Zhenfang Wu

Effects of DNMT1 and HDAC Inhibitors on Gene-Specific Methylation Reprogramming during Porcine Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) in mammalian cloning currently remains inefficient. Incomplete or erroneous epigenetic reprogramming of specialized donor somatic nuclear and resulting aberrant gene expression during development of cloned embryos is commonly believed as the main reason that causes the low efficiency of SCNT. Use of small molecular reprogramming modifiers to assist the somatic nucleus to mimic naturally occurring DNA methylation and chromatin remodeling in nucleus of fertilization-derived zygotes, has been widely attempted to improve cloning efficiency. However, impacts of these small modifiers on gene-specific methylation dynamics and their potential effects on methylation of imprinted gene have rarely been traced. Here, we attempted two relatively novel DNMT1 inhibitor (DNMTi) and histone deacetylase inhibitor (HDACi), scriptaid and RG108, and demonstrated their effects on dynamics of gene-specific DNA methylation and transcription of porcine SCNT embryos. We found that scriptaid and RG108 had synergetic effects on rescuing the disrupted methylation imprint of H19 during SCNT at least partially by repression over-expressed MBD3 in eight-cell cloned embryos. Furthermore, we firstly identified a differential methylation regions (DMRs) at 5′ flanking regions of XIST gene and found that scriptaid alone and its combination with RG108 modify the dynamics of both transcription and DNA methylation levels in cloned embryos, by different manners. Additionally, we found that scriptaid alone and its combination with RG108 can significantly promote the transcription of NANOG in cloned embryos and enhance their pre-implantation developmental capacity. Our results would contribute to uncovering the epigenetic reprogramming mechanisms underlying the effects of assisted small molecules on improvement of mammalian cloning efficiency.

Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase Based pmGENIE-3 Plasmids

ABSTRACT The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.

Genome Wide Association Analysis Reveals New Production Trait Genes in a Male Duroc Population.

In this study, 796 male Duroc pigs were used to identify genomic regions controlling growth traits. Three production traits were studied: food conversion ratio, days to 100 KG, and average daily gain, using a panel of 39,436 single nucleotide polymorphisms. In total, we detected 11 genome-wide and 162 chromosome-wide single nucleotide polymorphism trait associations. The Gene ontology analysis identified 14 candidate genes close to significant single nucleotide polymorphisms, with growth-related functions: six for days to 100 KG (WT1, FBXO3, DOCK7, PPP3CA, AGPAT9, and NKX6-1), seven for food conversion ratio (MAP2, TBX15, IVL, ARL15, CPS1, VWC2L, and VAV3), and one for average daily gain (COL27A1). Gene ontology analysis indicated that most of the candidate genes are involved in muscle, fat, bone or nervous system development, nutrient absorption, and metabolism, which are all either directly or indirectly related to growth traits in pigs. Additionally, we found four haplotype blocks composed of suggestive single nucleotide polymorphisms located in the growth trait-related quantitative trait loci and further narrowed down the ranges, the largest of which decreased by ~60 Mb. Hence, our results could be used to improve pig production traits by increasing the frequency of favorable alleles via artificial selection.

Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells

CRISPR/Cas9 is an efficient customizable nuclease to generate double-strand breaks (DSBs) in the genome. This process results in knockout of the targeted gene or knock-in of a specific DNA fragment at the targeted locus in the genome of various species. However, efficiency of knock-in mediated by homology-directed repair (HDR) pathway is substantially lower compared with the efficiency of knockout mediated by the nonhomologous end-joining (NHEJ) pathway. Suppressing NHEJ pathway or enhancing HDR pathway has been proven to enhance the nuclease-mediated knock-in efficiency in cultured cells and model organisms. We here investigated the effect of small molecules, Scr7, L755507 and resveratrol, on promoting HDR efficiency in porcine fetal fibroblasts. Results from eGFP reporter assay showed that these small molecules could increase the HDR efficiency by 2–3-fold in porcine fetal fibroblasts. When transfecting with the homologous template DNA and CRISPR/Cas9 plasmid and treating with small molecules, the rate of knock-in porcine fetal fibroblast cell lines with large DNA fragment integration could reach more than 50% of the screened cell colonies, compared with 26.1% knock-in cell lines in the DMSO-treated group. The application of small molecules offers a beneficial approach to improve the frequency of precise genetic modifications in primary somatic cells.

