confirm a single copy insert, two lines were bred with a Cre-deleter strain to remove the puromycin selection cassette but retaining the flp sites. Two lines achieved germ-line transmission, with line 2* being the basis of the experimental breeding and phenotyping (Fig. 1B and C).


Maternal transmission of a humanised Igf2r allele results in an Igf2 dependent hypomorphic and non-viable growth phenotype.

Hughes J, Frago S, Bühnemann C, Carter EJ, Hassan AB - PLoS ONE (2013)

Transgene targeting and genotyping.(A) Schematic diagram of Igf2r humanisation targeting strategy. Exons are represented as numbered boxes, loxP sites as small triangles, FRT sites as large triangles, human IGF2R mini-gene (Exons 3–48) as open box, mouse 3′ UTR as crossed box. WT, wild type; TV, targeting vector; TA, targeted allele; HA, humanised allele; P, PspOMI; N, NsiI; O1, oligo1; O2, oligo 2; O3, oligo 3; O4, oligo 4. (B) To detect correct homologous recombination at 5′, 5 clones of ES cells were digested with PspOMI and hybridised to probe 5e1 (left panel), 3 clones gave the expected band at 14.9 kb. For the 3′flank, cells were digested with NsiI and hybridised to probe 3e1 (central panel), all 5 gave the expected band at 8.7 kb. To detect single insertion, the clones were digested with NsiI and hybridised to probe Puro and all 5 gave a single band at the correct size of 8.7 kb (right panel). * denotes clone 2 chosen for breeding (A-D09). (C) Genotyping analysis was carried out by PCR on pups bred from clone 2 in (B). (Left panel) The 396 bp fragment (arrow) amplified by oligos 1 and 2 denotes the presence of heterozygous/homozygous humanised alleles. Wild type allele is not detected. (Right panel) The 167 bp fragment (arrow) amplified by oligos 3 and 4 denotes the presence of heterozygous and homozygous wild type alleles. In both PCR reactions, an internal control fragment of 585 bp was included. Marker bands of 15 bp and 7000 bp are shown. NC, negative control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585325&req=5

pone-0057270-g001: Transgene targeting and genotyping.(A) Schematic diagram of Igf2r humanisation targeting strategy. Exons are represented as numbered boxes, loxP sites as small triangles, FRT sites as large triangles, human IGF2R mini-gene (Exons 3–48) as open box, mouse 3′ UTR as crossed box. WT, wild type; TV, targeting vector; TA, targeted allele; HA, humanised allele; P, PspOMI; N, NsiI; O1, oligo1; O2, oligo 2; O3, oligo 3; O4, oligo 4. (B) To detect correct homologous recombination at 5′, 5 clones of ES cells were digested with PspOMI and hybridised to probe 5e1 (left panel), 3 clones gave the expected band at 14.9 kb. For the 3′flank, cells were digested with NsiI and hybridised to probe 3e1 (central panel), all 5 gave the expected band at 8.7 kb. To detect single insertion, the clones were digested with NsiI and hybridised to probe Puro and all 5 gave a single band at the correct size of 8.7 kb (right panel). * denotes clone 2 chosen for breeding (A-D09). (C) Genotyping analysis was carried out by PCR on pups bred from clone 2 in (B). (Left panel) The 396 bp fragment (arrow) amplified by oligos 1 and 2 denotes the presence of heterozygous/homozygous humanised alleles. Wild type allele is not detected. (Right panel) The 167 bp fragment (arrow) amplified by oligos 3 and 4 denotes the presence of heterozygous and homozygous wild type alleles. In both PCR reactions, an internal control fragment of 585 bp was included. Marker bands of 15 bp and 7000 bp are shown. NC, negative control.
Mentions: To generate a humanised allele of mouse Igf2r, we targeted the human IGF2R exons 3–48 cDNA into the intron 2 region by homologous recombination (Fig. 1). The targeting vector replaced the intron 2 Airn promoter region but retained the 3′ splice site of mouse exon 2, with the 5′ splice site of human exon 3 distal to a flp site flanked human cDNA and mouse derived 3′UTR. C57BL6 ES lines were transformed under positive (puromycin) and negative selection with gancyclovir. Following PCR verification of correct targeting, and Southern blot to confirm a single copy insert, two lines were bred with a Cre-deleter strain to remove the puromycin selection cassette but retaining the flp sites. Two lines achieved germ-line transmission, with line 2* being the basis of the experimental breeding and phenotyping (Fig. 1B and C).

Bottom Line: Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality.In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r.This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Tumour Growth Group, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The cation independent mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) functions in the transportation and regulation of insulin-like growth factor 2 (IGF2) and mannose 6-phosphate modified proteins. The relative and specific titration of IGF2 by high affinity binding of IGF2R represents a mechanism that supports the parental conflict theory of genomic imprinting. Imprinting of Igf2 (paternal allele expressed) and Igf2r (maternal allele expressed) arose to regulate the relative supply of both proteins. Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality. In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r. Here we show that the knock-in construct resulted in over-growth when the humanised Igf2r allele was maternally transmitted, a phenotype that was rescued by either paternal transmission of the humanised allele, expression of a wild-type paternal allele or loss of function of Igf2. We also show that expression of IGF2R protein was reduced to less than 50% overall in tissues previously known to be Igf2 growth dependent. This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele. The phenotype following maternal transmission of the humanised allele resulted in overgrowth of the embryo, heart and placenta with partial peri-natal lethality, suggesting that further generation of hypomorphic Igf2r alleles are likely to be at the borderline of maintaining Igf2 dependent viability.

Show MeSH
Related in: MedlinePlus