The Soluble Type 2 Insulin-like Growth Factor (IGF-II) Receptor Reduces Organ Size by IGF-II-mediated and IGF-II-independent Mechanisms

The soluble type 2 insulin-like growth factor (IGF) receptor or IGF-II/mannose 6-phosphate receptor (sIGF2R) is produced in vivo by proteolytic deletion of the transmembrane and intracellular domains of the cellular form of the receptor (IGF2R). There is evidence that sIGF2R is a negative regulator of growth. We have shown that transgenic mice expressing anIgf2r cDNA with a deleted transmembrane domain sequence (sΔIgf2r) show reduced local organ size. In the present study, we investigate whether sΔIGF2R can slow the growth induced by an excess of IGF-II and whether the biological activity of sΔIGF2R is due solely to its interactions with IGF-II. To this end, we crossed sΔIgf2r transgenics by mice overexpressing IGF-II (Blast line) or by mice carrying a disrupted paternal (active) allele of the Igf2 gene (Igf2 m+/p−). Analysis of the phenotypes revealed that the soluble IGF2R affects the size of some organs (colon and cecum) exclusively by reducing the biological activity of IGF-II, whereas in other organs (stomach and skin) the biological activity of the receptor is at least in part independent of IGF-II and must involve an interaction with other factor(s). The soluble type 2 insulin-like growth factor (IGF) receptor or IGF-II/mannose 6-phosphate receptor (sIGF2R) is produced in vivo by proteolytic deletion of the transmembrane and intracellular domains of the cellular form of the receptor (IGF2R). There is evidence that sIGF2R is a negative regulator of growth. We have shown that transgenic mice expressing anIgf2r cDNA with a deleted transmembrane domain sequence (sΔIgf2r) show reduced local organ size. In the present study, we investigate whether sΔIGF2R can slow the growth induced by an excess of IGF-II and whether the biological activity of sΔIGF2R is due solely to its interactions with IGF-II. To this end, we crossed sΔIgf2r transgenics by mice overexpressing IGF-II (Blast line) or by mice carrying a disrupted paternal (active) allele of the Igf2 gene (Igf2 m+/p−). Analysis of the phenotypes revealed that the soluble IGF2R affects the size of some organs (colon and cecum) exclusively by reducing the biological activity of IGF-II, whereas in other organs (stomach and skin) the biological activity of the receptor is at least in part independent of IGF-II and must involve an interaction with other factor(s). insulin-like growth factor-II peptide type 2 insulin-like growth factor receptor or IGF-II/mannose 6-phosphate receptor polypeptide soluble IGF2R polypeptide transmembrane domain-deleted transgenic sIGF2R transforming growth factor-β1 polymerase chain reaction base pair(s). The growth and survival factor insulin-like growth factor II (IGF-II)1 binds to at least three different receptors: the type 1 and 2 IGF receptors and IGF2R (also known as the mannose 6-phosphate/IGF-II receptor) and the insulin receptor. Type 1 IGF receptor and the insulin receptor are members of the tyrosine kinase receptors family and mediate most of the biological effects of IGF-II (1Stewart C.E.H. Rotwein P. Physiol. Rev. 1996; 76: 1005-1026Crossref PubMed Scopus (698) Google Scholar, 2Louvi A. Accili D. Efstradiatis A. Dev. Biol. 1997; 189: 33-48Crossref PubMed Scopus (327) Google Scholar). Genetic evidence suggests that the IGF2R gene encodes a negative regulator of growth. Many human tumors show loss of heterozygosity at the IGF2R locus frequently accompanied by mutations in the remaining allele (3Hankins G.R. De Souza A.T. Bentley R.C. Patel M.R. Marks J.R. Iglehart J.D. Jirtle R.L. Oncogene. 