Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer.
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ABSTRACT:
SUBMITTER: Rogers CS
PROVIDER: S-EPMC2265103 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:UnlabelledHereditary tyrosinemia type I (HT1) results in hepatic failure, cirrhosis, and hepatocellular carcinoma (HCC) early in childhood and is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (FAH). In a novel approach we used the chimeric adeno-associated virus DJ serotype (AAV-DJ) and homologous recombination to target and disrupt the porcine Fah gene. AAV-DJ is an artificial chimeric AAV vector containing hybrid capsid sequences from three naturally occurring serotypes (AAV2, 8, and 9). The AAV-DJ vector was used to deliver the knockout construct to fetal pig fibroblasts with an average knockout targeting frequency of 5.4%. Targeted Fah-null heterozygote fibroblasts were used as nuclear donors for somatic cell nuclear transfer (SCNT) to porcine oocytes and multiple viable Fah-null heterozygote pigs were generated. Fah-null heterozygotes were phenotypically normal, but had decreased Fah transcriptional and enzymatic activity compared to wildtype animals.ConclusionThis study is the first to use a recombinant chimeric AAV vector to knockout a gene in porcine fibroblasts for the purpose of SCNT. In using the AAV-DJ vector we observed targeting frequencies that were higher than previously reported with other naturally occurring serotypes. We expect that the subsequent generation of FAH-null homozygote pigs will serve as a significant advancement for translational research in the areas of metabolic liver disease, cirrhosis, and HCC.
| S-EPMC3184202 | biostudies-literature
Project description:Hyperacute rejection of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-alpha-1,3-galactose (Galalpha-1,3-Gal) epitopes expressed on the pig cell surface. Previously, we generated inbred miniature swine with a null allele of the alpha-1,3-galactosyltransferase locus (GGTA1) by nuclear transfer (NT) with gene-targeted fibroblasts. To expedite the generation of GGTA1 null pigs, we selected spontaneous null mutant cells from fibroblast cultures of heterozygous animals for use in another round of NT. An unexpectedly high rate of spontaneous loss of GGTA1 function was observed, with the vast majority of null cells resulting from loss of the WT allele. Healthy piglets, hemizygous and homozygous for the gene-targeted allele, were produced by NT by using fibroblasts that had undergone deletional and crossover/gene conversion events, respectively. Aside from loss of Galalpha-1,3-Gal epitopes, there were no obvious phenotypic differences between these null piglets and WT piglets from the same inbred lines. In fact, congenital abnormalities observed in the heterozygous NT animals did not reappear in the serially produced null animals.
| S-EPMC409919 | biostudies-literature
Project description:Imprinting disorders induced by somatic cell nuclear transfer (SCNT) usually lead to the abnormalities of cloned animals and low cloning efficiency. Histone deacetylase inhibitors have been shown to improve gene expression, genomic methylation reprogramming and the development of cloned embryos, however, the imprinting statuses in these treated embryos and during their subsequent development remain poorly studied. In this study, we investigated the dynamics of H19/Igf2 methylation and transcription in porcine cloned embryos treated with trichostatin A (TSA), and examined H19/Igf2 imprinting patterns in cloned fetuses and piglets. Our results showed that compared with the maintenance of H19/Igf2 methylation in fertilized embryos, cloned embryos displayed aberrant H19/Igf2 methylation and lower H19/Igf2 transcripts. When TSA enhanced the development of cloned embryos, the disrupted H19/Igf2 imprinting was largely rescued in these treated embryos, more similar to those detected in fertilized counterparts. Further studies displayed that TSA effectively rescued the disrupted imprinting of H19/Igf2 in cloned fetuses and piglets, prevented the occurrence of cloned fetus and piglet abnormalities, and enhanced the full-term development of cloned embryos. In conclusion, our results demonstrated that aberrant imprinting induced by SCNT led to the abnormalities of cloned fetuses and piglets and low cloning efficiency, and TSA rescued the disrupted imprinting in cloned embryos, fetuses and piglets, and prevented the occurrence of cloned fetus and piglet abnormalities, thereby improving the development of cloned embryos. This study would have important implications in improving cloning efficiency and the health of cloned animals.
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Project description:Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The most common mutation, a deletion of the phenylalanine at position 508 (DeltaF508), disrupts processing of the protein. Nearly all human CFTR-DeltaF508 is retained in the endoplasmic reticulum and degraded, preventing maturation to the plasma membrane. In addition, the F508 deletion reduces the activity of single CFTR channels. Human CFTR-DeltaF508 has been extensively studied to better understand its defects. Here, we adopted a cross-species comparative approach, examining human, pig, and mouse CFTR-DeltaF508. As with human CFTR-DeltaF508, the DeltaF508 mutation reduced the single-channel activity of the pig and mouse channels. However, the mutant pig and mouse proteins were at least partially processed like their wild-type counterparts. Moreover, pig and mouse CFTR-DeltaF508 partially restored transepithelial Cl(-) transport to CF airway epithelia. Our data, combined with earlier work, suggest that there is a gradient in the severity of the CFTR-DeltaF508 processing defect, with human more severe than pig or mouse. These findings may explain some previously puzzling observations in CF mice, they have important implications for evaluation of potential therapeutics, and they suggest new strategies for discovering the mechanisms that disrupt processing of human CFTR-DeltaF508.
