Transgenic Research

, Volume 18, Issue 4, pp 545–558

Hemizygous minipigs produced by random gene insertion and handmade cloning express the Alzheimer’s disease-causing dominant mutation APPsw

  • Peter M. Kragh
  • Anders Lade Nielsen
  • Juan Li
  • Yutao Du
  • Lin Lin
  • Mette Schmidt
  • Ingrid Brück Bøgh
  • Ida E. Holm
  • Jannik E. Jakobsen
  • Marianne G. Johansen
  • Stig Purup
  • Lars Bolund
  • Gábor Vajta
  • Arne Lund Jørgensen
Original Paper

Abstract

In an effort to develop a porcine model of Alzheimer’s disease we used handmade cloning to produce seven transgenic Göttingen minipigs. The donor fibroblasts had been stably transfected with a plasmid cassette containing, as transgene, the cDNA of the neuronal variant of the human amyloid precursor protein gene with the Swedish mutation preceded by beta-globin sequences to induce splicing and a human PDGFbeta promoter fragment to drive transcription. Transgene insertion had occurred only at the GLIS3 locus where a single complete copy of the transgene was identified in intronic sequences in opposite direction. Similar and robust levels of the transgene transcript were detected in skin biopsies from all piglets and the sequence of full-length transcript was verified. Consistent with PDGFbeta promoter function, high levels of transgene expression, including high level of the corresponding protein, was observed in brain tissue and not in heart or liver tissues. A rough estimate predicts that accumulation of the Aβ peptide in the brain may develop at the age of 1–2 years.