1996; 12: 2003-2009PubMed Google Scholar, 4Ouyang H. Shiwaku H.O. Hagiwara H. Miura K. Abe T. Kato Y. Ohtani H. Shiiba K. Souza R.F. Meltzer S.J. Horii A. Cancer Res. 1997; 57: 1851-1854PubMed Google Scholar, 5Yamada T. De Souza A.T. Finkelstein S. Jirtle R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10351-10355Crossref PubMed Scopus (190) Google Scholar). Furthermore, mice in which the Igf2r gene is disrupted are born 25–35% bigger than controls (6Wang Z-Q. Fung M, R. Barlow D.P. Wagner E.F. Nature. 1994; 372: 464-467Crossref PubMed Scopus (408) Google Scholar, 7Lau M.M.H. Stewart C.E.H. Liu Z. Bhatt H. Rotwein P. Stewart C.L. Genes Dev. 1994; 8: 2953-2963Crossref PubMed Scopus (475) Google Scholar, 8Ludwig T. Eggenschwiler J. Fisher P. D'Ercole A.J. Davenport M.L. Efstradiatis A. Dev. Biol. 1996; 177: 517-535Crossref PubMed Scopus (406) Google Scholar). IGF2R is a multifunctional protein that participates in the activation of TGF-β1, regulates lysosomal enzymes trafficking, and binds a number of ligands including proliferin, herpes simplex virus glycoprotein D, thyroglobulin, and retinoic acid (9Kornfeld S. Annu. Rev. Biochem. 1992; 61: 307-330Crossref PubMed Scopus (936) Google Scholar, 10Oka Y. Rozek L.M. Czech M.P. J. Biol. Chem. 1985; 260: 9435-9442Abstract Full Text PDF PubMed Google Scholar, 11Hille-Rehfeld A. Biochim. Biophys. Acta. 1995; 1241: 177-194Crossref PubMed Scopus (218) Google Scholar, 12Dahms N.M. Biochem. Soc. Trans. 1996; 24: 136-141Crossref PubMed Scopus (38) Google Scholar, 13Dennis P.A. Rifkin D.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 580-584Crossref PubMed Scopus (460) Google Scholar, 14Kang J.X. Li Y. Leaf A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13671-13676Crossref PubMed Scopus (117) Google Scholar). A soluble form of IGF2R (sIGF2R) is produced by proteolytic cleavage of the transmembrane and intracellular domains of the membrane form of the receptor and is present in the serum, amniotic fluid, and urine of rodents and humans. sIGF2R binds IGF-II with high affinity in vivo and can bind mannose 6-phosphate in vitro, suggesting that it shares at least some of its ligand specificity with the membrane IGF2R (15Kiess W. Greenstein L.A. White R.M. Lee L. Rechler M.M. Nissley S.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7720-7724Crossref PubMed Scopus (88) Google Scholar, 16Causin C. Waheed A. Braulke T. Junghans U. Maly P. Humbel R.E. von Figura K. Biochem. J. 1988; 252: 795-799Crossref PubMed Scopus (75) Google Scholar, 17MacDonald R.G. Tepper M.A. Clairmont K.B. Perregaux S.B. Czech M.P. J. Biol. Chem. 1989; 264: 3256-3261Abstract Full Text PDF PubMed Google Scholar, 18Xu Y. Papageorgiou A. Polychronakos C. J. Clin. Endocrinol. Metab. 1998; 83: 437-442PubMed Google Scholar, 19Valenzano K.J. Remmler J. Lobel P. J. Biol. Chem. 1995; 270: 16441-16448Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). There is evidence that sIGF2R is a biologically active molecule. First, sIGF2R can inhibit DNA synthesis induced by IGF-II and epidermal growth factor in cultured rat hepatocytes (20Scott C.D. Ballesteros M. Madrid J. Baxter R.C. Endocrinology. 