Kristin M. Whitworth

Disruption of the CFTR Gene Produces a Model of Cystic Fibrosis in Newborn Pigs

Almost two decades after CFTR was identified as the gene responsible for cystic fibrosis (CF), we still lack answers to many questions about the pathogenesis of the disease, and it remains incurable. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but the mutant mice do not develop the characteristic manifestations of human CF, including abnormalities of the pancreas, lung, intestine, liver, and other organs. Because pigs share many anatomical and physiological features with humans, we generated pigs with a targeted disruption of both CFTR alleles. Newborn pigs lacking CFTR exhibited defective chloride transport and developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF. The pig model may provide opportunities to address persistent questions about CF pathogenesis and accelerate discovery of strategies for prevention and treatment.

Production of CFTR -null and CFTR-ΔF508 heterozygous pigs by adeno-associated virus–mediated gene targeting and somatic cell nuclear transfer

Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (DeltaF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice.

Use of the CRISPR/Cas9 System to Produce Genetically Engineered Pigs from In Vitro-Derived Oocytes and Embryos

ABSTRACT Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.

Transcriptional Profiling of Pig Embryogenesis by Using a 15-K Member Unigene Set Specific for Pig Reproductive Tissues and Embryos

Abstract Differential mRNA expression patterns were evaluated between germinal vesicle oocytes (pgvo), four-cell (p4civv), blastocyst (pblivv), and in vitro-produced four-cell (p4civp) and in vitro-produced blastocyst (pblivp) stage embryos to determine key transcripts responsible for early embryonic development in the pig. Five comparisons were made: pgvo to p4civv, p4civv to pblivv, pgvo to pblivv, p4civv to p4civp, and pblivv to pblivp. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg false-discovery-rate multiple correction test on each comparison. A comparison of pgvo to p4civv, p4civv to pblivv, and pgvo to pblivv resulted in 3214, 1989, and 4528 differentially detected cDNAs, respectively. Real-time PCR analysis on seven transcripts showed an identical pattern of changes in expression as observed on the microarrays, while one transcript deviated at a single cell stage. There were 1409 and 1696 differentially detected cDNAs between the in vitro- and in vivo-produced embryos at the four-cell and blastocyst stages, respectively, without the Benjamini and Hochberg false-discovery-rate multiple correction test. Real-time polymerase chain reaction (PCR) analysis on four genes at the four-cell stage showed an identical pattern of gene expression as found on the microarrays. Real-time PCR analysis on four of five genes at the blastocyst stage showed an identical pattern of gene expression as found on the microarrays. Thus, only 1 of the 39 comparisons of the pattern of gene expression exhibited a major deviation between the microarray and the real-time PCR. These results illustrate the complex mechanisms involved in pig early embryonic development.

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

Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs

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

An Intact Sialoadhesin (Sn/SIGLEC1/CD169) Is Not Required for Attachment/Internalization of the Porcine Reproductive and Respiratory Syndrome Virus

We report using zinc finger nucleases (ZFNs) and somatic cell nuclear transfer (SCNT) to generate pigs with a knockout (KO) mutation of an enhanced green fluorescent protein (eGFP) transgene. ZFNs are synthetic modular proteins composed of a FokI endonuclease domain linked to a sequence-specific zinc finger DNA-binding domain. Pairs of ZFNs bind to the target region, allowing FokI dimerization and subsequent DNA cleavage. Mutation of an eGFP transgene by using ZFNs has been demonstrated in rats (Geurts et al., 2009); however, there are no reports of ZFN-mutated livestock offspring. Using ZFNs to increase the efficiency of gene modification may advance the production of agriculturally and clinically relevant animal models, particularly in species where modification is difficult. All animal procedures followed an approved IACUC protocol. Adult porcine ear fibroblasts hemizygous for the eGFP transgene (Whitworth et al., 2009), from a grandson of the founder animal, were cultured to 75% confluency, trypsinized, and cotransfected (Ross et al., 2010) with a pair of ZFN plasmids that bind to opposing strands at the eGFP target site (Sigma–Aldrich CompoZr®) and a red fluorescent CAG-tomato plasmid as a transient selectable fluorophore reporting transfection efficiency. Transfected fibroblasts were cultured for 96 h and selected for red fluorescence by automated cell sorting (FACS; Fig. 1A,B) and seeded into 96-well plates (100 cells/well) based on CAG-tomato expression (2% of total cells sorted). A second round of FACS using negative selection of eGFP fluorescence enriched for eGFP KO cells (~5% of sorted cells). PCR of genomic DNA from fibroblasts in wells containing predominantly non-green cells was used to amplify a fragment bracketing the ZFN targeting site. The PCR product was cloned into E. coli, and 16 colonies showed mutations by Sanger sequencing. These mutations at the ZFN cleavage site included a 6-bp deletion, a 333-bp deletion, and a 222-bp deletion replaced with a 113-bp inversion of the deleted sequence (Supplemental Information http://animalsciences.missouri.edu/faculty/prather/). Fibroblast colonies determined to carry ZFN-induced mutations were used as donor cells for SCNT, and embryo transfer (n = 3; Whitworth et al., 2009). One Day-12 embryo collection recovered 7 of 9 embryos that did not fluoresce. Quality sequence was obtained from four embryos, and confirmed that the two embryos that fluoresced had an intact eGFP and two embryos that did not fluoresce were mutated. One of seven piglets recovered via Cesarean section at term for the other two pregnancies fluoresced (Fig. 1C). DNA sequencing confirmed that this piglet had unaltered sequence at the predicted ZFN cut site, while the non-fluorescing piglets had various deletions and insertions. While a direct comparison to a 12% mutation rate in the rat made by microinjection (Geurts et al., 2009) cannot be made because of our FACS preselection and SCNT methods, six out of seven founders being mutated is consistent with their results. This study establishes ZFN-based gene modification in a large animal model. The methods used are anticipated to be useful in selective knockout of endogenous swine genes of agricultural and biomedical importance without introducing any transgenic sequence into the genome. Figure 1 A: Control porcine ear fibroblasts; eGFP and Hoechst DNA stain fluorescence. B: Porcine ear fibroblasts treated with eGFP-specific ZFNs; eGFP and Hoechst DNA stain fluorescence. GFP fluorescence is absent in a high proportion of the treated fibroblasts ...