Keywords

Animal models Porcine Transgenesis Handmade cloning Alzheimer’s disease 

Abbreviations

APP

Amyloid precursor protein

APP695sw

Neuronal splicevariant of APP with the Swedish mutation

Amyloid beta-protein

AD

Alzheimer’s disease

PSEN

Presenilin

NFT

Neurofibrillary tangles

HMC

Handmade cloning

SCNT

Somatic cell nuclear transfer

References

  1. Alexander LJ, Rohrer GA, Beattie CW (1996) Cloning and characterization of 414 polymorfic porcine microsatellites. Anim Genet 27:137–148PubMedCrossRefGoogle Scholar
  2. Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, Enos J, Forsythe T, Golueke P, Jurgella G, Koppang R, Lesmeister T, Mallon K, Mell G, Misica P, Pace M, Pfister-Genskow M, Strelchenko N, Voelker G, Watt S, Thompson S, Bishop M (2000) Production of cloned pigs from in vitro systems. Nat Biotechnol 18:1055–1059. doi:10.1038/80242 PubMedCrossRefGoogle Scholar
  3. Blechingberg J, Lykke-Andersen S, Jensen TH, Jørgensen AL, Nielsen AL (2007) Regulatory mechanisms for 3′-end alternative splicing and polyadenylation of the glial fibrillary acidic protein, GFAP, transcript. Nucleic Acids Res 35:7636–7650. doi:10.1093/nar/gkm931 PubMedCrossRefGoogle Scholar
  4. Book SA, Bustad LK (1974) The fetal and neonatal pig in biomedical research. J Anim Sci 38:997–1002PubMedGoogle Scholar
  5. Borchelt DR, Ratovitski T, van Lare J, Lee MK, Gonzales V, Jenkins NA, Copeland NG, Price DL, Sisodia SS (1997) Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron 19:939–945. doi:10.1016/S0896-6273(00)80974-5 PubMedCrossRefGoogle Scholar
  6. Chen X-H, Siman R, Iwata A, Meaney DH, Trojanowski JQ, Smith DH (2004) Long-term accumulation of amyloid-β, β-secretase, presenilin-1, and caspase-3 in damaged axons following brain trauma. Am J Pathol 165:357–371PubMedGoogle Scholar
  7. Chen K, Baxter T, Muir WM, Groenen MA, Schook LB (2007) Genetic resources, genome mapping and evolutionary genomics of the pig (Sus scrofa). Int J Biol Sci 3:153–165PubMedCrossRefGoogle Scholar
  8. Douglas WR (1972) Of pigs and men and research: a review of applications and analogies of the pig, Sus Scrofa, in human medical research. Space Life Sci 3:226–234. doi:10.1007/BF00928167 PubMedCrossRefGoogle Scholar
  9. Du Y, Kragh PM, Zhang X, Purup S, Yang H, Bolund L, Vajta G (2005) High overall in vitro efficiency of porcine handmade cloning (HMC) combining partial zona digestion and oocyte trisection with sequential culture. Cloning Stem Cells 7:199–205. doi:10.1089/clo.2005.7.199 PubMedCrossRefGoogle Scholar
  10. Du Y, Kragh PM, Zhang Y, Li J, Smidt M, Bøgh IB, Zhang X, Purup S, Jørgensen AL, Pedersen AM, Villemoes K, Yang H, Bolund L, Vajta G (2007) Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology 68:1104–1110. doi:10.1016/j.theriogenology.2007.07.021 PubMedCrossRefGoogle Scholar
  11. Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, Carr T, Clemens J, Donaldson T, Gillespie F, Guido T, Hagopian S, Johnson-Wood K, Khan K, Lee M, Leibowitz P, Lieberburg I, Little S, Masliah E, McConlogue L, Montoya-Zavala M, Mucke L, Paganini L, Penniman E, Power M, Schenk D, Seubert P, Snyder B, Soriano F, Tan H, Vitale J, Wadsworth S, Wolozin B, Zhao J (1995) Alzheimer-type neuropathology in transgenic mice overexpressing V717F β-amyloid precursor protein. Nature 373:523–527. doi:10.1038/373523a0 PubMedCrossRefGoogle Scholar
  12. Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297:353–356. doi:10.1126/science.1072994 PubMedCrossRefGoogle Scholar
  13. Holm IE, West MJ (1994) The hippocampus of the domestic pig: a stereological study of the subdivisional volumes and neuron number. Hippocampus 4:115–126. doi:10.1002/hipo.450040112 PubMedCrossRefGoogle Scholar
  14. Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G (1996) Correlative memory deficits, Aβ elevation, and amyloid plaques in transgenic mice. Science 274:99–102. doi:10.1126/science.274.5284.99 PubMedCrossRefGoogle Scholar
  15. Hyman BT, West HL, Rebeck GW, Lai F, Mann DM (1995) Neuropathological changes in Down’s syndrome hippocampal formation. Effect of age and apolipoprotein E genotype. Arch Neurol 52:373–378PubMedGoogle Scholar
  16. Jelsing J, Olsen AK, Cumming P, Gjedde A, Hansen AK, Arnfred S, Hemmingsen R, Pakkenberg B (2005) A volumetric screening procedure for the Göttingen minipig brain. Exp Brain Res 162:428–435. doi:10.1007/s00221-004-2026-7 PubMedCrossRefGoogle Scholar