1996; 137: 873-878Crossref PubMed Scopus (25) Google Scholar). Second, we have obtained transgenic mice expressing a soluble IGF2R by deletion of the transmembrane domain sequence (sΔIgf2r) and fused to the regulatory sequence of the keratin 10 promoter to target expression to the alimentary canal, skin, and uterus (K10sΔIgf2r transgene). Two lines of K10sΔIgf2r transgenic mice (Kipps and Krishna) showed a 9–20% reduction of wet weight, dry weight, and water content in the alimentary canal. The effects of sΔIGF2R expression were mainly local, because the organs negative for transgene expression were only marginally affected (21Zaina, S., Newton, R. V. S., Paul, M. R., and Graham, C. F. (1998) Endocrinology, in pressGoogle Scholar). The interpretation of the biological activity of sIGF2R is complicated by the heterogeneity of its ligands. To understand to what extent the biological activity of sΔIGF2R is due to interaction with IGF-II, we crossed K10sΔIgf2r transgenics by the following genetically modified mice: 1) transgenics expressing aK10Igf2 minigene and showing local organomegaly (Blast line; Ref. 22Ward A. Bates P. Fisher R. Richardson L. Graham C.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10365-10369Crossref PubMed Scopus (90) Google Scholar). If sΔIGF2R acts by reducing the activity of IGF-II, double transgenics Blast and K10sΔIgf2r should show an attenuation of organomegaly compared with Blast. 2) mice in which the paternal (active) allele of the Igf2 gene is disrupted (Igf2 +/p− ).Igf2 m+/p− mice show a growth deficiency phenotype and are fertile (23DeChiara T.M. Efstradiatis A. Robertson E.J. Nature. 1990; 345: 78-80Crossref PubMed Scopus (1410) Google Scholar). If sΔIGF2R acts exclusively by interacting with IGF-II, organ size should not be affected in K10sΔIgf2r transgenics that are alsoIgf2 m+/p− compared withIgf2 m+/p− mice. The results of the present work provide insights into the mechanism of organ size reduction by the soluble IGF2R. K10sΔIgf2r (linesKipps and Krishna) transgenic mice express a mutant mouse Igf2r cDNA in which the sequence encoding the transmembrane domain has been deleted to encode a soluble polypeptide (sΔIGF2R). The mutant cDNA is under the transcriptional control of the keratin 10 promoter (K10).Kipps is the line expressing theK10sΔIgf2r transgene at highest levels, and Krishna is the second best expressing line; their phenotype has been described in detail (21Zaina, S., Newton, R. V. S., Paul, M. R., and Graham, C. F. (1998) Endocrinology, in pressGoogle Scholar). K10Igf2(Blast) and Igf2 m+/p− mice have been described elsewhere (22Ward A. Bates P. Fisher R. Richardson L. Graham C.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10365-10369Crossref PubMed Scopus (90) Google Scholar, 23DeChiara T.M. Efstradiatis A. Robertson E.J. Nature. 1990; 345: 78-80Crossref PubMed Scopus (1410) Google Scholar). All mice used in this study were heterozygotes (indicated as K10sΔIgf2r/+, K10Igf2/+ and Igf2 m+/p−) in a mixed genetic background as the integrated transgenes were bred from a F1(C57Bl/6 × CBA) onto a 129J/Sv genetic background. In both crosses the K10sΔIgf2r transgene was transmitted maternally, because Blast females are usually not fertile, and the disrupted copy of the imprinted Igf2 gene has to be transmitted paternally (22Ward A. Bates P. Fisher R. Richardson L. Graham C.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10365-10369Crossref PubMed Scopus (90) Google Scholar, 23DeChiara T.M. Efstradiatis A. Robertson E.J. Nature. 1990; 345: 78-80Crossref PubMed Scopus (1410) Google Scholar). There is no evidence that the phenotype of K10sΔIgf2r/+ mice is altered by the sex of the parent, which transmits the transgene. Genotyping was performed by PCR using genomic DNA extracted from blood by using Isocode Stix (Schleicher & Schuell) according to manufacturer's instructions or from tail tissue (24Gemmell N.J. Akiyama S. Trends Genet. 