US20110197290A1 - Methods and materials for producing transgenic artiodactyls - Google Patents

Methods and materials for producing transgenic artiodactyls Download PDF

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US20110197290A1
US20110197290A1 US13/025,373 US201113025373A US2011197290A1 US 20110197290 A1 US20110197290 A1 US 20110197290A1 US 201113025373 A US201113025373 A US 201113025373A US 2011197290 A1 US2011197290 A1 US 2011197290A1
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swine
gene
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Scott C. Fahrenkrug
Daniel F. Carlson
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Recombinetics Inc
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Definitions

  • the field of the invention relates to the production of transgenic artiodactyls, for instance pigs. Some aspects of the field relate to the genes manipulated to make the transgenic animal, for instance the low density lipoprotein receptor (LDLR) gene, Duchene's Muscular Dystrophy (DMD) gene, and the Hairless (HR) gene. Other aspects of the field relate to techniques for transforming swine cells.
  • LDLR low density lipoprotein receptor
  • DMD Duchene's Muscular Dystrophy
  • HR Hairless
  • Swine are an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Heightened interest in such models for human disease and in the production of transgenic livestock for biomedical applications have increased the need for improved methods for transgenesis, as well as for particular models of various diseases.
  • transgenic artiodactyl cloning have generally lacked a consistent and robust technique for making stably transfected swine cells. Once a stably transfected cell is produced, transgenic swine may be made and then used to meet a growing need for uniform animal models of human pathologies.
  • One aspect of the method is that a first group of cells may be treated to introduce an exogenous gene and then mixed with a second group of cells that have not been treated. A series of collection and selection processes may be overlaid with this step being repeated.
  • This method is exemplified herein in the context of the production of swine cells with a knockout for the low density lipoprotein receptor (LDLR), Dystrophin gene (DMD), and Hairless gene (HR) in male and female domestic and miniature swine cells.
  • LDLR low density lipoprotein receptor
  • DMD Dystrophin gene
  • HR Hairless gene
  • Transgenic animals with knockouts for LDLR, and DMD gene are described herein. These animals are useful for modeling atherosclerosis and Duchenne's muscular dystrophy, respectively. Knockouts for HR are useful for providing components for medical devices, including dermal derived biomaterials and for the use of pigs as models for transdermal drug delivery, and other applications benefiting from denuded skin.
  • An embodiment of the invention is a transgenic swine comprising a genomic disruption of an endogenous gene chosen from the group consisting of a Low-Density Lipoprotein Receptor gene (LDLR), Duchene's Muscular Dystrophy (DMD) gene, and hairless gene (HR).
  • Said genomic disruption may be engineered for preventing expression of a functional protein and/or preventing expression on any protein.
  • the swine may be homozygous or heterozygous for said disrupted gene.
  • the swine may be free of a marker gene.
  • the swine may exhibit a phenotype chosen from the group consisting of hypercholesterolemia, atherosclerosis, and atherosclerotic lesions.
  • the disruption may be inducible upon administration of an induction agent.
  • the swine may be chosen from the group consisting of pig, miniature pig, and Ossabaw pig. Tissue recovered from such a pig is included, as well as methods of recovering said tissue.
  • An embodiment is a transfected somatic swine cell comprising a disrupted gene chosen from the group consisting of a Low-Density Lipoprotein Receptor gene (LDLR), Duchene's Muscular Dystrophy (DMD) gene, and a hairless gene (HR).
  • the cell may be chosen from the group consisting of embryonic blastomere, fetal fibroblast, adult ear fibroblast, and granulosa cell.
  • a transgenic swine may be prepared by nuclear transfer of such a cell.
  • An embodiment is a method of introducing an exogenous nucleic acid into a swine cell in vitro comprising exposing a first group of swine cells to a transfection agent that comprises an exogenous nucleic acid during a first culture time period and subsequently adding a second group of swine cells to the first group for a second culture time period, wherein the second group of cells have not been exposed to the transfection agent.
  • the first group of cells may be chosen from the group consisting of primary fetal swine cells and swine fibroblasts.
  • a ratio of the second group of cells to the first group of cells may be between 1:1 and 20:1.
  • the exogenous nucleic acid may disrupts a target gene chosen from the group consisting of a Low-Density Lipoprotein Receptor gene (LDLR), Duchene's Muscular Dystrophy (DMD) gene, and a hairless gene (HR).
  • LDLR Low-Density Lipoprotein Receptor gene
  • DMD Duchene's Muscular Dystrophy
  • HR hairless gene
  • FIG. 1 depicts the LDLR knockout strategy and PCR results.
  • Panel A Schematic of wildtype (wt) and correctly targeted (Targeted) LDLR locus at exon 4 after homologous recombination with the rAAV-LDLR E4-stop replacement cassette.
  • the rAAV-LDLR E4-stop cassette contains a floxed PGK-Neo selection cassette inserted within exon 4 flanked by a 5′ homology arm of 0.9 kb and a 3′ homology arm of 0.3 kb.
  • FIG. 2 depicts the restriction analysis of the 3′-end of recombinant amplicons in the strategy of FIG. 1 .
  • a second set of PCR primers (blue and red triangle) also confirmed accurate targeting at the LDLR locus.
  • PCR amplicons were TOPO-cloned and subjected to restriction analysis and sequencing (data not shown) to confirm homology recombination. The liberation of a 0.7 Kb band from clones 1, 2, 3, and 5 confirmed the identity of the recombinant colonies.
  • FIG. 3 depicts the results of a Southern analysis of recombinant colonies. Eight colonies amplified by WGA were subjected to Southern analysis with the probe indicated in FIG. 1 panel A. The expected endogenous (E), positive control (+), and targeted band (clones marked with a asterisk) were observed, although the majority (-80%) of cells appear in each clone appear to be wild type.
  • E expected endogenous
  • (+) positive control (+
  • targeted band clones marked with a asterisk
  • FIG. 4 Confirmation of round 1 and 3 positives by PCR/Restriction analysis.
  • Panel (A) shows a correctly targeted (Targeted) LDLR locus at exon 4 after homologous recombination with the rAAV-LDLR E4 stop cassette. PCR primers for screening 5′ (open triangles) and 3′ (filled triangles) junctions are shown.
  • Panel (B) depicts 5′ junction PCR performed on WGA DNA from 7 and 8 colonies identified in the primary PCR screen of round 1 and 3, respectively.
  • PCR from correctly targeted clones was expected to produce the 1735 by and its identity is verified by restriction digest with EcoRI (labeled “E” in panel A) resulting in 3 fragments (748, 607 and 380 bp) of which the 748 by band is indicated. WGA DNA from a negative colony was used as the negative control ( ⁇ ).
  • Panel (C) depicts 3′ PCR from correctly targeted colonies that produced a band of 999 by and with its identity as verified by restriction digest with XhoI resulting in 2 fragments of 842 (indicated) and 157 bp. Two positive (++ ⁇ 150 copies, +15 copies) and two negative controls (WGA DNA from a negative colony) are shown.