Influence of embryo handling and transfer method on pig cloning efficiency

The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipients ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipients pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs.

Possible introgression of the VRTN mutation increasing vertebral number, carcass length and teat number from Chinese pigs into European pigs

Vertnin (VRTN) variants have been associated with the number of thoracic vertebrae in European pigs, but the association has not been evidenced in Chinese indigenous pigs. In this study, we first performed a genome-wide association study in Chinese Erhualian pigs using one VRTN candidate causative mutation and the Illumina Porcine 60K SNP Beadchips. The VRTN mutation is significantly associated with thoracic vertebral number in this population. We further show that the VRTN mutation has pleiotropic and desirable effects on teat number and carcass (body) length across four diverse populations, including Erhualian, White Duroc × Erhualian F2 population, Duroc and Landrace pigs. No association was observed between VRTN genotype and growth and fatness traits in these populations. Therefore, testing for the VRTN mutation in pig breeding schemes would not only increase the number of vertebrae and nipples, but also enlarge body size without undesirable effects on growth and fatness traits, consequently improving pork production. Further, by using whole-genome sequence data, we show that the VRTN mutation was possibly introgressed from Chinese pigs into European pigs. Our results provide another example showing that introgressed Chinese genes greatly contributed to the development and production of modern European pig breeds.

Transgenic overexpression of bone morphogenetic protein 11 propeptide in skeleton enhances bone formation.

Bone morphogenetic protein 11 (BMP11) is a key regulatory protein in skeletal development. BMP11 propeptide has been shown to antagonize GDF11 activity in vitro. To explore the role of BMP11 propeptide in skeletal formation in vivo, we generated transgenic mice with skeleton-specific overexpression of BMP11 propeptide cDNA. The mice showed a transformation of the seventh cervical vertebra into a thoracic vertebra in our previous report. Presently, further characterizations of the transgenic mice indicated that ossification in calvatia was dramatically enhanced in transgenic fetuses at 16.5 dpc in comparison with their wild-type littermates. At 10 weeks of age, bone mineral content and bone mineral density were significantly (P<0.05) higher in transgenic mice than that in their wild-type littermates based on dual energy X-ray absorptiometry analysis. The relative trabecular bone volume measured by histological analysis was dramatically increased in transgenic mice compared with their wild-type littermates. The enhanced bone formations in the transgenic mice appear to result from increase osteoblast activities as the expressions of four osteoblast markers - α1 type 1 collagen, osteocalcin, alkaline phosphatase and phex were significantly higher in transgenic fetuses than that in their wild-type littermates. These results suggest that over-expression of BMP11 propeptide stimulates bone formation by increasing osteoblast cell functions.

Unraveling the Fecal Microbiota and Metagenomic Functional Capacity Associated with Feed Efficiency in Pigs

Gut microbiota plays fundamental roles in energy harvest, nutrient digestion, and intestinal health, especially in processing indigestible components of polysaccharides in diet. Unraveling the microbial taxa and functional capacity of gut microbiome associated with feed efficiency can provide important knowledge to improve pig feed efficiency in swine industry. In the current research, we studied the association of fecal microbiota with feed efficiency in 280 commercial Duroc pigs. All experimental pigs could be clustered into two enterotype-like groups. Different enterotypes showed the tendency of association with the feed efficiency (P = 0.07). We further identified 31 operational taxonomic units (OTUs) showing the potential associations with porcine feed efficiency. These OTUs were mainly annotated to the bacteria related to the metabolisms of dietary polysaccharides. Although we did not identify the RFI-associated bacterial species at FDR < 0.05 level, metagenomic sequencing analysis did find the distinct function capacities of gut microbiome between the high and low RFI pigs (FDR < 0.05). The KEGG orthologies related to nitrogen metabolism, amino acid metabolism, and transport system, and eight KEGG pathways including glycine, serine, and threonine metabolism were positively associated with porcine feed efficiency. We inferred that gut microbiota might improve porcine feed efficiency through promoting intestinal health by the SCFAs produced by fermenting dietary polysaccharides and improving the utilization of dietary protein. The present results provided important basic knowledge for improving porcine feed efficiency through modulating gut microbiome.

CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic losses to current swine production worldwide. Highly pathogenic PRRSV (HP‐PRRSV), originated from a genotype 2 PRRSV, is more virulent than classical PRRSV and further exacerbates the economic impact. HP‐PRRSV has become the predominant circulating field strain in China since 2006. CD163 is a cellular receptor for PRRSV. The depletion of CD163 whole protein or SRCR5 region (interaction site for the virus) confers resistance to infection of several PRRSV isolates in pigs or cultured host cells. In this study, we described the generation of a CD163 knockout (KO) pig in which the CD163 protein was ablated by using CRISPR/Cas9 gene targeting and somatic cell nuclear transfer (SCNT) technologies. Challenge with HP‐PRRSV TP strain showed that CD163 KO pigs are completely resistant to viral infection manifested by the absence of viremia, antibody response, high fever or any other PRRS‐associated clinical signs. By comparison, wild‐type (WT) controls displayed typical signs of PRRSV infection and died within 2 weeks after infection. Deletion of CD163 showed no adverse effects to the macrophages on immunophenotyping and biological activity as hemoglobin–haptoglobin scavenger. The results demonstrated that CD163 knockout confers full resistance to HP‐PRRSV infection to pigs without impairing the biological function associated with the gene. HighlightsWe establish knockout pigs devoid of CD163 expression via CRISPR/Cas9 gene editing combined with SCNT.The modified pigs are completely protected from challenge with highly pathogenic PRRSV infection.Deletion of CD163 shows no adverse effects to the other biological functions associated with this gene.

Genome-wide association analysis reveals genetic loci and candidate genes for feeding behavior and eating efficiency in Duroc boars

Efficient use of feed resources is a challenge in the pork industry because the largest variability in expenditure is attributed to the cost of fodder. Efficiency of feeding is directly related to feeding behavior. In order to identify genomic regions controlling feeding behavior and eating efficiency traits, 338 Duroc boars were used in this study. The Illumina Porcine SNP60K BeadChip was used for genotyping. Data pertaining to individual daily feed intake (DFI), total daily time spent in feeder (TPD), number of daily visits to feeder (NVD), average duration of each visit (TPV), mean feed intake per visit (FPV), mean feed intake rate (FR), and feed conversion ratio (FCR) were collected for these pigs. Despite the limited sample size, the genome-wide association study was acceptable to detect candidate regions association with feeding behavior and eating efficiency traits in pigs. We detected three genome-wide (P < 1.40E-06) and 11 suggestive (P < 2.79E-05) single nucleotide polymorphism (SNP)-trait associations. Six SNPs were located in genomic regions where quantitative trait loci (QTLs) have previously been reported for feeding behavior and eating efficiency traits in pigs. Five candidate genes (SERPINA3, MYC, LEF1, PITX2, and MAP3K14) with biochemical and physiological roles that were relevant to feeding behavior and eating efficiency were discovered proximal to significant or suggestive markers. Gene ontology analysis indicated that most of the candidate genes were involved in the development of the hypothalamus (GO:0021854, P < 0.0398). Our results provide new insights into the genetic basis of feeding behavior and eating efficiency in pigs. Furthermore, some significant SNPs identified in this study could be incorporated into artificial selection programs for Duroc-related pigs to select for increased feeding efficiency.