Kevin D. Wells

Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs

Galactose-α1,3-galactose (α1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig α1,3-galactosyltransferase (α1,3GT) by homologous recombination is a means to completely remove the α1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig α1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig α1,3GT allele.

Genetically enhanced cows resist intramammary Staphylococcus aureus infection

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 mg/ml in their milk were produced. In vitro assays demonstrated the milks ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 mg/ml of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15–30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln125,232-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5′-flanking region of the ovine β-lactoglobulin gene directed the secretion of Gln125,232-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

Bovine Blastocyst-Derived Trophectoderm and Endoderm Cell Cultures: Interferon Tau and Transferrin Expression as Respective In Vitro Markers

Abstract Continuous cultures of bovine trophectoderm (CT-1 and CT-5) and bovine endoderm (CE-1 and CE-2) were initiated and maintained on STO feeder cells. CT-1 and CT-5 were derived from the culture of intact, 10- to 11-day in vitro-produced blastocysts. CE-1 and CE-2 were derived from the culture of immunodissected inner cell masses of 7- to 8-day in vitro-produced blastocysts. The cultures were routinely passaged by physical dissociation. Although morphologically distinct, the trophectoderm and endoderm both grew as cell sheets of polarized epithelium (dome formations) composed of approximately cuboidal cells. Both cell types, particularly the endoderm, grew on top of the feeder cells for the most part. Trophectoderm cultures grew faster, relative to endoderm, in large, rapidly extending colonies of initially flat cells with little or no visible lipid. The endoderm, in contrast, grew more slowly as tightly knit colonies with numerous lipid vacuoles in the cells at the colony centers. Ultrastructure analysis revealed that both cell types were connected by desmosomes and tight junctional areas, although these were more extensive in the trophectoderm. Endoderm was particularly rich in rough endoplasmic reticulum and Golgi apparatus indicative of cells engaged in high protein production and secretion. Interferon tau expression was specific to trophectoderm cultures, as demonstrated by reverse transcription-polymerase chain reaction, Western blot, and antiviral activity; and this property may act as a marker for this cell type. Serum protein production specific to endoderm cultures was demonstrated by Western blot; this attribute may be a useful marker for this cell type. This simple coculture method for the in vitro propagation of bovine trophectoderm and endoderm provides a system for assessing their biology in vitro.

Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus

VOLUME 34 NUMBER 1 JANUARY 2016 NATURE BIOTECHNOLOGY To the Editor: Porcine reproductive and respiratory syndrome (PRRS) is the most economically important disease of swine in North America, Europe and Asia, costing producers in North America more than

Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs.

600 million annually1. The disease syndrome was first recognized in the United States in 1987 and described in 1989 (ref. 2). The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), was subsequently isolated and characterized in Europe in 1991 (ref. 3). Vaccines have been unable to control the disease. It has been suggested that CD163 is the receptor for entry of PRRSV into cells4. Thus, we hypothesized that pigs with defective CD163 would be immune to PRRSV. Previously we used CRISPRCas9 to generate pigs lacking functional CD163 (ref. 5). Here we demonstrate that these animals are resistant to the PRRSV isolate NVSL 97-7895, a well-characterized, relatively virulent viral isolate that is commonly used in experimental PRRSV infection trials. After infection, they showed no clinical signs (fever or respiratory signs), lung pathology, viremia or antibody response and remained healthy for the 35 d after infection measured in this study. Because CD163 was edited using CRISPR-Cas9, the pigs challenged in this study do not contain any transgenes5. PRRSV is a member of the mammalian arterivirus group, which also includes murine lactate dehydrogenase-elevating virus, simian hemorrhagic fever virus and equine arteritis virus. The arteriviruses share important pathogenesis properties, including macrophage tropism and the capacity to cause both severe disease and persistent infection. In young pigs, infection with PRRSV results in respiratory disease, including cough and fever and reduced growth performance. In pregnant sows, PRRSV infection can result in reproductive failure, as well as persistently infected and low birth weight piglets.The virus is associated with polymicrobial disease syndromes, including porcine respiratory Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus

Cell Donor Influences Success of Producing Cattle by Somatic Cell Nuclear Transfer

Abstract:  Background:  The presence of multiple copies of porcine endogenous retrovirus (PERV) within the pig genome, and the demonstration that replication competent PERV, that infect human cells in culture, can be isolated from pig cells, directly impacts the drive towards the development of pigs for xenotransplantation. The development of technology to produce pigs that do not propagate PERV has the potential to facilitate the development of xenotransplantation products for human use, and as such, is the focus of this investigation. The shear number of PERV loci, most of which are defective or pseudogenes, renders conventional gene targeting impractical, if not impossible, to inactivate all PERV provirus within the pig genome, including potential replication competent PERV arising from spontaneous recombination. The recently developed RNA interference (RNAi) technology to knockdown/silence post‐transcriptional gene expression, offers a promising alternative to achieving this goal.