  • FIG. 5 Confirmation of round 1 and 3 positives by Southern Blotting: Candidates identified by PCR ( FIG. 4 ) were subjected to WGA/Southern blotting. Restriction digest with EcoRI releases a fragment of 2.8 kb in wild type (Wt) cells, and a 4.0 kb fragment for correctly targeted (Targeted) cells (see FIG. 1 for schematic). Each colony identified confirmed positive by 5′ and 3′ junction PCR ( FIG. 4 ) displayed signal characteristic of a correctly targeted clone. Some variation in signal indicates not all colonies are pure, however, clones 8, 10, 13 and 15 appear to contain a majority of heterozygous LDLR knockout cells.
  • FIG. 6 DMD Exon 7 Replacement.
  • Panel (A) is a schematic that shows both wild type (Wt) and a correctly targeted (Targeted) DMD locus at exon 7 after homologous recombination with the rAAV-DMD E7R replacement cassette.
  • the rAAV-DMD E7R cassette contains a floxed PGK-Neo selection cassette flanked by approximately 1kb homology arms upstream and downstream of exon 7. Homologous recombination between the DMD locus and rAAV-DMD E7R will result in the complete ablation of exon 7 and a frame shift in the full length dystrophin isoform.
  • Panel (B) shows that G-418 resistant colonies were screened for gene targeting by amplification of junctions between the DMD locus and the PGK-Neo cassette. Both 5′ and 3′ junctions could be detected with separate primer pairs (panel A) 5′ primers open triangles, 3′ primers filled triangles, allowing for confirmation of replacement rather than insertion at exon 7. Several positive signals are observed for both the 5′ and 3′ ends, often from identical wells confirming the presence of correctly targeted cells. Both positive (++ ⁇ 3,000 copies, +30 copies) and negative ( ⁇ wild type genomic DNA only) controls are shown in the bottom right corner of each gel. Panel (C) shows candidates identified by PCR (panel B) and subjected to WGA/Southern blotting.
  • Clones correspond to the numbering in panel (B) (clone 1 not shown), and lanes labeled 1′ and 2′ are simply replicates created with half volume WGA reactions. Restriction digest with EcoRI and NcoI (indicated as “E” and “N” in panel A respectively) will release a fragment of 6.2 kb in wild type cells and a 3.3 kb fragment for correctly targeted cells.
  • the strong band observed in for wells 1, 2 and 4 above the 3.3 kb “targeted” band is the predicted size of head to tail concatemers of the rAAV-DMD E7R construct. Each well contains at some signal at 3.3 kb while wells 3 and 5 contain mostly targeted cells.
  • FIG. 7 Confirmation of round 2 positives by PCR/Restriction analysis:
  • the panel (A) schematic shows a correctly targeted (Targeted) dystophin locus at exon 7 after homologous recombination with the rAAV-DMD E7R replacement cassette. PCR primers for screening 5′ (open triangles) and 3′ (filled triangles) junctions are shown.
  • Panel (B) shows a 5′ junction PCR that was performed on WGA DNA from 11 colonies identified in the primary PCR screen. PCR from correctly targeted clones will produce the expected 1201 by and its identity is verified by restriction digest with Spel (labeled “S” in panel A) resulting in 3 fragments of which the 947 by band is indicated.
  • FIG. 8 Confirmation of round 2 positives by Southern Blotting.
  • Panel (A) is a schematic of gene targeting at exon 7 of the DYSTROPHIN locus.
  • Panel (B) shows results for candidates identified by PCR ( FIG. 7 ) and subjected to WGA/Southern blotting. Eleven male colonies (7-16) and one female colony (17) are shown. Restriction digest with EcoRI and NcoI (indicated as “E” and “N” in panel A respectively) will release a fragment of 6.2 kb in wild type cells and a 3.3 kb fragment for correctly targeted cells. Each colony, with the exception of 8 and 11 (indicated with arrows) gave positive signal for both 5′ and 3′ junctions.
  • FIG. 9 The porcine Hairless gene (HR) and knockout strategy.
  • the wild type (Wt) HR gene is comprised of 18 exons, and is located on chromosome 14. The area surrounding exon 2 is highlighted and enlarged.
  • a premature stop codon (TGA) was introduced into exon 2 by rAAV-Homologous recombination to ablate full length HR protein in pigs by truncation of the protein.
  • the pAAV-HR TGA vector includes the majority of exon 2 and homology arms both up and downstream of exon 2.
  • two version of the HR TGA have been constructed, one with a neomycin (Neo) resistance cassette, another with a puromycin (Puro) resistance cassette.
  • Panel (B) This schematic shows the structure of the targeted HR TGA allele.
  • the HR TGA allele will interfere with full length HR production in two ways; 1) translation will be terminated at the engineered TGA stop codon 2) skipping of exon 3 by alternative splicing between exons 1 and 3 will cause a frame shift mutation.
  • FIG. 10 LDLR Partial CDS from MARC library est sequenced by Applicant. (SEQ ID NO:1).
  • FIG. 11 LDLR HinDIII Subclone sequence (includes exons 2-5). (SEQ ID NO:2).
  • One embodiment of the invention is a method of transfecting an artiodactyl cell.
  • a first group of artiodactyl cells may be treated to introduce an exogenous gene and then mixed with a second group of artiodactyl cells that have not been so treated. This process has been observed to produce significant efficiencies and reproducibility.
  • a series of working examples are set forth below, followed by a more detailed overview of this embodiment.
  • the working examples are also embodiments of the invention. These examples describe stably transfected swine cells made with various transformations. These cells may be used to make transgenic animals, which are useful for many purposes including animal models of human diseases and conditions and sources of tissue.
  • Hypercholesterolemia Swine Model Transgenic Pigs with LDLR Gene Modification
  • Cardiovascular disease is a leading cause of death and dysfunction in the United States, with coronary artery disease being a major contributor.
  • Animal models are fundamental to understanding the mechanisms of atherosclerosis.
  • the development of new therapies relies heavily on the use of these models.
  • Unfortunately there is a lack of suitable large-animal models for studying new therapies or testing them.
  • several stent-drug combinations have been successful in animal studies but failed in subsequent human clinical trials.
  • Trials that are successful in the animal models that subsequently fail in human trials may be explained by unfaithful replication of human disease pathology in the animal models.
  • Hypercholesterolemia is a principal cause of atherosclerosis. Rabbits, swine and rhesus monkeys with genetic mutations linked to hypercholesterolemia have been used to study atherosclerosis and recent research has focused on genetically modified mice. However, genetically modified mice that manifest hypercholesterolemia do not exhibit lesions typical of atherosclerosis in humans. Some inbred swine with defective Low-Density Lipoprotein Receptors (LDLR) do develop lesions but do not show a consistent and predictable manifestation of the disease.
  • the Low-Density Lipoprotein Receptor (LDLR) is a cell surface receptor that mediates the endocytosis of cholesterol-rich low density lipoprotein (LDL).
  • the LDLR protein is encoded by the LDLR gene and was implicated as having a role in familial hypercholesterolemia (Brown M S, Goldstein J L (1984). “How LDL Receptors Influence Cholesterol and Atherosclerosis”. Scientific American 251: 52-60.).