  17. Jelsing J, Nielsen R, Olsen AK, Grand N, Hemmingsen R, Pakkenberg B (2006) The postnatal development of neocortical neurons and glial cells in the Göttingen minipig and domestic pig brain. J Exp Biol 209:1454–1462. doi:10.1242/jeb.02141 PubMedCrossRefGoogle Scholar
  18. Johnson-Wood K, Lee M, Motter R, Hu K, Gordon G, Barbour R, Khan K, Gordon M, Tan H, Games D, Lieberburg I, Schenk D, Seubert P (1997) Amyloid precursor protein processing and Aβ42 deposition in a transgenic mouse model of Alzheimer disease. Proc Natl Acad Sci USA 94:1550–1555. doi:10.1073/pnas.94.4.1550 PubMedCrossRefGoogle Scholar
  19. Kragh PM, Vajta G, Corydon TJ, Purup S, Bolund L, Callesen H (2004) Production of transgenic blastocysts by handmade cloning. Reprod Fertil Dev 16:315–318. doi:10.1071/RD04007 PubMedCrossRefGoogle Scholar
  20. Lai L, Kolber-Simonds D, Park K-W, Cheong H-T, Greenstein JL, Im G-S, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS (2002) Production of α-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295:1089–1092. doi:10.1126/science.1068228 PubMedCrossRefGoogle Scholar
  21. Lai L, Kang JX, Li R, Wang J, Witt WT, Yong HY, Hao Y, Wax DM, Murphy CN, Rieke A, Samuel M, Linville ML, Korte SW, Evans RW, Starzl TW, Prather RS, Dai Y (2006) Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat Biotechnol 24:435–436. doi:10.1038/nbt1198 PubMedCrossRefGoogle Scholar
  22. Lewis J, McGowan E, Rockwool J, Melrose H, Nacharaju P, Van Slegtenhorst M, Gwinn-Hardy K, Murphy MP, Baker M, Yu X, Duff K, Hardy J, Corral A, Lin W-L, Yen S-H, Dickson DW, Davies P, Hutton M (2000) Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein. Nat Genet 25:402–405. doi:10.1038/78078 PubMedCrossRefGoogle Scholar
  23. McGowan E, Erikson J, Hutton M (2006) A decade of modelling Alzheimer’s disease in transgenic mice. Trends Genet 22:281–288. doi:10.1016/j.tig.2006.03.007 PubMedCrossRefGoogle Scholar
  24. Mullan M, Crawford F, Axelman K, Houlden H, Lilius L, Winblad B, Lannfelt L (1992) A pathogenic mutation for probable Alzheimer’s disease in the APP gene at the N-terminus of β-amyloid. Nat Genet 1:345–347. doi:10.1038/ng0892-345 PubMedCrossRefGoogle Scholar
  25. Neve RL, Finch EA, Dawes LR (1988) Expression of the Alzheimer amyloid precursor gene transcripts in the human brain. Neuron 1:669–677. doi:10.1016/0896-6273(88)90166-3 PubMedCrossRefGoogle Scholar
  26. Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, Metherate R, Mattson MP, Akbari Y, Le Ferla FM (2003) Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracellular Aβ and synaptic dysfunction. Neuron 39:409–421. doi:10.1016/S0896-6273(03)00434-3 PubMedCrossRefGoogle Scholar
  27. Oerum MA, Bendixen C, Madsen LB, Larsen K (2006) Porcine APP cDNAs: molecular cloning and characterization, expression analysis, chromosomal localization and SNP analysis. Biochim Biophys Acta 175:378–384Google Scholar
  28. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry ACF (2000) Pig cloning by microinjection of fetal fibroblast nuclei. Science 289:1188–1190. doi:10.1126/science.289.5482.1188 PubMedCrossRefGoogle Scholar
  29. Pillay P, Manger PR (2007) Order-specific quantitative patterns of cortical gyrification. Eur J NeuroSci 25:2705–2712. doi:10.1111/j.1460-9568.2007.05524.x PubMedCrossRefGoogle Scholar
  30. Polejaeva IA, Chen S-H, Vaught TD, Page RL, Mullins J, Ball S, Dal Y, Boone J, Walker S, Ayares DL, Colman A, Campbell KHS (2000) Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86–90. doi:10.1038/35024082 PubMedCrossRefGoogle Scholar
  31. Pond WG, Boleman SL, Fiorotto ML, Ho H, Knabe DA, Mersmann HJ, Savell JW, Su DR (2000) Perimatal ontogeny of brain growth in the domestic pig. Proc Soc Exp Biol Med 223:102–108. doi:10.1046/j.1525-1373.2000.22314.x PubMedCrossRefGoogle Scholar
  32. Rockenstein EM, McConlogue L, Tan H, Power M, Masliah E, Mucke L (1995) Levels and alternative splicing of amyloid β protein precursor (APP) transcripts in brains of APP transgenic mice and humans with Alzheimer’s disease. J Biol Chem 270:28257–28267. doi:10.1074/jbc.270.47.28257 PubMedCrossRefGoogle Scholar
  33. Rogers CS, Hao Y, Rokhlina T, Samuel M, Stoltz DA, Li Y, Petroff E, Vermeer DW, Kabel AC, Yan Z, Spate L, Wax D, Murphy CN, Rieke A, Whitworth K, Linville ML, Korte SW, Engelhardt JF, Welsh MJ, Prater RS (2008a) Production of CFTR-null and CFTRF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer. J Clin Invest 118:1571–1577. doi:10.1172/JCI34773 PubMedCrossRefGoogle Scholar