1996; 12: 338-339Abstract Full Text PDF PubMed Scopus (243) Google Scholar). The following protocols were used. 1) K10sΔIgf2r(Kipps and Krishna lines): forward primer, 5′-ACGAGACCGCTGACTGCCAGTACC-3′, and reverse primer, 5′-AGCCATTCTGTCTCATTCTCATCTGTCTCC-3′. These two primers amplify the region of the Igf2r cDNA containing the deletion. The K10sΔIgf2r transgene produced a unique 256-bp fragment. The conditions were 94 °C for 3 min, 60 °C for 2 min, and 72 °C for 2 min for 35 cycles. 2) K10Igf2(Blast line): forward primer, 5′-TTCAAAACCAAACATATTGGGGCTGG-3′ (corresponding to a sequence 140 bp 5′ to the TATA box of the keratin 10 promoter), and reverse primer, 5′-TGAAGTAGAAGCCGCGGTCCGAACAG-3′ (corresponding to the 3′ end of exon 4 in the Igf2 gene). The conditions were 94 °C for 3 min, 53 °C for 2 min, and 72 °C for 2 min for 35 cycles. TheBlast transgene produced a unique 357-bp fragment. 3)Igf2 m+/p−: forward primer, 5′-GTGTTCCGGCTGTCAGCGCA-3′, and reverse primer, 5′-GTCCTGATAGCGGTCCGCCA-3′, both corresponding to sequences in the neo r gene. The conditions were 94 °C for 1 min and 70 °C for 4 min for 35 cycles. Theneo r gene produced a unique 555-bp fragment. Analysis of organ weights was performed in 90-day-old mice produced by crossing heterozygotesK10sΔIgf2r/+ (Kipps orKrishna line) by K10Igf2/+ orIgf2 m+/p− mice. Live weight was recorded, and then anesthetized mice were bled by decapitation and dissected, and the total wet weight of organs was measured after removal of fat and mesenteries. The contents of the alimentary canal were removed by gentle scraping in phosphate-buffered saline followed by blotting in tissue paper. Organs were always dissected in the same order to normalize wet weight loss due to evaporation. Each organ was cut in two or three parts, and the parts were weighed. This made it possible to calculate the total organ dry weight, water content, DNA, and detergent-soluble protein content after assaying different parts. For dry weight measurement, tissue fragments were dried at 65 °C for 6 days. Detergent-soluble protein was measured by the Coomassie Blue method (Bradford reagent, Sigma) after tissue homogenization in 20 mm Tris, pH 7.5, 10 mm EDTA, 0.1% Tween 20, 0.15 m NaCl. DNA content was measured in the same extract usingt the Hoechst 33258 fluorochrome, following addition of NaCl to 2m final concentration and brief sonication (25Labarca C. Paigen K. Anal. Biochem. 1980; 102: 344-352Crossref PubMed Scopus (4553) Google Scholar). For all measured parameters, relative values (e.g. per mg wet weight) were first obtained by dividing the value obtained for a given tissue part by the weight of that tissue part. This relative value was then multiplied by the total organ wet weight to obtain the total value (e.g. for the whole organ). Total water content was calculated by subtracting the total dry weight from the total wet weight. Total water content was divided by the total dry weight to obtain the water content/unit dry weight. All comparisons were by paired t test of pairs of mice matched for litter, age, and sex. The number of paired comparisons used in each t test was in some cases lower than the total number of animals displayed in each column of genotypes (Tables Table I, Table II, Table III, Table IV). The reason was that in comparing two given groups, only mice that could be matched were used, and this was frequently a subset of one of the two groups. Thus the description of the phenotype for each genotype given in