  • transgenic swine cells were made with a defect in LDLR expression.
  • Transgenic animals may be made from these cells using any of a variety of standard technique known to artisans in these fields.
  • the production of a functional LDLR gene product was disrupted by introduction of a stop cassette within LDLR exon 4 by Adeno-associated virus (rAAV) homologous recombination (HR).
  • An rAAV HR cassette (rAAV-LDLR-E4-stop) was generated with a PGK-Neo selection cassette inserted within LDLR exon 4 at the XhoI restriction site ( FIG. 1 ).
  • the second transduction was performed as with the first transduction with a few exceptions, as follows. PFF cells were plated on 6-well plates 24 hours prior to transduction to achieve a density of 30% confluence. Twenty-five microliters of rAAV-LDLR E4-stop viral supernatant was added to 1 ml growth medium and were plated onto five 96-well plates at the density of 1,000 cells/well 24 hours later. Cells were allowed to recover overnight prior to selection in medium containing 300 ug/m1 G418. On day 15, most wells were 100% confluent and cells were split and plated on replicate 96-well plate wells. When wells were 100% confluent, cells from one replicate were collected in lx PCR lysis buffer.
  • PCR analysis was conducted on the 5 ⁇ l and 25 ⁇ l transduction plates from the first transduction and the 25 ⁇ l transduction plates from the second transduction using primers designed to amplify across the 3′ junction ( FIG. 1 Panel A). Approximately 50% of wells were confluent in the first transduction 5 ⁇ l transduction plates, therefore, it would be expected that most wells would harbor one G-418 resistant colony. Unfortunately, no positive wells were detected from the 5 ⁇ l plates suggesting that homologous recombination at the LDLR locus had occurred in less than 1 in 250 G-418 resistant colonies.
  • PCR products from 4 of 5 of the strong positives were confirmed by restriction digestion ( FIG. 2 ). These clones were further analyzed by Southern hybridization ( FIG. 3 ) and identified the expected band for the correctly targeted locus. However, in pure knockout colonies, a 50:50 ratio of intensity between knockout and wild type alleles were expected, thus clones with the expected knockout allele appear to be confounded by the presence of wt cells, ⁇ 80% based on signal intensity ( FIG. 3 ).
  • LDLR knockouts were made with LDLR knockouts. Specifically, the following clones contained cells with heterozygous knockout of the LDLR locus; M: clone 1 & F: clones 8, 10, 11, 13, 14, and 15. In addition, the male clones 1 and 7 may contain the knockout, but failed to be verified by WGA: Southern analysis. The cells may be cloned into male and female pigs by Somatic Cell nuclear transfer, Chromatin transfer or other suitable techniques. These founders may then be bred to create pigs homozygous for knockout of the LDLR gene.
  • These techniques may be used to produce animals that are homozygous or heterozygous for the disrupted gene; cells that have a marker gene or are free of a marker gene, a swine that exhibits a phenotype chosen from the group consisting of hypercholesterolemia, atherosclerosis, and atherosclerotic lesions (including any combination thereof), wherein the disrupted LDLR gene is disrupted at exon 4, and wherein all of the LDLR genes in the swine are disrupted.
  • apolipoprotein E Apolipoprotein E gene that are associated with a predisposition for high plasma LDL-cholesterol in patients, i.e., they encode arginine residues at positions 112 and 158 that correspond to the deleterious ApoE4 isoform.
  • Rapacz identified “naturally” occurring hypercholesterolemia in farm pigs leading to atherosclerotic plaques in aged pigs.
  • the causative mutations were identified in two familial hypercholesterolemic pig lines including an alternative apolipoprotein B (ApoB) allele and a missense mutation in exon 4 of the low density lipoprotein receptor (LDLR) (R84C).
  • Ossabaw pigs are an animal model of hypercholesterolemia; while useful, genetic engineering of LDDR defects in this breed and others will be have a number of advantages for improving these models.
  • One advantage is a severe and rapid onset of dyslipidemia, considering the conservation of LDLR in pigs and humans, as well as the predicted predisposition to high plasma LDL-cholesterol.
  • the motifs known for truncating, ablating, or otherwise disrupting LDDR in swine, humans, and mice may accordingly be applied in the creation of a transfected cell and a transgenic swine.
  • SEQ ID NO:1 was used to generate a probe for BAC library screening and recovery of a genomic clone containing the pig LDLR gene.
  • SEQ ID NO:2 is a HinD III subclone from the LDLR BAC that encompasses exons 2-5. Once available, this sequence was verified by comparison to GenBank: FP102365.2.
  • dystrophin a 427 kDa rod-shaped protein having four domains: an N-terminal actin binding domain, 24 triple helix spectrin-like repeats with four hinge regions, a cysteine-rich domain with two potential calcium binding motifs, and a unique C-terminal domain (Koenig et al.).
  • dystrophin forms a linkage between the cytoskeletal actin and a group of membrane proteins, as well as with a number of non-membranal proteins (collectively called dystrophin associated proteins; DAPs) (Yoshida et al. , Ervasti et al.).
  • the N-terminal domain binds to the cytoskeletal actin and the association with the DAPs is mediated mainly by the cysteine-rich and C-terminal domains of dystrophin (Suzuki et al., Jung et al.).
  • DAPs a-dystroglycan
  • this complex links the cytoskeleton, the sarcolemma and the extracellular matrix (Ahn et al., Campbell, Ozawa et al.).
  • the Dystrophin gene also codes for two non-muscle isoforms of dystrophin, each controlled by a different promoter located in the 5′ end region of the gene; the brain type dystrophin (Nudel et al., Barnea et al., Boyce et al.) and Purkinje cell type dystrophin (Gorecki et al.).
  • brain type dystrophin Nudel et al., Barnea et al., Boyce et al.
  • Purkinje cell type dystrophin (Gorecki et al.).
  • internal promoters located within downstream introns for the dystrophin gene regulate the expression of smaller products.
  • Dp71 a 70.8 kDa protein, consists of only the cysteine-rich and C-terminal domains of dystrophin (Bar et al., Lederfein et al.).
  • Dp71 is found in the brain (Rapaport et al., Greenberg et al.).
  • the other known small products of the dystrophin gene consist of the cysteine-rich and C-terminal domains with various extensions into the spectrin-like repeats domain. These products are: Dp116 (Byers et al.), Dp140 (Lidov et al.), and Dp260 (D'Souza et al.), which are expressed mainly in Schwann cells, brain, and retina, respectively, and have molecular weights of 116, 140 and 260 kDa.
  • the functions of the non-muscle dystrophins and of the smaller products of the dystrophin gene are not known.
  • Rodent models of dystrophin have proven invaluable in defining the complexity of muscle disease, and enabled the development of several promising therapeutic strategies for DMD.
  • muscle degeneration in the mdx mouse model is mild in comparison to DMD patients.
  • mdx mice are mobile, they do not have significant fibrosis or joint contractures, and the skeletal myofibers are only partially replaced by adipose cells later in life.
