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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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Maternal and paternal transmission of Igf2r+m/HU.p(A) Total number of neonates per litter at postnatal day 10 resulting from either paternal transmission (wild-type females crossed to Igf2r+m/HUp males, 8 pairs, 20 litters, open squares) or maternal transmission (Igf2r+m/HUp females crossed to wild-type males, 4 pairs, 17 litters, closed squares). Litter sizes were significantly reduced following maternal transmission. (B) Number of Igf2r+m/HUp pups present at postnatal day 10 in the litters described in (A) indicating that maternal transmission results reduced numbers of pups with the humanised allele. (C) Adult body weights (measured at 10 weeks) of offspring resulting from paternal transmission where there was no associated lethality (wild-type females crossed to Igf2r+m/HUp males, progeny; Igf2r+m/HUp males n = 16, wild-type males n = 21, Igf2r+m/HUp females n = 21, wild-type females n = 9). ***p<0.0001. Whiskers ± standard deviation. Paired t-test.
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pone-0057270-g002: Maternal and paternal transmission of Igf2r+m/HU.p(A) Total number of neonates per litter at postnatal day 10 resulting from either paternal transmission (wild-type females crossed to Igf2r+m/HUp males, 8 pairs, 20 litters, open squares) or maternal transmission (Igf2r+m/HUp females crossed to wild-type males, 4 pairs, 17 litters, closed squares). Litter sizes were significantly reduced following maternal transmission. (B) Number of Igf2r+m/HUp pups present at postnatal day 10 in the litters described in (A) indicating that maternal transmission results reduced numbers of pups with the humanised allele. (C) Adult body weights (measured at 10 weeks) of offspring resulting from paternal transmission where there was no associated lethality (wild-type females crossed to Igf2r+m/HUp males, progeny; Igf2r+m/HUp males n = 16, wild-type males n = 21, Igf2r+m/HUp females n = 21, wild-type females n = 9). ***p<0.0001. Whiskers ± standard deviation. Paired t-test.

Mentions: Maternal transmission of the humanised allele (HUm) resulted in reduced litter sizes compared to paternal transmission (HUp) (Fig. 2A). Genotyping with allele specific primers revealed that very few Igf2rHUm/+p pups survived following maternal transmission with wild-type males (Table 1). Examination of twenty litters from eight breeding pairs resulted in only nine viable Igf2rHUm/+p progeny compared to sixty two Igf2r+m/+p littermate controls (χ2, P<0.0001) (Fig. 2B and Table 1). Following paternal transmission, all pups were viable and Igf2r+m/HUp embryos were the same as wild-type littermates in weight (Table 1 and Fig. 2C).


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)

Maternal and paternal transmission of Igf2r+m/HU.p(A) Total number of neonates per litter at postnatal day 10 resulting from either paternal transmission (wild-type females crossed to Igf2r+m/HUp males, 8 pairs, 20 litters, open squares) or maternal transmission (Igf2r+m/HUp females crossed to wild-type males, 4 pairs, 17 litters, closed squares). Litter sizes were significantly reduced following maternal transmission. (B) Number of Igf2r+m/HUp pups present at postnatal day 10 in the litters described in (A) indicating that maternal transmission results reduced numbers of pups with the humanised allele. (C) Adult body weights (measured at 10 weeks) of offspring resulting from paternal transmission where there was no associated lethality (wild-type females crossed to Igf2r+m/HUp males, progeny; Igf2r+m/HUp males n = 16, wild-type males n = 21, Igf2r+m/HUp females n = 21, wild-type females n = 9). ***p<0.0001. Whiskers ± standard deviation. Paired t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585325&req=5

pone-0057270-g002: Maternal and paternal transmission of Igf2r+m/HU.p(A) Total number of neonates per litter at postnatal day 10 resulting from either paternal transmission (wild-type females crossed to Igf2r+m/HUp males, 8 pairs, 20 litters, open squares) or maternal transmission (Igf2r+m/HUp females crossed to wild-type males, 4 pairs, 17 litters, closed squares). Litter sizes were significantly reduced following maternal transmission. (B) Number of Igf2r+m/HUp pups present at postnatal day 10 in the litters described in (A) indicating that maternal transmission results reduced numbers of pups with the humanised allele. (C) Adult body weights (measured at 10 weeks) of offspring resulting from paternal transmission where there was no associated lethality (wild-type females crossed to Igf2r+m/HUp males, progeny; Igf2r+m/HUp males n = 16, wild-type males n = 21, Igf2r+m/HUp females n = 21, wild-type females n = 9). ***p<0.0001. Whiskers ± standard deviation. Paired t-test.
Mentions: Maternal transmission of the humanised allele (HUm) resulted in reduced litter sizes compared to paternal transmission (HUp) (Fig. 2A). Genotyping with allele specific primers revealed that very few Igf2rHUm/+p pups survived following maternal transmission with wild-type males (Table 1). Examination of twenty litters from eight breeding pairs resulted in only nine viable Igf2rHUm/+p progeny compared to sixty two Igf2r+m/+p littermate controls (χ2, P<0.0001) (Fig. 2B and Table 1). Following paternal transmission, all pups were viable and Igf2r+m/HUp embryos were the same as wild-type littermates in weight (Table 1 and Fig. 2C).

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