the tables does not always indicate the mean value of the phenotype character that was involved in paired t tests.Table ICrosses K10Igf2/+ × K10sΔIgf2r/+: body weights and parameters of organs coexpressing the K10Igf2 and K10sΔIgf2r transgenesABCWild typeK10Igf2/+ (Blast)K10Igf2/+/K10sΔIgf2r/+KippsKrishnaBody weight1-aWeight expressed in grams.25.6 ± 0.7 (13)28.3 ± 0.8w* (13)26.2 ± 0.5 (11)31.1 ± 1.4 (9)Stomach Wet weight158.9 ± 5.6 (13)185.4 ± 5.7w** (12)146.5 ± 5.2w*/B** (13)152.7 ± 8.3B* (6) Dry weight32.9 ± 1.3 (12)41.8 ± 0.9w** (10)32.4 ± 1.6B* (12)32.6 ± 1.7B* (6) Water content1-bWater content/unit dry weight.3.7 ± 0.2 (12)3.5 ± 0.1 (7)3.6 ± 0.2 (12)3.7 ± 0.1 (6) DNA0.24 ± 0.05 (10)0.27 ± 0.04 (10)0.20 ± 0.06 (7)0.20 ± 0.05 (8) Protein1-cDetergent-soluble protein.4.3 ± 1.7 (11)4.8 ± 1.2 (11)5.3 ± 1.4 (7)7.4 ± 1.5 (8)Small intestine Wet weight964.9 ± 30.7 (13)1141.4 ± 27.5w** (9)982.1 ± 48.5B** (11)998.2 ± 36.9 (6) Dry weight207.1 ± 10.0 (12)252.8 ± 11.6w* (11)194.7 ± 8.1B* (12)211.6 ± 10.6B* (6) Water content1-bWater content/unit dry weight.3.7 ± 0.3 (12)3.8 ± 0.1 (8)3.9 ± 0.2 (11)3.7 ± 0.1 (8) DNA3.2 ± 0.7 (10)4.3 ± 0.9 (10)2.6 ± 0.9 (7)1.8 ± 1.2B* (8) Protein1-cDetergent-soluble protein.29.2 ± 3.9 (11)28.5 ± 7.2 (11)32.0 ± 6.2 (7)41.1 ± 2.3 (8)Cecum Wet weight158.7 ± 6.3 (14)282.4 ± 20.6w*** (10)202.0 ± 9.1w***/B** (12)205.0 ± 5.1w***/B* (6)Colon Wet weight447.3 ± 24.2 (14)969.1 ± 59.9w*** (10)641.1 ± 46.9w***/B* (12)834.9 ± 36.1w***/B* (7) Dry weight87.0 ± 3.3 (12)168.1 ± 7.5w*** (11)121.7 ± 3.4w***/B** (12)145.2 ± 4.5w***/B* (6) Water content1-bWater content/unit dry weight.4.1 ± 0.1 (11)4.3 ± 0.1 (7)4.3 ± 0.4 (12)4.6 ± 0.2 (6) DNA0.9 ± 0.2 (10)2.0 ± 0.4w* (10)1.4 ± 0.6 (7)0.8 ± 0.4B* (8) Protein1-cDetergent-soluble protein.21.4 ± 3.7 (11)28.7 ± 2.0 (11)31.7 ± 8.9 (7)28.5 ± 2.6 (8)Skin Wet weight1-aWeight expressed in grams.2.7 ± 0.21 (13)4.3 ± 0.36w*** (9)3.8 ± 0.28w**/B** (11)3.2 ± 0.23w* (5)Uterus Wet weight91.2 ± 10.2 (6)433.3 ± 24.1w*** (6)404.6 ± 95.2w* (6)502.4 ± 59.8w** (5)Values are mean weights (mg) ± S.E. in wild type (column A), heterozygotes overexpressing a K10Igf2 transgene (K10Igf2/+, Blast line, column B), and double transgenics overexpressing both the K10Igf2and the K10sΔIgf2r(K10sΔIgf2r/+, Kipps, and Krishna lines, column C) transgenes. Figures in parentheses are the number of samples. Levels of significance are given for comparisons with wild type (w) and K10Igf2/+ (B). Comparisons were by pairedt test of sex, litter, and age matched mice. *,p < 0.05; **, p < 0.01; ***,p < 0.001; no symbol, not significant.1-a Weight expressed in grams.1-b Water content/unit dry weight.1-c Detergent-soluble protein. Open table in a new tab Table IICrosses K10Igf2/+ × K10sΔIgf2r/+: parameters of organs not expressing the transgenesABCWild typeK10Igf2/+ (Blast)K10Igf2/+/K10sΔIgf2r/+KippsKrishnaLiver Wet weight1277.2 ± 77.7 (12)1295.8 ± 64.7 (12)1146.2 ± 62.5 (8)1329.9 ± 70.3 (6)Kidneys Wet weight334.1 ± 14.0 (13)353.4 ± 13.2 (13)334.5 ± 12.2 (11)415.9 ± 36.5 (6) Dry weight79.8 ± 3.9 (12)89.7 ± 3.1 (11)85.0 ± 3.0 (11)101.0 ± 9.4w* (6) Water content2-aWater content/unit dry weight.3.1 ± 0.04 (12)3.1 ± 0.03 (8)2.9 ± 0.04 (11)3.1 ± 0.04 (6) DNA1.92 ± 0.45 (10)2.19 ± 0.52 (10)1.64 ± 0.58 (7)1.76 ± 0.66 (8) ± 5.6 ± ± ± Wet ± ± ± ± (6) Dry ± 3.1 ± 0.9 ± 0.6 ± 1.5 (6) Water content2-aWater content/unit dry weight.3.1 ± 0.04 ± 0.04 ± 0.03 ± 0.06 (6) ± 0.03 ± 0.05 ± 0.05 ± 0.03 (8) ± (11)4.8 ± ± ± Wet ± ± ± ± values and of Table Water content/unit dry Detergent-soluble protein. Open table in a new tab Table × K10sΔIgf2r/+: body weight and parameters of organs expressing the K10sΔIgf2r expressed in ± 0.9 ± ± ± ± Wet ± ± ± ± ± Dry ± 1.3 ± ± ± (6) Water content/unit dry ± 0.2 ± 0.1 ± 0.1 ± 0.4 (6) ± 0.04 ± ± ± (6) ± 0.7 ± ± ± Wet ± ± ± ± ± Wet ± ± 20.6w*** ± ± ± Dry ± ± ± ± (6) Water content/unit dry ± 0.3 ± 0.2 ± 0.2 ± 0.1 (6) ± ± ± ± (6) ± 1.2 ± ± ± 0.9w** Wet expressed in grams.2.7 ± ± ± ± ± are mean weights (mg) ± S.E. in wild type (column A), mice carrying a disrupted paternal Igf2 allele (Igf2 column B), heterozygotes carrying a K10sΔIgf2r transgene line, column and mice with both genetic (Kipps and Krishna lines, column Figures in parentheses are the number of samples. Each value is followed by the of significance of comparisons with wild type m+/p− and K10sΔIgf2r/+ Comparisons were by paired t test of sex, litter, and age matched mice. For a given r is the the of in body weight and the of in organ wet weight, both compared with wild of r 1 indicate that organ size is than from body weight. *,p < 0.05; **, p < 0.01; ***,p < 0.001; no symbol, not Weight expressed in Water content/unit dry Detergent-soluble protein. Open table in a new tab Table × K10sΔIgf2r/+: parameters of organs not expressing the K10sΔIgf2r Wet ± ± ± ± ± Wet ± ± ± ± ± Dry ± ± ± ± (6) Water content/unit dry weight.3.1 ± 0.4 (11)3.2 ± 0.4 ± 0.2 ± 0.3 (6) ± ± ± ± (6) soluble ± 3.0 ± 3.0 ± ± Wet ± ± ± ± ± values and of Table Water content/unit dry soluble protein. Open table in a new tab are mean weights (mg) ± S.E. in wild type (column A), heterozygotes overexpressing a K10Igf2 transgene (K10Igf2/+, Blast line, column B), and double transgenics overexpressing both the K10Igf2and the K10sΔIgf2r(K10sΔIgf2r/+, Kipps, and Krishna lines, column C) transgenes. Figures in parentheses are the number of samples. Levels of significance are given for comparisons with wild type (w) and K10Igf2/+ (B). Comparisons were by pairedt test of sex, litter, and age matched mice. *,p < 0.05; **, p < 0.01; ***,p < 0.001; no symbol, not For values and of Table are mean weights (mg) ± S.E. in wild type (column A), mice carrying a disrupted paternal Igf2 allele (Igf2 column B), heterozygotes carrying a K10sΔIgf2r transgene line, column and mice with both genetic (Kipps and Krishna lines, column Figures in parentheses are the number of samples. Each value is followed by the of significance of comparisons with wild type m+/p− and K10sΔIgf2r/+ Comparisons were by paired t test of sex, litter, and age matched mice. For a given r is the the of in body weight and the of in organ wet weight, both compared with wild of r 1 indicate that organ size is than from body weight. *,p < 0.05; **, p < 0.01; ***,p < 0.001; no symbol, not For values and of Table Igf2 was by reverse 1 of total was by using the according to the manufacturer's instructions cDNA was by PCR using the primers and These primers sequences in exon 4 of the Igf2 gene and a 22Ward A. Bates P. Fisher R. Richardson L. Graham C.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10365-10369Crossref PubMed Scopus (90) Google and To control for in and primers for the mouse cDNA were in the same PCR The conditions for were 94 °C for 3 min, 53 °C for 2 min, and 72 °C for 2 min for 35 cycles. were in a and with To whether the soluble receptor could growth by excess local IGF-II, two transgenes were crossed into the same mice. The of both transgenes was by the same keratin 10 and their of organ expression The to double transgenics mice with one or the other of the transgenes. The phenotype of the transgenic is first described to provide the base line for with the double transgenic mice expressing the Igf2 gene under the transcriptional control of the keratin 10 promoter (Blast line, showed local organ as described (22Ward A. Bates P. Fisher R. Richardson L. Graham C.