  • the myotendinous junctions are severely impaired in DMD patients (Bell, C. D. and Conen, Hasegawa et al., Nagao et al.), but only have minor alterations in maturation and maintenance in mdx mice (Law and Tidbal).
  • mdx mice One explanation for the mild phenotype of mdx mice is that the functional requirement of dystrophin to transmit muscle forces may be minimal given their small and weak stature in comparison to humans. Satellite cells also retain their regenerative potential better in mdx mice than in DMD patients, so may more actively repair damaged tissue. Another possibility is that homologous proteins (such as utrophin) can compensate more effectively for the absence of dystrophin in mice. Consistent with this hypothesis, two independent laboratories generated mice lacking both dystrophin and utrophin to generate a more severe model of DMD (Deconinck et al., Grady et al.).
  • mdx:utrn ⁇ / ⁇ mice are smaller than wild-type mice, develop severe kyphosis, and become less mobile with age (Deconinck et al., Grady et al.) and they develop an inflammatory response in the skeletal musculature (Deconinck et al., Grady et al. In these double knockout mice, many of the muscle fibers are replaced by fibrotic tissue that contributes to joint contractures (Deconinck et al., Grady et al.).
  • clear differences in the size, stem cell dynamics, and requirement for dystrophin function argue against the continued reliance on rodent models.
  • Some of the muscles have high concentrations of crystalline calcium and hyaline (Cooper et al., Nguyen et al.) and muscle fibers begin to be replaced by fibrotic tissue and adipose cells at approximately 2 months of age (Nguyen et al.). Joint contractures are prominent by 6 months and mobility is severely impaired. The muscles are atrophic, weaker, and more susceptible to contraction-induced injury (Nguyen et al., Childers et al.). GRMD dogs develop cardiomyopathy (Chetboul and Carlos, et al., Chetboul and Escriou et al.) and respiratory distress that can lead to death (Valentine et al., 1991).
  • GRMD dogs display a mosaic expression of truncated dystrophins with deletions from exons 2-10 and 4-13 (Schatzberg et al.), although expression becomes somewhat more uniform with age (Cooper et al.). These truncated dystrophins lack part of the N-terminal actin binding domain, hinge 1, spectrin repeat 1, and part of spectrin repeat 2.
  • the N-terminal actin-binding domain of dystrophin is important for dystrophin expression and function (Banks et al., Beggs et al., Le et al., Chelley et al., Le et al., Matsumra et al., Muntoni et al., Novakic et al., Prior et al., Takeshima et al., Winnard et al.).
  • This method is described in detail herein in the context of the production of swine cells with a knockout for the dystrophin gene in male and female domestic and miniature swine cells.
  • These cells may be used in nuclear transfer to produce DMD ⁇ /+founder animals that are bred and expanded through breeding and then used to meet a growing need of medical device and pharmaceutical companies for uniform animal models of human pathologies that can help predict the outcome of human therapeutic interventions.
  • the porcine dystrophin gene was disrupted by recombinant Adeno-associated virus (rAAV) homologous recombination to produce a model of muscular dystrophy in swine. Homologous recombination between the rAAV cassette and the dystrophin gene would result in the replacement of exon 7 with a PGK-Neo selection cassette ( FIG. 6 ). The absence of exon 7 creates a frame shift in the full length dystrophin transcript eliminating the production of the Dp427 dystrophin isoform.
  • rAAV Adeno-associated virus
  • rAAV-DMD E7R rAAV replacement cassette
  • FIG. 6 Panel A The experimental approach involved creation of a rAAV replacement cassette (rAAV-DMD E7R) for targeting of DMD exon 7 using a fusion PCR technique described in Kohil et al., 2004 ( FIG. 6 Panel A).
  • Viral packaging was conducted by co-transfection of AAV-293 cells with plasmids: rAAV-DMD E7R, pAAV-RC, and pAAV-helper. Two days after transfection, cells from one 100 mm plate were lysed in 1 ml of growth media by 3x freeze thaw cycles and stored at ⁇ 80° C. in 300 microliter aliquots.
  • Viral transduction methods Early passage pig fetal fibroblasts (PFF) were plated at a density of 30,000 cells/cm 2 in a single well of a six-well plate to achieve 70-80% confluence within 24 hours. Media was changed 1 hour prior to transduction and replaced with 1 ml of fresh growth medium. One hundred-fifty microliters of viral lysate was added to a single well and incubated under standard growing conditions. After a 24 hour incubation, cells were washed 3x with PBS, trypsinized and plated onto 96 well plates at densities ranging from 250 cells/well to 2,000 cells/well. Plates seeded at low density were adjusted to 1,000 cells per well with wild type fibroblasts to enhance plating efficiency.
  • PFF fetal fibroblasts
  • medium containing 300 ⁇ g/m1 G-418 was added and changed 3 ⁇ in the course of two weeks.
  • the surface area in the 96-well plate in this and other experiments was about 0.33 cm 2 per well, so that a density of 1000 cells per well is equal to about 3,000 cells per cm 2 , so that densities ranged from about 800 to about 6000 cells per cm 2 .
  • PCR Screen methods After two weeks of selection in G-418, cells were trypsinized and divided 50:50 between a 96-well PCR plate and a 96-well growth plate. Cells in the PCR plate were pelleted and resuspended in 25 ⁇ l of lysis buffer while the growth plate was returned to the incubator. PCR was conducted between the PGK-Neo cassette and primers located outside of both the 5′ and 3′ homology arms ( FIG. 6 Panel B). PCR positive wells were allowed to grow to confluence prior to trypsinization followed by removal of cells for Whole Genome Amplification (WGA)/Southern blotting and cryopreservation.
  • WGA Whole Genome Amplification
  • Second Transduction A second infection was conducted in both male and female PFF. Transduced cells were plated at densities of 100/well, 200/well, and 500/well on 96-well plates (5 replicates for each density and sex), supplemented with wild type cells to a total of 1,000 cells per well, and selected in G-418 for two weeks. Neomycin resistant colonies appeared in approximately 40, 60, and greater than 90 percent of wells in the 100, 200 and 500 plates respectively (Table 5). Primary PCR screening was performed on the 100 and 200 plates resulting in 13 and 2 positive wells for male and female cells respectively (Table 6). The healthy (11 male and 1 female) colonies were cryopreserved and a portion was set aside for WGA.
  • the results show a successful knockout of the dystrophin gene in male and female fibroblasts.
  • These cells are a suitable resource for Somatic cell nuclear transfer and may be used to create founders.
  • the founders and transgenic wine progeny may have a disrupted DMD gene and exhibit a muscular dystrophy phenotype.
  • the DMD gene may be disrupted, for instance, at exon 7, or at other sites that are known to disrupt production of a functional DMD gene product or which have already been established to produce a muscular dystrophy phenotype in other animals. Some or all of the DMD genes may be disrupted.
  • the gene disruption may be performed to prevent expression of a functional Dp427 dystrophin isoform.