Somatic cell nuclear transfer efficiency: How can it be improved through nuclear remodeling and reprogramming?

ABSTRACT Surface expression of SIGLEC1, also known as sialoadhesin or CD169, is considered a primary determinant of the permissiveness of porcine alveolar macrophages for infection by porcine reproductive and respiratory syndrome virus (PRRSV). In vitro, the attachment and internalization of PRRSV are dependent on the interaction between sialic acid on the virion surface and the sialic acid binding domain of the SIGLEC1 gene. To test the role of SIGLEC1 in PRRSV infection, a SIGLEC1 gene knockout pig was created by removing part of exon 1 and all of exons 2 and 3 of the SIGLEC1 gene. The resulting knockout ablated SIGLEC1 expression on the surface of alveolar macrophages but had no effect on the expression of CD163, a coreceptor for PRRSV. After infection, PRRSV viremia in SIGLEC1 −/− pigs followed the same course as in SIGLEC1 −/+ and SIGLEC1 +/+ littermates. The absence of SIGLEC1 had no measurable effect on other aspects of PRRSV infection, including clinical disease course and histopathology. The results demonstrate that the expression of the SIGLEC1 gene is not required for infection of pigs with PRRSV and that the absence of SIGLEC1 does not contribute to the pathogenesis of acute disease.

Scriptaid Corrects Gene Expression of a Few Aberrantly Reprogrammed Transcripts in Nuclear Transfer Pig Blastocyst Stage Embryos

Fertile offspring from somatic cell nuclear transfer (SCNT) is the goal of most cloning laboratories. For this process to be successful, a number of events must occur correctly. First the donor nucleus must be in a state that is amenable to remodeling and subsequent genomic reprogramming. The nucleus must be introduced into an oocyte cytoplasm that is capable of facilitating the nuclear remodeling. The oocyte must then be adequately stimulated to initiate development. Finally the resulting embryo must be cultured in an environment that is compatible with the development of that particular embryo. Much has been learned about the incredible changes that occur to a nucleus after it is placed in the cytoplasm of an oocyte. While we think that we are gaining an understanding of the reorganization that occurs to proteins in the donor nucleus, the process of cloning is still very inefficient. Below we will introduce the procedures for SCNT, discuss nuclear remodeling and reprogramming, and review techniques that may improve reprogramming. Finally we will briefly touch on other aspects of SCNT that may improve the development of cloned embryos. Mol. Reprod. Dev. 77:1001–1015, 2010.

Method of Oocyte Activation Affects Cloning Efficiency in Pigs

Nuclear transfer efficiency in the pig is low and is thought to be caused by inadequate nuclear reprogramming. The objective of this study was to identify differentially represented transcripts in pig in vivo derived (BLIVV), in vitro fertilized (BLIVF), or nuclear transfer derived (NT, three different activation methods) blastocyst stage embryos and the donor cell line by microarray analysis, and to determine if treatment of reconstructed embryos with the histone deacetylase inhibitor, Scriptaid (NTS), for 14 h postactivation would correct gene expression in a subset of the identified aberrantly reprogrammed transcripts. There were 1481 differentially expressed transcripts when comparing all six treatment groups (p < 0.05). Transcripts that were different between BLIVV and NT (p < 0.20) and significantly different from donor cells (p < 0.05) were classified as being aberrantly reprogrammed (179 transcripts). Fourteen transcripts were chosen to determine the effect of Scriptaid treatment. After real-time PCR relative gene expression was compared among BLIVV, NT pool, cells, and NTS by the comparative Ct method, statistical analysis was performed in SAS 9.1 (p < 0.05). NTS embryos had three transcripts returning to the same level as BLIVV (H3F3A, CAPG, and SEPT7). Half of the transcripts (7/14) were not affected by NTS treatment, for example, SIRT1 and H1F0. Scriptaid treatment resulted high expression of COX5A and very low expression of GPD1L, EIF3E, and GSTA3. Scriptaid also reduced the number of 5-Methylcytidine-positive nuclei in blastocyst stage embryos (p < 0.0003). Scriptaid treatment significantly affected gene expression in 7 of the 14 transcripts evaluated and returned 3 genes to BLIVV levels.