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10365-10369Crossref PubMed Scopus (90) Google Scholar). wet weight was in the alimentary canal, skin, and uterus compared with whereas wet weight was in organs negative for transgene expression (column A column in and Total dry weight followed a in the organs (column A and of water content relative to dry content has been shown in mice with of IGF-II T. Eggenschwiler J. Fisher P. D'Ercole A.J. Davenport M.L. Efstradiatis A. Dev. Biol. 1996; 177: 517-535Crossref PubMed Scopus (406) Google Scholar). The water content relative to dry weight was in Blast in all organs A and DNA and detergent-soluble protein contents not in of the organs with the of the as (22Ward A. Bates P. Fisher R. Richardson L. Graham C.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10365-10369Crossref PubMed Scopus (90) Google Scholar). In this organ the total DNA content was by a extent as wet and dry weights I, column A column The phenotype of K10sΔIgf2r/+ mice was a reduced wet weight of the alimentary canal (21Zaina, S., Newton, R. V. S., Paul, M. R., and Graham, C. F. (1998) Endocrinology, in pressGoogle Scholar). The of the × Blast was to the extent of organomegaly in double transgenics with wild type and For two the phenotype of K10sΔIgf2r/+ mice be described in the of the K10sΔIgf2r/+ m+/p− the column in and IV). In the double the soluble receptor transgene was in the same mouse as the transgene that expressed excess IGF-II A reduction of organomegaly was in most of the organs coexpressing the two transgenes. wet weight was reduced in double transgenics compared with Blast in the alimentary canal (column column Table This reduction from than in the to in the and and was in double transgenics (column Table This is with the relative of the expression in the two lines (21Zaina, S., Newton, R. V. S., Paul, M. R., and Graham, C. F. (1998) Endocrinology, in pressGoogle Scholar). In the skin, a reduction of organomegaly was only in double transgenics (column Table of sΔIGF2R expression was the wet weight of the uterus (column column Table of the two transgenes produced a in organ dry weight with the in wet weight and not DNA content or detergent-soluble protein content (column column Table The in dry weight was in than in transgenics (column Table DNA content was reduced in the intestine and in compared with Blast (column Table in the organ parameters measured was in the which not express the transgene (column column Table carrying a paternal allele (Igf2 m+/p−) were than wild type at in a mixed genetic background and were fertile as 23DeChiara T.M. Efstradiatis A. Robertson E.J. Nature. 1990; 345: 78-80Crossref PubMed Scopus (1410) Google column column Table The of IGF-II and peptide in Igf2 m+/p− mice were the in three independent First, no IGF-II was in the at 10 by reverse not Second, obtained m+/p− mice was in a IGF-II as a negative control the of the K10sΔIgf2r/+ transgenics (21Zaina, S., Newton, R. V. S., Paul, M. R., and Graham, C. F. (1998) Endocrinology, in pressGoogle Scholar). IGF-II peptide were the of the in an independent study in the and intestine of Igf2 m+/p− mice were shown to no Igf2 by a in Analysis of organs revealed that the wet weight of most organs was to the whole body weight of r to column in and IV). The and the were be