  • a pig model of DMD may be derived from the introduction of mutant alleles most common amongst DMD patients into the pig dystrophin locus by homologous recombination, which are know, e.g., see DYSTROPHIN; DMD-OMIM, which is hereby incorporated herein by reference for all purposes; in case of conflict, the specification is controlling (also Tuffery-Giraud, 2009).
  • 1 ⁇ 3 of the cases of DMD result from a de novo mutation, for which neither parent is a carrier, as is known, see DYSTROPHIN; DMDallelic variants-OMIM, which is hereby incorporated herein by reference for all purposes; in case of conflict, the specification is controlling.
  • Pig hair is a problematic contaminant for both meat production and derivation of skin derived products. Whereas wild species of swine require hair for protection from the sun, hair is not required for the well-being of modern commercial swine.
  • the hairless (HR) gene encodes a nuclear receptor corepressor that is required for hair growth. Humans and rodents lacking a functional HR gene are born with hair, but are unable to regenerate hair follicles resulting in congenital hair loss early in life (Thompson 2009).
  • swine cells were made with a disrupted porcine hairless gene (HR) using recombinant Adeno-associated virus (rAAV) homologous recombination. The resultant transfected cells may be used to produce pigs lacking hair by somatic cell nuclear transfer or chromatin transfer.
  • An rAAV replacement cassette (pAAV-ssHRTGA) was created for targeting of swine HR exon 2 using a fusion PCR technique described in Kohil et al. 2004.
  • Viral packaging was conducted by co-transfection of AAV-293 cells with plasmids: pAAV-ssHRTGA, pAAV-RC, and pAAV-helper. Two days after transfection, cells from one 100 mm plate were lysed in 1 ml of growth media by 3 ⁇ freeze thaw cycles and stored at ⁇ 80° C. in 300 microliter aliquots.
  • FIG. 9 depicts the porcine Hairless gene (ssHR) and knockout strategy.
  • Panel (A) depicts the wild type (Wt) ssHR gene, which is comprised of 18 exons, and is located on chromosome 14. The area surrounding exon 2 is highlighted and enlarged.
  • a premature stop codon (TGA) was introduced into exon 2 by rAAV-Homologous recombination to ablate full length ssHR protein in pigs by truncation of the protein.
  • the pAAV-ssHR TGA vector includes the majority of exon 2 and homology aims both up and downstream of exon 2.
  • Panel (B) is a schematic that shows the structure of the targeted ssHR TGA allele.
  • the ssHR TGA allele may be used to interfere with full length ssHR production in two ways; by (1) translation terminated at the engineered TGA stop codon and by (2) skipping of exon 3 by alternative splicing between exons 1 and 3 to cause a frame shift mutation.
  • Embodiments include a swine wherein the disrupted endogenous gene is ssHR.
  • the swine may exhibit a phenotype chosen from the group consisting of hairlessness and reduced hair (one or both).
  • the swine HR gene may be disrupted at ssHR exon 2.
  • the HR gene may also be mutated at other locations as in humans (artisans are able to identify suitable sequences as at, e.g., HAIRLESS, MOUSE, HOMOLOG OF; HR-OMIM, which is hereby incorporated herein by reference for all purposes; in case of conflict, the specification is controlling) by homologous recombination, ZFN, TALENs, to create point mutations, frame shift mutation or early termination resulting in varying levels of severity.
  • swine cells may be conveniently transfected.
  • a first group of artiodactyl cells may be transfected and then mixed with a second group of artiodactyl cells that have not been so treated.
  • Conventional approaches rely on treating as many cells as possible to enhance the odds that a cell with a desired genetic trait can be found.
  • the presence of the untreated cells is believed to produce autocrine and/or paracrine factors that enhance cell survival or cell phenotype, e.g., activation of more preferable DNA repair pathways.
  • a first embodiment of the method involves introducing an exogenous nucleic acid into a swine cell in vitro comprising exposing a first group of swine cells to a vector that comprises an exogenous nucleic acid during a first culture time period and subsequently adding a second group of swine cells to the first group for a second culture time period, wherein the second group of cells have not been exposed to the vector.
  • the first group and the second group after being combined, are referred to as a collection, or mixed collection.
  • the mixed collection may be subjected to another round of transfection.
  • One method involves exposing the first group of cells to transfection agents and then splitting the group into a plurality of cultures.
  • the cultures of the first cell group may be prepared at various seeding densities and allowed to grow for a time period and/or until a desired level of confluence is achieved.
  • a second group of cells may be added to the first group to achieve an overall seeding density and/or after seeding to achieve a desired cell concentration.
  • This second group may be cells that have not been exposed to the transfection agents, and may be wild type cells.
  • the wild type cells may be from the same animal as the first group of cells, or from a different animal of the same or different species. Any of the groups may also be from a pool of animals, for instance a plurality of swine fetuses.
  • the wild type cells may also be from a culture of cells, or a primary or secondary cell culture line. Accordingly, the term wild-type in this specific context of mixing with a group of transfected cells is broad and includes cells transfected by other means.
  • the term native wild-type refers to wild-type cells that have never been modified.
  • the ratio of wild-type cells to the first group of cells may be, for example, between 0.1:1 and 100:1, or between 0.5:1 and 10:1, or between about 1:1 and about 20:1; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated.
  • the first group of cells may be seeded at a first seeding density and wild-type cells co-cultured to achieve a total density or confluence.
  • a group of cells may be exposed to transfection agents and then seeded into a plurality of cultures at a seeding density (referring to a concentration per area of cells), e.g., from about 100 to about 10,000 cells per cm 2 ; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., about 500 or about 8,000 to about 7,000 or about 10,000. Wild-type cells may then be added to bring the cells to a predetermined concentration, e.g., to a value between 1000 and 100,000 cells/cm 2 .
  • the wild-type cells may be added before, during, or after the seeding of the first group of cells. Accordingly, embodiments include seeding the first group of cells and the wild type cells within a 24-hour time period, or at essentially the same time. Embodiments also include seeding the wild-type cells at a time between about 1 day and about 1 week before introduction of the first group of cells exposed to the transfection agents. And embodiments also include seeding the wild-type cells at a time between about 1 day and two weeks after seeding of the first group of cells. Artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated.
  • the cells may be somatic or germ cells.
  • the cells may be an artiodactyl cell, e.g., pig, miniature pig, Ossabow pig.
  • the cells may be adult, juvenile, or fetal, and from any of a variety of tissue sources, e.g., fibroblasts, dermal fibroblasts, dermal, epidermal, mesodermal, mesenchymal, endothelial, vascular, hepatocyte.
  • transfection techniques may be used to transfect a cell with an exogenous nucleic acid that disrupts a target gene, e.g., by introduction of a stop codon or by way of other techniques commonly available to an artisan skilled in the art of preventing expression of a nucleic acid in a cell.
  • the genes in the cell may be modified, e.g., LDLR, DMD, and HR.
  • transgenic artiodactyls e.g., pigs, sheep, goats, and cows.
  • the nucleated cells of the transgenic artiodactyls contain a nucleic acid construct.
  • transgenic artiodactyl includes founder transgenic artiodactyls as well as progeny of the founders, progeny of the progeny, and so forth, provided that the progeny retain the nucleic acid construct.
