PHuntington’s disease is an autosom

PHuntington’s disease is an autosomal dominant, progressive neurodegenerative disorder involving the premature loss of specific neurons. It is associated with an expansion of a CAG trinucleotide repeat in the 5′ region of the huntingtin gene (HTT) which results in a lengthened polyglutamine tract of the protein. The CAG repeat number is polymorphic, ranging from 6 to 35 units in normal alleles and from 36 to 120 units in alleles associated with Huntington’s disease. The 12.8-kb HTT transcript of Göttingen minipigs codes for a 345-kDa protein (3,139 amino acids) [33]. The 8.2-kb transgene used for DNA microinjection into minipig embryos consisted of the 4-kb rat neuronspecific enolase (Nse) promoter, a 3.3-kb 5 ′ minipig huntingtin cDNA which was mutated by insertion of 75 CAG repeats into the triplet region of exon 1, and a 0.9-kb SV40 polyadenylation signal. Five transgenic founder pigs were produced each harboring one to three different integration sites with variable copy numbers and indication of genetic mosaicism [34]. To date, no follow-up publication describing mutant phenotypes appeared.
656 J Mol Med (2010) 88:653–664
Retinitis pigmentosa (RP) typically causes night blindness early in life due to loss of rod photoreceptors. The remaining cone photoreceptors slowly degenerate leading ultimately to blindness. Various genes and loci are associated with the
disease. Both transgenic and knockout rodent models of retinal dystrophy contributed to the analysis of the disease. Compared to humans, rodent models are limited by two disadvantages, the small number and different distribution of
neopromoter coding sequence Expressing transgenic pigs
Selectable expression vector
Transfection
Selection Embryo transfer
Nuclear transfer I
Nuclear transfer II
Establishment of cell culturesEmbryo transfer
Fetuses or offspring
Screening (integration, expression)
Fig. 2 Efficient production of transgenic pigs by using somatic cell nuclear transfer. An expression vector carrying a removable selection cassette is transfected into nuclear donor cells. After selection, the resulting transgenic cells are pooled and used for nuclear transfer. Pooling of cell colonies reduces the time in culture and allows the generation of independent founder fetuses/offspring in one litter. Cloned embryos are transferred to synchronized recipients. Depending
on the expected onset and tissue specificity of transgene expression, pregnancies may be terminated to recover fetuses, or birth and early development of offspring is awaited. Fetuses or tissues from born offspring are processed for transgene integration and expression studies, while individual cell cultures are established for re-cloning of the fetuses/offspring with the most suitable integration/expression pattern
Table 1 Transgenic pigs as disease models
Human disease Transgene expression References
Alzheimer’s disease Expression of mutant human APPsw in the brain Kragh PM et al. 2009 [32] Huntington’s disease Transgenic animals with mutant pig HTT Uchida M et al. 2001 [34] Retinitis pigmentosa Retinal expression of mutant pig RHOP347L or RHOP347S Petters RM et al. 1997, Kraft TW et al. 2005 [36, 37] Cardiovascular disease Endothelial over-expression of pig eNOS Hao YH et al. 2006 [40] Cystic fibrosis CFTR knockout or mutant pig CFTRdeltaF508 knockin Rogers CS et al. 2008 [28, 44] Type 2 diabetes mellitus Beta-cell expression of mutant dominant-negative human GIPRdn Renner S et al. 2010 [56] Type 3 of maturity-onset diabetes of the young Beta-cell expression of mutant dominant-negative human HNF1AP291fsinsC Umeyama K et al. 2009 [59]
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photoreceptors in the retina and the small eyes [35]. Therefore, transgenic pigs expressing a mutant porcine rhodopsin (RHO; P347L or P347S) were produced by additive gene transfer. A 12.5-kb porcine genomic DNA containing 4-kb 5′ flanking sequences, the coding sequences for the 348 amino acid protein and 2.9-kb 3′ flanking sequences of the porcine RHO gene was used for the introductionofthemutationCCA(Pro)→CTA(Leu)orTCA (Ser) in codon 347. DNA microinjection of the expression vectors for mutant rhodopsin resulted in the generation of transgenic lines. Retinal RNA expression of the mutant transgene exceeded the expression of the wild-type endogenous gene. Like human patients with the same mutation, the transgenic pigs showed early and severe loss of rod photoreceptors, and the surviving cone photoreceptors slowly degenerated. The phenotypes of mutant RHO transgenic pigs and of RP patients are comparable. Therefore, this novel animal model is intensely used for studying the pathogenesis of retinitis pigmentosa as well as for preclinical treatment trials [36, 37]. Furthermore, production of a transgenic pig model for spinal muscular atrophy, an autosomal recessive disorder characterized by the degeneration of motor neurons of the spinal cord leading to muscle atrophy, has been announced [38].