| S-EPMC1976592 | biostudies-literature
Project description:Somatic cell nuclear transfer (SCNT) is a useful tool for animal cloning, but the efficiency of producing viable offspring by SCNT is very low. To improve this efficiency in the production of cloned pigs, it is critical to understand the interactions between uterine function and cloned embryos during implantation. Lysophosphatidic acid (LPA) is a lipid mediator that plays an important role in the establishment of pregnancy in pigs; however, LPA production in the uterine endometrium of pigs carrying SCNT-cloned conceptuses has not been determined. Therefore, we investigated expression of ENPP2, an LPA-generating enzyme, in the uterine endometrium of gilts with conceptuses derived from SCNT during the implantation period. Uterine endometrial tissue and uterine flushing were obtained from gilts carrying SCNT-derived conceptuses and from gilts carrying conceptuses resulting from natural mating on d 12 of pregnancy. Our results demonstrated no difference in the level of ENPP2 mRNA expression in the uterine endometrium between gilts carrying SCNT-derived conceptuses and gilts carrying naturally-conceived conceptuses, but secretion of ENPP2 protein into the uterine lumen did decrease significantly in pigs with SCNT-derived conceptuses. These results indicate that expression and secretion of ENPP2, which are critical for appropriate LPA production and successful pregnancy, are dysregulated in the uterine endometrium of pigs carrying SCNT-derived conceptuses.
| S-EPMC4093402 | biostudies-literature
Project description:BackgroundPigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate.MethodsTranscription activator-like effector nucleases (TALENs) were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO) cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT) for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting.ResultsThe luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19) were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean section after 38 days of gestation for genotyping. Finally, six live piglets and one stillborn piglet were collected from two recipients by caesarean section. Sequencing analyses of the target site confirmed the P53 biallelic knockout in all fetuses and piglets, consistent with the genotype of the donor cells. The qPCR analysis showed that the expression of the P53 mRNA had significant reduction in various tissues of the knockout piglets. Furthermore, confocal microscopy and western blotting analyses demonstrated that the fibroblast cells of Diannan miniature piglets with a P53 biallelic knockout were defective in mediating DNA damage when incubated with doxorubicin.ConclusionTALENs combined with SCNT was successfully used to generate P53 KO Diannan miniature pigs. Although these genetically engineered Diannan miniature pigs had no tumorigenic signs, the P53 gene was dysfunctional. We believe that these pigs will provide powerful new resources for preclinical oncology and basic cancer research.
| S-EPMC5670695 | biostudies-literature
Project description:Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1-5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.
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Project description:The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature DeltaRI/DeltaF508 had a T(1/2) of approximately 14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of DeltaF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 degrees C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.
| S-EPMC4361937 | biostudies-literature
Project description:The most common mutation in cystic fibrosis (CF) is DeltaF508, which is associated with failure of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) to traffic to the plasma membrane. By a still unknown mechanism, the loss of correctly trafficked DeltaF508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane. ENaC trafficking is known to be regulated by a signaling pathway involving the glucocorticoid receptor, the serum- and glucocorticoid-regulated kinase SGK1, and the ubiquitin E3 ligase Nedd4-2. We show here that dexamethasone rescues functional expression of DeltaF508-CFTR. The half-life of DeltaF508-CFTR is also dramatically enhanced. Dexamethasone-activated DeltaF508-CFTR rescue is blocked either by the glucocorticoid receptor antagonist RU38486 or by the phosphatidylinositol 3-kinase inhibitor LY294002. Co-immunoprecipitation studies indicate that Nedd4-2 binds to both wild-type- and DeltaF508-CFTR. These complexes are inhibited by dexamethasone treatment, and CFTR ubiquitination is concomitantly decreased. We further show that knockdown of Nedd4-2 by small interfering RNA also corrects DeltaF508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated DeltaF508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells.
| S-EPMC2757227 | biostudies-literature
Project description:Heterologous expression of the cystic fibrosis transmembrane conductance regulator (CFTR) provided evidence that the major cystic fibrosis (CF) mutation DeltaF508 leads to defective protein folding in the endoplasmic reticulum, which prevents its processing and targeting to the cell surface. In this study, we investigated endogenous CFTR expression in skin biopsies and respiratory and intestinal tissue specimens from DeltaF508 homozygous and non-CF patients, using immunohistochemical and immunoblot analyses with a panel of CFTR antibodies. CFTR expression was detected at the luminal surface of reabsorptive sweat ducts and airway submucosal glands, at the apex of ciliated cells in pseudostratified respiratory epithelia and of isolated cells of the villi of duodenum and jejunum, and within intracellular compartments of intestinal goblet cells. In DeltaF508 homozygous patients, expression of the mutant protein proved to be tissue specific. Whereas DeltaF508 CFTR was undetectable in sweat glands, the expression in the respiratory and intestinal tracts could not be distinguished from the wild-type by signal intensity or localization. The tissue-specific variation of DeltaF508 CFTR expression from null to apparently normal amounts indicates that DeltaF508 CFTR maturation can be modulated and suggests that determinants other than CFTR mislocalization should play a role in DeltaF508 CF respiratory and intestinal disease.
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