  • a transgenic founder animal can be used to breed additional animals that contain the nucleic acid construct.
  • Transgenic pigs are particularly useful.
  • Embodiments of the invention include a tissue obtained from the transgenic artiodactyls (e.g., transgenic pigs) and cells derived from the transgenic artiodactyls (e.g., transgenic pigs).
  • the term derived from indicates that the cells can be isolated directly from the animal or can be progeny of such cells.
  • an embodiment of the invention is a brain, lung, liver, pancreas, islets, heart and heart valves, muscle, kidney, thyroid, corneal, skin, blood vessel or other connective tissue obtained from a transgenic artiodactyl (e.g., transgenic pig).
  • Blood and hematopoietic cells Islets of Langerhans, beta cells, brain cells, hepatocytes, kidney cells, and cells from other organs and body fluids, for example, also can be derived from transgenic artiodactyls (e.g., transgenic pigs).
  • transgenic artiodactyls e.g., transgenic pigs
  • nucleic acid constructs into non-human animals to produce founder lines, in which the nucleic acid construct is integrated into the genome.
  • Such techniques include, without limitation, pronuclear microinjection (U.S. Pat. No. 4,873,191), retrovirus mediated gene transfer into germ lines (Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6148-1652), gene targeting into embryonic stem cells (Thompson et al. (1989) Cell 56, 313-321), electroporation of embryos (Lo (1983) Mol. Cell. Biol. 3, 1803-1814), sperm mediated gene transfer (Lavitrano et al. (2002) Proc.
  • somatic cells such as cumulus or mammary cells, or adult, fetal, or embryonic stem cells, followed by nuclear transplantation (Wilmut et al. (1997) Nature 385, 810-813; and Wakayama et al. (1998) Nature 394, 369-374).
  • Pronuclear microinjection, sperm mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques.
  • a nucleic acid construct is introduced into a fertilized egg; 1 or 2 cell fertilized eggs are used as the pronuclei containing the genetic material from the sperm head and the egg are visible within the protoplasm.
  • Pronuclear staged fertilized eggs can be obtained in vitro or in vivo (i.e., surgically recovered from the oviduct of donor animals).
  • In vitro fertilized eggs can be produced as follows. For example, swine ovaries can be collected at an abattoir, and maintained at 22-28° C. during transport.
  • Ovaries can be washed and isolated for follicular aspiration, and follicles ranging from 4-8 mm can be aspirated into 50 mL conical centrifuge tubes using 18 gauge needles and under vacuum. Follicular fluid and aspirated oocytes can be rinsed through pre-filters with commercial TL-HEPES (Minitube, Verona, Wis.).
  • Oocytes surrounded by a compact cumulus mass can be selected and placed into TCM-199 OOCYTE MATURATION MEDIUM (Minitube, Verona, Wis.) supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid, 50 ⁇ M 2-mercaptoethanol, 0.5 mg/ml cAMP, 10 ng/mL each of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) for approximately 22 hours in humidified air at 38.7° C. and 5% CO 2 .
  • PMSG pregnant mare serum gonadotropin
  • hCG human chorionic gonadotropin
  • the oocytes can be moved to fresh TCM-199 maturation medium which will not contain cAMP, PMSG or hCG and incubated for an additional 22 hours. Matured oocytes can be stripped of their cumulus cells by vortexing in 0.1% hyaluronidase for 1 minute.
  • Mature oocytes can be fertilized in 500 ⁇ l Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, Wis.) in Minitube 5-well fertilization dishes.
  • IVF in vitro fertilization
  • freshly-collected or frozen boar semen can be washed and resuspended in PORCPRO IVF Medium to 4 ⁇ 10 5 sperm.
  • Sperm concentrations can be analyzed by computer assisted semen analysis (SPERMVISION, Minitube, Verona, Wis.).
  • Final in vitro insemination can be performed in a 10 ⁇ l volume at a final concentration of approximately 40 motile sperm/oocyte, depending on boar.
  • Linearized nucleic acid constructs can be injected into one of the pronuclei then the injected eggs can be transferred to a recipient female (e.g., into the oviducts of a recipient female) and allowed to develop in the recipient female to produce the transgenic animals.
  • a recipient female e.g., into the oviducts of a recipient female
  • in vitro fertilized embryos can be centrifuged at 15,000 ⁇ g for 5 minutes to sediment lipids allowing visualization of the pronucleus.
  • the embryos can be injected with approximately 5 picoliters of the transposon/transposase cocktail using an Eppendorf FEMTOJET injector and can be cultured until blastocyst formation ( ⁇ 144 hours) in NCSU 23 medium (see, e.g., PCT Publication No. 2006/036975). Rates of embryo cleavage and blastocyst formation and quality can be recorded.
  • Embryos can be surgically transferred into uteri of asynchronous recipients.
  • anesthesia can be induced with a combination of the following: ketamine (2 mg/kg); tiletamine/zolazepam (0.25 mg/kg); xylazine (1 mg/kg); and atropine (0.03 mg/kg) (all from Columbus Serum).
  • the recipients While in dorsal recumbency, the recipients can be aseptically prepared for surgery and a caudal ventral incision can be made to expose and examine the reproductive tract.
  • 100-200 (e.g., 150-200) embryos can be deposited into the ampulla-isthmus junction of the oviduct using a 5.5-inch TOMCAT® catheter.
  • a transgenic artiodactyl cell e.g., a transgenic pig cell
  • a transgenic artiodactyl cell such as an embryonic blastomere, fetal fibroblast, adult ear fibroblast, or granulosa cell that includes a nucleic acid construct described above
  • Oocytes can be enucleated by partial zona dissection near the polar body and then pressing out cytoplasm at the dissection area.
  • an injection pipette with a sharp beveled tip is used to inject the transgenic cell into an enucleated oocyte arrested at meiosis 2.
  • oocytes arrested at meiosis 2 are termed “eggs.”
  • the porcine embryo After producing a porcine embryo (e.g., by fusing and activating the oocyte), the porcine embryo is transferred to the oviducts of a recipient female, about 20 to 24 hours after activation. See, for example, Cibelli et al. (1998) Science 280, 1256-1258 and U.S. Pat. No. 6,548,741.
  • recipient females can be checked for pregnancy approximately 20-21 days after transfer of the embryos.
  • Standard breeding techniques can be used to create animals that are homozygous for the target nucleic acid from the initial heterozygous founder animals. Homozygosity may not be required, however.
  • Transgenic pigs described herein can be bred with other pigs of interest.
  • a nucleic acid of interest and a selectable marker can be provided on separate transposons and provided to either embryos or cells in unequal amount, where the amount of transposon containing the selectable marker far exceeds (5-10 fold excess) the transposon containing the nucleic acid of interest.
  • Transgenic cells or animals expressing the nucleic acid of interest can be isolated based on presence and expression of the selectable marker. Because the transposons will integrate into the genome in a precise and unlinked way (independent transposition events), the nucleic acid of interest and the selectable marker are not genetically linked and can easily be separated by genetic segregation through standard breeding. Thus, transgenic animals can be produced that are not constrained to retain selectable markers in subsequent generations, an issue of some concern from a public safety perspective.