Cardiovascular diseases
Endothelial cell nitric oxide synthase (eNOS) regulates vascular function by releasing nitric oxide [39]. Transgenic pigs were produced for the analysis of the cardiovascular regulation by eNOS. The 7.3 kb transgene consisted of the 3.6-kb Yucatan pig eNOS cDNA and a V5 epitope and polyhistidine tag (V5-His tag) to discriminate between endogenous and transgenic eNOS that was cloned between the 2-kb TIE2 promoter and 1.7-kb TIE2 intron/enhancer elements for the endothelial cell-specific expression. The transgene was used for co-electroporation with a neomycin resistance gene expression cassette into Yucatan pig fetal fibroblasts for additive gene transfer. Four cloned transgenic pigs derived by SCNTof transgene-positive cells expressed the fusion protein which was localized to the endothelial cells of placental vasculature from the conceptuses as did the endogenous eNOS. The predicted size of the recombinant eNOS (1,242 amino acids) was 138 kDa, compared to 133 kDa of endogenous eNOS (1,205 amino acids). Localization of endogenous and transgenic eNOS revealed the expression in the endothelium. The transgenic pigs are further used to analyze the function of eNOS in regulating muscle metabolism and in the cardiorespiratory system [40]. In addition, a complementary knockout model of eNOS has been announced [41].
Cystic fibrosis
Alterations of the cystic fibrosis transmembrane conductance regulator (1,480 amino acids) were identified to cause the autosomal recessive cystic fibrosis which still remains incurable. Mice with a disrupted Cftr gene failed to develop the lung and pancreatic disease causing most of the morbidity and mortality in human patients [42]. Porcine lungs share many anatomical, histological, biochemical, and physiological features with human lungs [43]. Mutant pigs were produced using SCNT and fetal fibroblasts with the CFTR gene either disrupted or containing the most common cystic fibrosis-associated mutation (deltaF508). Therefore, recombinant adeno-associated virus (rAAV) vectors were used to target CFTR in male fetal fibroblasts ofoutbreddomesticpigs.The4.5-kbknockoutgeneconstruct disrupted exon 10 encoding a portion of nucleotide-binding domain1withastopcodonatposition508(F508X)followed by a floxed neomycin resistance gene driven by the phosphoglycerate kinase (PGK) promoter, whereas the deltaF508 knockin gene construct harbors the three nt deletion in exon 10 leading to deltaF508 followed by a floxed neomycin resistance gene driven by the PGK promoter in the downstream intronic region. Using successfully targeted cells without viral vector sequences for SCNT, heterozygous mutant male piglets were generated with each mutation [28, 44]. Newborn piglets with a targeted disruption of both CFTR alleles exhibited similar defects as seen in newborn human patients, i.e., meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis. Thus, the novel disease models may improve the analysis of the pathogenesis as well as the development of treatment strategies for cystic fibrosis [28, 44]. In addition, preliminary conference reports announced the production of transgenic pigs for the suppression of CFTR expression by RNA interference.
Diabetes mellitus
Rodent models for diabetes mellitus have been developed by the structural and/or functional modification of candidate genes [45] or by random mutagenesis programs [46]. Transgenic pigs were recently established as large animal models. In the context of type 2 diabetes mellitus, a chronic metabolic disorder of multiple etiologies characterized by uncontrolled hyperglycemia caused by both insulin resistance and progressive pancreatic beta-cell dysfunction [47], the two incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) attracted particular attention. GIP and GLP-1 are secreted by enteroendocrine cells in response to nutrients like fat and glucose and enhance glucose-induced insulin secretion [48]. In type 2 diabetic patients, the insulinotropic action of GIP is