David Wax

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.

Generation of cloned transgenic pigs rich in omega-3 fatty acids

Meat products are generally low in omega-3 (n-3) fatty acids, which are beneficial to human health. We describe the generation of cloned pigs that express a humanized Caenorhabditis elegans gene, fat-1, encoding an n-3 fatty acid desaturase. The hfat-1 transgenic pigs produce high levels of n-3 fatty acids from n-6 analogs, and their tissues have a significantly reduced ratio of n-6/n-3 fatty acids (P < 0.001).

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.

Cloned Transgenic Swine Via In Vitro Production and Cryopreservation

Abstract Ithas been notoriously difficult to successfully cryopreserve swine embryos, a task that has been even more difficult for in vitro-produced embryos. The first reproducible method of cryopreserving in vivo-produced swine embryos was after centrifugation and removal of the lipids. Here we report the adaptation of a similar process that permits the cryopreservation of in vitro-produced somatic cell nuclear transfer (SCNT) swine embryos. These embryos develop to the blastocyst stage and survive cryopreservation. Transfer of 163 cryopreserved SCNT embryos to two surrogates produced 10 piglets. Application of this technique may permit national and international movement of cloned transgenic swine embryos, storage until a suitable surrogate is available, or the long-term frozen storage of valuable genetics.

Method of Oocyte Activation Affects Cloning Efficiency in Pigs

The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken β‐actin/rabbit β‐globin hybrid promoter (pCAGG‐EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca2+ followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst‐stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P < 0.05). There was no significant difference in mean nuclear number. Pregnancy rate for NT2 was 100% (n = 19) at all stages collected and was significantly higher than NT1 (71.4%, n = 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n = 23; P < 0.15). Recovery rates were calculated based on the number of embryos, conceptuses, fetuses, or piglets present at the time of collection, divided by the number of embryos transferred to the recipient gilts. Recovery rates between the three groups were not significantly different at any of the stages collected (P > 0.05). All fusion/activation treatments produced live, pCAGG‐EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer. Mol. Reprod. Dev. 76: 490–500, 2009.

Production of Piglets after Cryopreservation of Embryos Using a Centrifugation-Based Method for Delipation Without Micromanipulation

Abstract It is still difficult to successfully cryopreserve in vitro-produced (IVP) swine embryos, as they are sensitive to chilling due to the abundance of intracellular lipids. Mechanical delipation through micromanipulation is successful, but this method increases the potential of pathogen transmission because of the damage inflicted upon the zona pellucida during micromanipulation, and it is labor intensive. Reported here is a method to remove the lipid of IVP porcine embryos, without significantly compromising the zona pellucida, by trypsin treating the embryos or exposing the embryo to a high-osmolality solution to enlarge the perivitelline space so that the lipid could be polarized and separated completely after subsequent centrifugation without micromanipulation. The procedures work both for nuclear transfer-derived embryos and in vitro-fertilized embryos. Both methods provide a high-throughput process that leaves the zona pellucida intact (or relatively intact for the trypsin treatment) to aid in preventing disease transmission. It is also demonstrated that this procedure results in viable piglets, a claim that could not be made in many previous reports. Although the efficiencies of cryopreservation have not been dramatically improved, these procedures allow a single person to process very large numbers of embryos without the necessity of manipulating each individual embryo on a micromanipulator. Such high-throughput processing overcomes the lack of high efficiency (i.e., the system can be overloaded with embryos for transfer to surrogates).

Porcine Skin-Derived Stem Cells Can Serve as Donor Cells for Nuclear Transfer

Although transgenic animal production through somatic cell nuclear transfer (SCNT) has been successful, the process is still inefficient. One major limitation is the use of somatic donor cells that have a finite life span. Identification and isolation of a cell type capable of rapid proliferation while possessing immortal or prolonged life span in culture and is capable of being genetically modified would be very valuable for utilization in the production of genetically modified pigs. Here we report the birth of live piglets after cloning by using porcine skin-derived stem cells (SSC) as a donor cell type. In the present study, cell cycle analysis indicates that the porcine SSC proliferate rapidly in vitro. The porcine SSC are capable of producing live offspring and can be genetically modified with positive selection. Utilization of porcine SSC may prove to be an excellent cell type for genetic modification followed by nuclear transfer for the production of transgenic pigs.

The Nuclear Mitotic Apparatus (NuMA) protein is contributed by the donor cell nucleus in cloned porcine embryos.