  • PCR Polymerase chain reaction
  • PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA.
  • Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. PCR is described in, for example PCR Primer: A Laboratory Manual , ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.
  • Nucleic acids also can be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplified. See, for example, Lewis (1992) Genetic Engineering News 12,1; Guatelli et al. (1990) Proc. Natl. Acad.
  • embryos can be individually processed for analysis by PCR, Southern hybridization and splinkerette PCR (see, e.g., Dupuy et al. Proc Natl Acad Sci USA (2002) 99(7):4495-4499).
  • RNA expression of a nucleic acid sequence encoding a polypeptide in the tissues of transgenic pigs can be assessed using techniques that include, for example, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, Western analysis, immunoassays such as enzyme-linked immunosorbent assays, and reverse-transcriptase PCR (RT-PCR).
  • Northern blot analysis of tissue samples obtained from the animal in situ hybridization analysis
  • Western analysis Western analysis
  • immunoassays such as enzyme-linked immunosorbent assays
  • RT-PCR reverse-transcriptase PCR
  • nucleic acids may be introduced into the swine cells, for knockout purposes, or to obtain expression of a gene for other purposes.
  • Nucleic acid constructs that can be used to produce transgenic animals include a target nucleic acid sequence.
  • nucleic acid includes DNA, RNA, and nucleic acid analogs, and nucleic acids that are double-stranded or single-stranded (i.e., a sense or an antisense single strand).
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid.
  • Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and 5-bromo-2′-doxycytidine for deoxycytidine.
  • Modifications of the sugar moiety include modification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained.
  • deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
  • the target nucleic acid sequence can be operably linked to a regulatory region such as a promoter.
  • Regulatory regions can be porcine regulatory regions or can be from other species.
  • operably linked refers to positioning of a regulatory region relative to a nucleic acid sequence in such a way as to permit or facilitate transcription of the target nucleic acid.
  • promoter can be operably linked to a target nucleic acid sequence.
  • tissue-specific promoters include, without limitation, tissue-specific promoters, constitutive promoters, and promoters responsive or unresponsive to a particular stimulus.
  • tissue specific promoters can result in preferential expression of a nucleic acid transcript in ⁇ cells and include, for example, the human insulin promoter.
  • tissue specific promoters can result in preferential expression in, for example, hepatocytes or heart tissue and can include the albumin or alpha-myosin heavy chain promoters, respectively.
  • a promoter that facilitates the expression of a nucleic acid molecule without significant tissue- or temporal-specificity can be used (i.e., a constitutive promoter).
  • a beta-actin promoter such as the chicken &-actin gene promoter, ubiquitin promoter, miniCAGs promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or 3-phosphoglycerate kinase (PGK) promoter can be used, as well as viral promoters such as the herpes virus thymidine kinase (TK) promoter, the SV40 promoter, or a cytomegalovirus (CMV) promoter.
  • TK herpes virus thymidine kinase
  • CMV cytomegalovirus
  • a fusion of the chicken ⁇ actin gene promoter and the CMV enhancer is used as a promoter. See, for example, Xu et al. (2001) Hum. Gene Ther. 12(5):563-73; and Kiwaki et al. (1996) Hum. Gene Ther. 7(7):821-30.
  • an inducible promoter is the tetracycline (tet)-on promoter system, which can be used to regulate transcription of the nucleic acid.
  • tet tetracycline
  • a mutated Tet repressor (TetR) is fused to the activation domain of herpes simplex VP 16 (transactivator protein) to create a tetracycline-controlled transcriptional activator (tTA), which is regulated by tet or doxycycline (dox).
  • tetR mutated Tet repressor
  • tTA tetracycline-controlled transcriptional activator
  • dox tetracycline-controlled transcriptional activator
  • Alternative inducible systems include the ecdysone or rapamycin systems.
  • Ecdysone is an insect molting hormone whose production is controlled by a heterodimer of the ecdysone receptor and the product of the ultraspiracle gene (USP). Expression is induced by treatment with ecdysone or an analog of ecdysone such as muristerone A.
  • the agent that is administered to the animal to trigger the inducible system is referred to as an induction agent.
  • Additional regulatory regions that may be useful in nucleic acid constructs, include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, inducible elements, or introns. Such regulatory regions may not be necessary, although they may increase expression by affecting transcription, stability of the mRNA, translational efficiency, or the like. Such regulatory regions can be included in a nucleic acid construct as desired to obtain optimal expression of the nucleic acids in the cell(s). Sufficient expression, however, can sometimes be obtained without such additional elements.
  • Signal peptides can be used such that an encoded polypeptide is directed to a particular cellular location (e.g., the cell surface).
  • selectable markers include puromycin, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphtransferase, thymidine kinase (TK), and xanthin-guanine phosphoribosyltransferase (XGPRT). Such markers are useful for selecting stable transformants in culture.
  • Other selectable markers include fluorescent polypeptides, such as green fluorescent protein or yellow fluorescent protein.
  • a sequence encoding a selectable marker can be flanked by recognition sequences for a recombinase such as, e.g., Cre or Flp.
  • the selectable marker can be flanked by loxP recognition sites (34 by recognition sites recognized by the Cre recombinase) or FRT recognition sites such that the selectable marker can be excised from the construct.
  • loxP recognition sites 34 by recognition sites recognized by the Cre recombinase
  • FRT recognition sites such that the selectable marker can be excised from the construct.
  • a transposon containing a Cre- or Flp-activatable transgene interrupted by a selectable marker gene also can be used to obtain transgenic animals with conditional expression of a transgene.
  • a promoter driving expression of the marker/transgene can be either ubiquitous or tissue-specific, which would result in the ubiquitous or tissue-specific expression of the marker in FO animals (e.g., pigs).
  • Tissue specific activation of the transgene can be accomplished, for example, by crossing a pig that ubiquitously expresses a marker-interrupted transgene to a pig expressing Cre or Flp in a tissue-specific manner, or by crossing a pig that expresses a marker-interrupted transgene in a tissue-specific manner to a pig that ubiquitously expresses Cre or Flp recombinase. Controlled expression of the transgene or controlled excision of the marker allows expression of the transgene.
  • the target nucleic acid encodes a polypeptide.
  • a nucleic acid sequence encoding a polypeptide can include a tag sequence that encodes a “tag” designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., to facilitate localization or detection).
  • Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide.
  • Non-limiting examples of encoded tags include glutathione S-transferase (GST) and FLAGTM tag (Kodak, New Haven, Conn.).
  • the target nucleic acid sequence induces RNA interference against a target nucleic acid such that expression of the target nucleic acid is reduced.
  • the target nucleic acid sequence can induce RNA interference against a nucleic acid encoding a cystic fibrosis transmembrane conductance regulatory (CFTR) polypeptide.
  • CFTR cystic fibrosis transmembrane conductance regulatory
  • siRNA double-stranded small interfering RNA
  • shRNA small hairpin RNA
  • Constructs for siRNA can be produced as described, for example, in Fire et al. (1998) Nature 391:806-811; Romano and Masino (1992) Mol. Microbiol.
