Cheng-Du Cui

Effect of Demecolcine-Assisted Enucleation on the MPF Level and Cyclin B1 Distribution in Porcine Oocytes

Demecolcine (DEM) treatment of oocytes induces formation of a membrane protrusion containing a mass of condensed maternal chromosomes, which can be removed with minimal damage prior to somatic cell nuclear transfer (SCNT). However, the effect of this method on the distribution of maturation-promoting factor (MPF) in porcine oocytes has not been reported. Here, the level of MPF and the distribution of cyclin B1 were assessed in porcine oocytes following DEM treatment. In addition, the efficiencies of DEM-assisted and mechanical enucleation were compared, as were the development (in vitro and in vivo) of these oocytes following SCNT. MPF was uniformly distributed in oocytes that had been treated with 0.4 μg/ml DEM for 1 h. Immunofluorescence microscopy showed that in untreated oocytes, cyclin B1, the regulatory subunit of MPF, accumulated around the spindle, and was lowly detected in the cytoplasm. DEM treatment disrupted spindle microtubules, induced chromosome condensation, and reduced the level of cyclin B1 in the nuclear region. Cyclin B1 was uniformly distributed in DEM-treated oocytes and the level of MPF was increased. The potential of embryos generated from DEM-treated oocytes to develop in vivo was significantly greater than that of embryos generated from mechanically enucleated oocytes. This is the first study to report the effects of DEM-assisted enucleation of porcine oocytes on the distribution of cyclin B1. MPF in mature oocytes is important for the development of reconstructed embryos and for efficient SCNT.

Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer

Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein–protein interactions in vitro in real‐time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP‐derived markers. We generated a pCX‐mRFP1‐pgk‐neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX‐mRFP1‐pgk‐neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture. genesis 51:575–586.© 2013 Wiley Periodicals, Inc.

Generation of cloned adult muscular pigs with myostatin gene mutation by genetic engineering

Skeletal muscle is the most economically valuable tissue in meat-producing animals and enhancing muscle growth in these species may enhance the efficiency of meat production. Skeletal muscle mass is negatively regulated by myostatin (MSTN), and non-functional mutations of the MSTN gene in various animal species have led to dramatic hypermuscularity. This study was designed to assess the characteristics of male MSTN-knockout (KO) pigs. A transcription activator-like effector nuclease (TALEN) pair targeting exon 1 of the swine MSTN gene was constructed and used to transfect porcine fetal fibroblasts (PFFs). We obtained a cell line consisting of a 2-bp deletion in one allele and a 4-bp deletion in the other allele, and this was used as a donor to generate cloned pigs via SCNT, and delivered 18 live piglets. They developed and grew normally to sexual maturity. These MSTN-KO boars grew normally to adulthood and showed visually-clear hypermuscular characteristics, increased carcass dressing percentage and loin eye size, and decreased backfat thickness. These pigs may show greater meat production, as well as being used in animal models of human diseases.

Production of Cloned Wuzhishan Miniature Pigs and Application for Alloxan Toxicity Test

Wuzhishan miniature pig is one of the four most important pig breeds in China and has many major economic characteristics. Herein, we successfully used SCNT to clone Wuzhishan miniature pig. First, ear fibroblasts were isolated from a 2-year-old female Wuzhishan miniature piglet to be used as the donor cell. Second, good-quality COCs were selected from ovaries obtained from pigs at a local slaughterhouse and cultured. Mature eggs with the first polar body and ear fibroblasts were applied SCNT. Lastly, we in total produced 12 piglets with 7 piglets surviving to adults. Next, we used these pigs to test alloxan toxicity and to build T I D diabetes type. We know that diabetes mellitus is a chronic heterogeneous metabolic disease characterized by a high blood glucose level and abnormal insulin secretion. In this study, T I D (type I diabetes) was experimentally induced in cloned Wuzhishan miniature pigs with alloxan. In brief, an intravenous injection of alloxan (group B: 170 mg/kg, n = 3) was administered to pigs weighing between 27 and 39 kg. Sterile saline was administered to control pigs (n = 3). We determined the glycometabolism related index, performed an intravenous glucose tolerance test, and carried out immunohistochemistry experiments. There were no significant differences in body weight, blood glucose, and serum insulin in all groups, before treatment. The level of blood glucose was significantly higher (P < 0.05) in group B (12.18 ± 0.70 mmol/L) than in the control (2.93 ± 0.39 mmol/L). By contrast, the level of serum insulin was lower in group B (5.641 ± 0.573 μIU/mL) than in the control (7.578 ± 0.539 μIU/mL). Histological studies by hematoxylin and eosin (H&E) revealed a loss of β-cells in the pancreas from pigs treated with 170 mg/kg alloxan. Immunolocalization studies showed a decrease in insulin reactivity in this treatment group as well. To conclude, our model holds promise in future studies of diabetes drug testing and islet xenotransplantation.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment

We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

Apancreatic pigs cloned using Pdx1-disrupted fibroblasts created via TALEN-mediated mutagenesis

Pancreatic and duodenal homeobox 1 (PDX1) plays a crucial role in pancreas development, β-cell differentiation, and maintenance of mature β-cell function. In this study, we designed a strategy to produce PDX1-knockout (KO) pigs. A transcription activator-like effector nuclease (TALEN) pair targeting exon 1 of the swine PDX1 gene was constructed. Porcine fetal fibroblasts (PFFs) were transfected with the TALEN plasmids plus a surrogate reporter plasmid. PDX1-mutated PFFs were enriched by magnetic separation and used to produce homozygous PDX1-KO pigs via a two-step somatic cell nuclear transfer (SCNT) cloning process. In the first SCNT step, we obtained eight fetuses, established PFF cell lines, and analyzed PDX1 gene mutations by T7 endonuclease 1 assays and Sanger sequencing. Five fetuses showed mutations at the PDX1 loci with two biallelic mutations and three monoallelic mutations (mutation rate of 62.5%). In the second step, a PDX1 biallelic mutant PFF cell line with a 2 bp deletion in one allele and a 4 bp insertion in the other allele was used as a donor to generate cloned pigs via SCNT. From 462 cloned embryos transferred into two surrogates, nine live piglets were delivered. These piglets at birth were not clearly distinguishable phenotypically from wild-type piglets, but soon developed severe diarrhea and vomiting and all died within 2 days after birth. Dissection of PDX1-KO piglets revealed that the liver, gallbladder, spleen, stomach, common bile duct, and other viscera were present and normal, but the pancreas was absent in all cases.