Large offspring syndrome, a bovine model for the human loss-of-imprinting overgrowth syndrome Beckwith-Wiedemann

Abstract To assess sources of variation in nuclear transfer efficiency, bovine fetal fibroblasts (BFF), harvested from six Jersey fetuses, were cultured under various conditions. After transfection, frozen-thawed lung or muscle BFF donor cells were initially cultured in DMEM in 5% CO2 and air and some were transferred to MEM, with 5% or 20% O2 or 0.5% or 10% serum and G418 for 2–3 wk. Selected clonal transfected fibroblasts were fused to enucleated oocytes. Fused couplets (n = 4007), activated with ionomycin and 6-dimethylaminopurine, yielded 927 blastocysts, and 650 were transferred to 330 recipients. Fusion rate was influenced by oxygen tension in a fetus-dependent manner (P < 0.001). Blastocyst development was influenced in a number of ways. Hip fibroblast generated more blastocysts when cultured in MEM (P < 0.001). The influence of serum concentration was fetus dependent (P < 0.001) and exposing fibroblast to low oxygen was detrimental to blastocyst development (P < 0.001). Cells from two of the six fetuses produced embryos that maintained pregnancies to term, resulting in eight viable calves. Pregnancy rates 56 days after transfer for the two productive donor fetuses, was at least double that of other recipients and may provide a fitness indicator of BFF cell sources for nuclear transfer. We conclude that a significant component in determining somatic cell nuclear transfer success is the source of the nuclear donor cells.

Analysis of bovine mammary gland EST and functional annotation of the Bos taurus gene index

Beckwith-Wiedemann syndrome (BWS) is a human loss-of-imprinting syndrome primarily characterized by macrosomia, macroglossia, and abdominal wall defects. BWS has been associated with misregulation of two clusters of imprinted genes. Children conceived with the use of assisted reproductive technologies (ART) appear to have an increased incidence of BWS. As in humans, ART can also induce a similar overgrowth syndrome in ruminants which is referred to as large offspring syndrome (LOS). The main goal of our study is to determine if LOS shows similar loss-of-imprinting at loci known to be misregulated in BWS. To test this, Bos taurus indicus × Bos taurus taurus F1 hybrids were generated by artificial insemination (AI; control) or by ART. Seven of the 27 conceptuses in the ART group were in the > 97th percentile body weight when compared with controls. Further, other characteristics reported in BWS were observed in the ART group, such as large tongue, umbilical hernia, and ear malformations. KCNQ1OT1 (the most-often misregulated imprinted gene in BWS) was biallelically-expressed in various organs in two out of seven overgrown conceptuses from the ART group, but shows monoallelic expression in all tissues of the AI conceptuses. Furthermore, biallelic expression of KCNQ1OT1 is associated with loss of methylation at the KvDMR1 on the maternal allele and with downregulation of the maternally-expressed gene CDKN1C. In conclusion, our results show phenotypic and epigenetic similarities between LOS and BWS, and we propose the use of LOS as an animal model to investigate the etiology of BWS.

Blastomeres from Somatic Cell Nuclear Transfer Embryos Are Not Allocated Randomly in Chimeric Blastocysts

Functional genomic studies of the mammary gland require an appropriate collection of cDNA sequences to assess gene expression patterns from the different developmental and operational states of underlying cell types. To better capture the range of gene expression, a normalized cDNA library was constructed from pooled bovine mammary tissues, and 23,202 expressed sequence tags (EST) were produced and deposited into GenBank. Assembly of these EST with sequences in the Bos taurus Gene Index (BtGI) helped to form 5751 of the current 23,883 tentative consensus (TC) sequences. The majority (87%) of these 5751 assemblies contained only one to three mammary-derived EST. In contrast, 18% of the mammary EST assembled with TC sequences corresponding to 12 genes. These results suggest library normalization was only partially effective, because the reduction in EST for genes abundantly transcribed during lactation could be attributed to pooling. For better assessment of novel content in the mammary library and to add to existing annotation of all bovine sequence elements, gene ontology assignments, and comparative sequence analyses against human genome sequence, human and rodent gene indices, and an index of orthologous alignments of genes across eukaryotes (TOGA) were performed, and results were added to existing BtGI annotation. Over 35,000 of the bovine elements significantly matched human genome sequence, and the positions of some alignments (3%) were unique relative to those using human expressed sequences. Because 3445 TC sequences had no significant match with any data set, mammary-derived cDNA clones representing 23 of these elements were analyzed further for expression and novelty. Only one clone met criteria suggesting the corresponding gene was a divergent ortholog or expressed sequence unique to cattle. These results demonstrate that bovine sequence expression data serve as a resource for characterizing mammalian transcriptomes and identifying those genes potentially unique to ruminants.