  34. Rogers CS, Stoltz DA, Meyerholtz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Wohlford-Lenane CL, Davis GJ, Hanfland RA, Smith TL, Samuel M, Wax D, Murphy CN, Rieke A, Whitworth K, Uc A, Starner TD, Brogden KA, Shilyanski J, McCray PB Jr, Zabner J, Prather RS, Welsh MJ (2008b) Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 321:1837–1841. doi:10.1126/science.1163600 PubMedCrossRefGoogle Scholar
  35. Rohrer GA, Alexander LJ, Keele JW, Smith TP, Beattie CW (1994) A microsatellite linkage map of the porcine genome. Genetics 136:231–245PubMedGoogle Scholar
  36. Sasahara M, Fries JWU, Raines EW, Gown AM, Westrum LE, Frosch MP, Bonthron DT, Ross R, Collins T (1991) PDGF B-chain in neurons of the central nervous system, posterior pituitary, and in a transgenic model. Cell 64:217–227. doi:10.1016/0092-8674(91)90223-L PubMedCrossRefGoogle Scholar
  37. Scheuner D, Eckman C, Jensen M, Song X, Citron M, Suzuki N, Bird TD, Haardy J, Hutton M, Kukull W, Larson E, Levy-Lehad E, Viitanen M, Peskind E, Poorkaj P, Schellenberg G, Tanzi R, Wasco W, Lannfelt L, Selkoe D, Younkin S (1996) Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer’s disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer’s disease. Nat Med 2:864–870. doi:10.1038/nm0896-864 PubMedCrossRefGoogle Scholar
  38. Senée V, Chelala C, Duchatelet S, Feng D, Blanc H, Cossec JC, Charon C, Nicolino M, Boileau P, Cavener DR, Bougnères P, Taha D, Julier C (2006) Mutations in GLIS3 are responsible for a rare syndrome with neonatal diabetes mellitus and congenital hypothyroidism. Nat Genet 38:682–687. doi:10.1038/ng1802 PubMedCrossRefGoogle Scholar
  39. Smith DH, Chen XH, Nonaka M, Trojanowski JQ, Lee VM, Saatman KE, Leoni MJ, Xu BN, Wolf JA, Meaney DF (1999) Accumulation of amyloid beta and tau and the formation of neurofilament inclusions following diffuse brain injury in the pig. J Neuropathol Exp Neurol 58:982–992. doi:10.1097/00005072-199909000-00008 PubMedCrossRefGoogle Scholar
  40. Takeuchi A, Irizarry MC, Duff K, Saido TC, Hsiao Ashe K, Hasegawa M, Mann DM, Hyman BT, Iwatsubo T (2000) Age-related amyloid beta deposition in transgenic mice overexpressing both Alzheimer mutant presenilin 1 and amyloid beta precursor protein Swedish mutant is not associated with global neuronal loss. Am J Pathol 157:331–339PubMedGoogle Scholar
  41. Vajta G (2007) Handmade cloning: the future way of nuclear transfer? Trends Biotechnol 25:250–253. doi:10.1016/j.tibtech.2007.04.004 PubMedCrossRefGoogle Scholar
  42. Vajta G, Lewis IM, Hyttel P, Thouas GA, Trounson AO (2001) Somatic cell cloning without micromanipulation. Cloning 3:89–95. doi:10.1089/15204550152475590 PubMedCrossRefGoogle Scholar
  43. Vajta G, Lewis IM, Trounson AO, Purup S, Maddox-Hyttel P, Schmidt M, Pedersen HG, Greve T, Callesen H (2003) Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro. Biol Reprod 68:571–578. doi:10.1095/biolreprod.102.008771 PubMedCrossRefGoogle Scholar
  44. Vodicka P, Smetana K Jr, Dvorankova B, Emerick T, Xu YZ, Ourednik J, Ourednik V, Motlik J (2005) The miniature pig as an animal model in biomedical research. Ann N Y Acad Sci 1049:161–171. doi:10.1196/annals.1334.015 PubMedCrossRefGoogle Scholar
  45. Watanabe H, Andersen F, Simonsen CZ, Evans SE, Gjedde A, Cumming P, the DaNeX Study Group (2001) MR-based statistical atlas of the Göttingen minipig brain. Neuroimage 14:1089–1096. doi:10.1006/nimg.2001.0910 Google Scholar
  46. Yoshioka K, Suzuki C, Tanaka A, Anas IM-K, Iwamura S (2002) Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 66:112–119. doi:10.1095/biolreprod66.1.112 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Peter M. Kragh
    • 1
    • 2
  • Anders Lade Nielsen
    • 1
  • Juan Li
    • 2
  • Yutao Du
    • 2
  • Lin Lin
    • 2
  • Mette Schmidt
    • 3
  • Ingrid Brück Bøgh
    • 3
    • 4
  • Ida E. Holm
    • 5
  • Jannik E. Jakobsen
    • 1
  • Marianne G. Johansen
    • 1
  • Stig Purup
    • 6
  • Lars Bolund
    • 1
  • Gábor Vajta
    • 2
    • 7
  • Arne Lund Jørgensen
    • 1
  1. 1.Department of Human Genetics, Faculty of Health Sciences, The Bartholin BuildingAarhus UniversityAarhus CDenmark
  2. 2.Department of Genetics and Biotechnology, Faculty of Agricultural SciencesAarhus UniversityTjeleDenmark
  3. 3.Department of Veterinary Reproduction and Obstetrics, Faculty of Life SciencesUniversity of CopenhagenFrederiksberg CDenmark
  4. 4.MåløvDenmark
  5. 5.Department of Pathology, Aalborg HospitalAarhus University HospitalAalborgDenmark
  6. 6.Department of Animal Health, Welfare and Nutrition, Faculty of Agricultural SciencesAarhus UniversityTjeleDenmark
  7. 7.Leederville, PerthAustralia

Personalised recommendations