The Nuclear Mitotic Apparatus (NuMA) protein is a multifunctional protein that is localized to the nucleus in interphase and to the poles of the mitotic apparatus during mitosis. In unfertilized porcine oocytes, NuMA is localized to the meiotic spindle. NuMA is removed along with the meiotic spindle during the enucleation process before reconstructing the egg by introducing the donor cell nucleus to produce cloned embryos. Questions have been raised regarding the source for NuMA in cloned embryos, as the enucleated oocyte does not contain detectable NuMA in the cytoplasm. To determine the source of NuMA in porcine nuclear transfer (NT) embryos, we conducted an immunofluorescence microscopy study with antibodies against NuMA to investigate the appearance and distribution of NuMA before and after reconstructing NT embryos with porcine skin fibroblasts. We used donor cells from a confluent culture with all cells in interphase. For comparative studies, we also determined the immunofluorescence pattern of NuMA, gamma-tubulin, and alpha-tubulin in porcine fibroblasts, parthenogenetic embryos and in vitro fertilized (IVF) embryos. Results show that NuMA was localized in nuclei of 33.5% (163/456) of the serum-deprived fibroblasts used as donor cells. No NuMA staining was detected in enucleated pig oocytes. Immediately after nuclear transfer, NuMA staining was absent in all donor cell fibroblast nuclei (0 h) but staining was detected by 6 h within the reconstructed eggs, at which time the transferred somatic cell nucleus swelled in most cells (19/27) and became a pronucleus-like structure. NuMA was localized exclusively within the pronucleus-like structures (15/27). At 25 h, NuMA was detected inside the nucleus (16/25) either in one-cell or in 2-cell stage embryos. Interestingly, in parthenogenetic embryos, NuMA staining was not detected in all 42 eggs examined at 1 h, and evident NuMA staining was only detected inside a few (4/51 at 6 h; 6/48 at 25 h) of the nuclei. In IVF embryos, NuMA was detected within the nucleus at 6 h (5/20) and 25 h (13/16). These results show that the donor cell nucleus contains NuMA that is contributed to the reconstructed embryo and possibly activated by mechanisms in the oocytes cytoplast.

Altered gene expression profiles in the brain, kidney, and lung of one-month-old cloned pigs.

Although numerous mammalian species have been successfully cloned by somatic cell nuclear transfer (SCNT), little is known about gene expression of cloned pigs by SCNT. In the present study, expression profiles of 1-month-old cloned pigs generated from fetal fibroblasts (n = 5) were compared to those of age-matched controls (n = 5) using a 13K oligonucleotide microarray. The brain, kidney, and lung were chosen for microarray analysis to represent tissues from endoderm, mesoderm, and ectoderm in origin. In clones, 179 and 154 genes were differentially expressed in the kidney and the lung, respectively (fold change >2, p < 0.05, false discovery rate = 0.05), whereas only seven genes were differentially expressed in the brain of clones. Functional analysis of the differentially expressed genes revealed that they were enriched in diabetic nephropathy in the kidney, delayed alveologenesis as well as downregulated MAPK signaling pathways in the lung, which was accompanied with collapsed alveoli in the histological examination of the lung. To evaluate whether the gene expression anomalies are associated with changes in DNA methylation, global concentration of the methylated cytosine was measured in lung DNA by HPLC. Clones were significantly hypermethylated (5.72%) compared to the controls (4.13%). Bisulfite-pyrosequencing analyses of the promoter regions of differentially expressed genes, MYC and Period 1 (PER1), however, did not show any differences in the degree of DNA methylation between controls and clones. Together, these findings demonstrate that cloned pigs have altered gene expression that may potentially cause organ dysfunction.

Altered Gene Expression Profiles in the Brain, Kidney, and Lung of Deceased Neonatal Cloned Pigs

Limited studies have been published analyzing the gene expression patterns of cloned pigs. We compared the expression profiles of brain, kidney, and lung tissues, representing each of the three germ layers, of deceased neonatal cloned pigs with those of age-matched controls using a 13K oligonucleotide microarray. We found 42 (0.7% of total genes analyzed), 178 (2.9%), and 121 (1.9%) genes differentially expressed in the brain, kidney, and lung of clones, respectively, when compared with the corresponding organs from controls (fold change >1.5, p < 0.05, false discovery rate (FDR) = 0.05). These expression aberrations could potentially cause the following pathological anomalies in clones: diabetic nephropathy in the kidney and dysregulated surfactant homeostasis in the lung. Interestingly, upregulated expression of genes belonging to the MAPK pathway was observed in all three organs. To investigate whether the differences in levels of gene expression were caused by differential DNA methylation, the global DNA methylation level was measured by high-performance liquid chromatography. In controls, global concentration of methylated cytosine was 5.35%, whereas clones had significantly hypomethylated genomic DNA (4.57%). Bisulfite-pyrosequencing analyses of the promoter regions of differentially expressed candidate genes, c-MYC, Period 1 (PER1), Cathepsin L (CTSL), and Follistatin (FS), however, did not show any differences in the degree of DNA methylation between controls and clones. Our findings demonstrate that deceased neonatal cloned pigs have considerable gene expression abnormalities, which may have contributed to the death of the animals.