  • shRNAs are transcribed as a single-stranded RNA molecule containing complementary regions, which can anneal and form short hairpins.
  • Nucleic acid constructs can be methylated using an SssI CpG methylase (New England Biolabs, Ipswich, Mass.).
  • the nucleic acid construct can be incubated with S-adenosylmethionine and SssI CpG-methylase in buffer at 37° C. Hypermethylation can be confirmed by incubating the construct with one unit of HinP1I endonuclease for 1 hour at 37° C. and assaying by agarose gel electrophoresis.
  • Nucleic acid constructs can be introduced into embryonic, fetal, or adult porcine cells of any type, including, for example, germ cells such as an oocyte or an egg, a progenitor cell, an adult or embryonic stem cell, a kidney cell such as a PK-15 cell, an islet cell, a beta cell, a liver cell, or a fibroblast such as a dermal fibroblast, using a variety of techniques.
  • germ cells such as an oocyte or an egg
  • a progenitor cell such as an adult or embryonic stem cell
  • a kidney cell such as a PK-15 cell
  • an islet cell a beta cell
  • a liver cell or a fibroblast such as a dermal fibroblast
  • Non-limiting examples of techniques include the use of transposon systems, recombinant viruses that can infect cells, or liposomes or other non-viral methods such as electroporation, microinjection, or calcium phosphate precipitation, that are capable of delivering nucle
  • transposon systems the transcriptional unit of a nucleic acid construct, i.e., the regulatory region operably linked to a target nucleic acid sequence, is flanked by an inverted repeat of a transposon.
  • transposon systems including, for example, Sleeping Beauty (see, U.S. Pat. No. 6,613,752 and U.S. Publication No. 2005/0003542); Frog Prince (Miskey et al. (2003) Nucleic Acids Res. 31(23):6873-81); Tol 2 (Kawakami (2007) Genome Biology 8(Suppl.1):S7; Minos (Pavlopoulos et al.
  • a transposase can be encoded on the same nucleic acid construct as the target nucleic acid, can be introduced on a separate nucleic acid construct, or provided as an mRNA (e.g., an in vitro transcribed and capped mRNA).
  • Insulator elements also can be included in a nucleic acid construct to maintain expression of the target nucleic acid and to inhibit the unwanted transcription of host genes. See, for example, U.S. Publication No. 2004/0203158.
  • an insulator element flanks each side of the transcriptional unit and is internal to the inverted repeat of the transposon.
  • Non-limiting examples of insulator elements include the matrix attachment region (MAR) type insulator elements and border-type insulator elements. See, for example, U.S. Pat. Nos. 6,395,549, 5,731,178, 6,100,448, and 5,610,053, and U.S. Publication No. 2004/0203158.
  • Nucleic acids can be incorporated into vectors.
  • a vector is a broad term that includes any specific DNA segment that is designed to move from a carrier into a target DNA.
  • a vector may be referred to as an expression vector, or a vector system, which is a set of components needed to bring about DNA insertion into a genome or other targeted DNA sequence such as an episome, plasmid, or even virus/phage DNA segment.
  • Vector systems such as viral vectors (e.g., retroviruses, adeno-associated virus and integrating phage viruses), and non-viral vectors (e.g., transposons) used for gene delivery in animals have two basic components: 1) a vector comprised of DNA (or RNA that is reverse transcribed into a cDNA) and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and a DNA target sequence and inserts the vector into the target DNA sequence.
  • Vectors most often contain one or more expression cassettes that comprise one or more expression control sequences, wherein an expression control sequence is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence or mRNA, respectively.
  • Plasmids and viral vectors are known.
  • Mammalian expression plasmids typically have an origin of replication, a suitable promoter and optional enhancer, and also any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking non-transcribed sequences.
  • vectors include: plasmids (which may also be a carrier of another type of vector), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., modified HIV-1, SIV or FIV), retrovirus (e.g., ASV, ALV or MoMLV), and transposons (e.g., Sleeping Beauty, P-elements, Tol-2, Frog Prince, piggyBac).
  • plasmids which may also be a carrier of another type of vector
  • adenovirus e.g., adeno-associated virus (AAV)
  • lentivirus e.g., modified HIV-1, SIV or FIV
  • retrovirus e.g., ASV, ALV or MoMLV
  • transposons e.g., Sleeping Beauty, P-elements, Tol-2, Frog Prince, piggyBac.
  • nucleic acid refers to both RNA and DNA, including, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, as well as naturally occurring and chemically modified nucleic acids, e.g., synthetic bases or alternative backbones.
  • a nucleic acid molecule can be double-stranded or single-stranded (i.e., a sense or an antisense single strand).
  • transgenic is used broadly herein and refers to a genetically modified organism or genetically engineered organism whose genetic material has been altered using genetic engineering techniques. A knockout artiodactyl is thus transgenic regardless of whether or not exogenous genes or nucleic acids are expressed in the animal or its progeny.
  • Hasler-Rapacz J Ellegren H, Fridolfsson AK, et al. Identification of a mutation in the low density lipoprotein receptor gene associated with recessive familial hypercholesterolemia in swine. Am J Med Genet 1998;76(5):379-86.
  • Rotational atherectomy does not reduce recurrent in-stent restenosis: results of the angioplasty versus rotational atherectomy for treatment of diffuse in-stent restenosis trial (ARTIST). Circulation 2002;105(5):583-8.
  • Apolipoprotein E modulates clearance of apoptotic bodies in vitro and in vivo, resulting in a systemic proinflammatory state in apolipoprotein E-deficient mice. J Immunol 2004;173(10):6366-75.
  • Rapacz J Hasler-Rapacz J. Animal Models: The Pig. In: Sparkes RS, Lusis A J, eds. Genetic factors in atherosclerosis: approaches and model systems. Basel ; New York: Karger, 1989: 139-169.
  • Duchenne muscular dystrophy gene product is not identical in muscle and brain. Nature, 337, 76-78.
  • Dystrophin is transcribed in brain from a distant upstream promoter. Proc Natl Acad Sci U S A, 88, 1276-1280.
  • a 71-kilodalton protein is a major product of the Duchenne muscular dystrophy gene in brain and other nonmuscle tissues. Proc Natl Acad Sci USA, 89, 5346-5350.
  • Dp140 a novel 140 kDa CNS transcript from the dystrophin locus. Hum Mol Genet, 4, 329-335.
  • Dp71 can restore the dystrophin-associated glycoprotein complex in muscle but fails to prevent dystrophy. Nature genetics, 8, 333-339.
  • Exogenous Dp71 is a dominant negative competitor of dystrophin in skeletal muscle. Neuromuscul Disord, 12, 836-844.
  • Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma. Faseb J, 21, 2195-2204.

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WO2011100505A2 (fr) 2011-08-18
BR112012020257A2 (pt) 2016-11-16
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US20120220037A1 (en) 2012-08-30
US20160273000A1 (en) 2016-09-22

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