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The article offers information on in vitro culture and gene editing of porcine spermatogonial stem cells. Topics discussed include application of gene editing in pig cloning technology; better serve animal husbandry, improve livestock fecundity, and enhance potential clinical use; and requirement of identification of key growth factors, component finite medium, and appropriate feeder cells for porcine spermatogonial stem cells.

The extremely low full-term developmental efficiency of cloned pig embryos limits the practical application of pig cloning techniques. Maternal dietary supplementation of the nutritionally important amino acid, arginine, can enhance prenatal developmental rate of in vivo fertilization-derived pig embryos. It was hypothesized that maternal dietary addition of arginine can also improve the developmental capacity of cloned pig embryos. To test this hypothesis, there was a comparison of the reproductive performance between recipient sows fed an L-arginine-supplemented diet (L-Arg group) and those fed the control diet (control group). There was a subsequent comparison of the developmental indexes of cloned piglets farrowed in the L-Arg and control groups of surrogate sows. Dietary supplementation of L-arginine during gestation days 14–75 increased the plasma concentrations of arginine and arginine metabolites, including nitric oxide, spermidine, and putrescine in recipient sows of transferred cloned pig embryos. Although maternal arginine addition did not affect the birth weight and placental development indexes of newborn cloned piglets, it significantly increased the ratio of total cloned piglets born to total transferred cloned pig embryos by increasing the pregnancy rate of recipient sows. The results of this study suggest that nutritional management of recipient sows is an effective approach to improve the developmental rate of cloned pig embryos. [ABSTRACT FROM AUTHOR]

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells. [ABSTRACT FROM AUTHOR]

Many studies have proved that DNA methylation can regulate gene expression and further affect skeletal muscle growth and development of pig, whereas the mechanisms of how DNA methylation or gene expression alteration ultimately lead to phenotypical differences between the cloned and natural mating pigs remain elusive. This study aimed to investigate genome-wide gene expression and DNA methylation differences between abnormally cloned and normally natural mating piglets and identify molecular markers related to skeletal muscle growth and development in pig. The DNA methylation and genome-wide gene expression in the two groups of piglets were analysed through methylated DNA immunoprecipitation binding high-throughput sequencing and RNA sequencing respectively. We detected 1493 differentially expressed genes between the two groups, of which 382 genes were also differentially methylated. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative and monotonic correlation with gene expression levels around the transcription start site of genes. By contrast, no notable monotonic correlation was observed in other regions. Furthermore, we identified some interesting genes and signalling pathways ( e.g. myosin, heavy chain 7 and mammalian target of rapamycin) which possibly play essential roles in skeletal muscle growth and development. The results of this study provide insights into the relationship of DNA methylation with gene expression in newborn piglets and into the mechanisms in abnormally cloned animals through somatic cell nuclear transfer. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) exhibited self-renewal and less differentiation, making the MSCs promising candidates for adult somatic cell nuclear transfer (SCNT). In this article, we tried to produce genome identical pigs through hand-made cloning (HMC), with MSCs and adult skin fibroblasts as donor cells. MSCs were derived from either adipose tissue or peripheral blood (aMSCs and bMSCs, respectively). MSCs usually showed the expression pattern of CD29, CD73, CD90, and CD105 together with lack of expression of the hematopoietic markers CD34and CD45. Flow cytometry results demonstrated high expression of CD29 and CD90 in both MSC lines, while CD73, CD34, and CD45 expression were not detected. In contrary, in reverse transcription-polymerase chain reaction (RT-PCR) analysis, CD73 and CD34 were detected indicating that human antibodies CD73 and CD34 were not suitable to identify porcine cell surface markers and porcine MSC cellular surface markers of CD34 might be different from other species. MSCs also had potential to differentiate successfully into chondrocytes, osteoblasts, and adipocytes. After HMC, embryos reconstructed with aMSCs had higher blastocyst rate on day 5 and 6 than those reconstructed with bMSCs and fibroblasts (29.6% ± 1.3% and 41.1% ± 1.4% for aMSCs vs. 23.9% ± 1.2% and 35.5% ± 1.6% for bMSCs and 22.1% ± 0.9% and 33.3% ± 1.1% for fibroblasts, respectively). Live birth rate per transferred blastocyst achieved with bMSCs (1.59%) was the highest among the three groups. This article was the first report to compare the efficiency among bMSCs, aMSCs, and fibroblasts for boar cloning, which offered a realistic perspective to use the HMC technology for commercial breeding. [ABSTRACT FROM AUTHOR]

Human α-lactalbumin (HLA) has very high nutritional value and important physiological functions during the neonatal period. The peptides derived from HLA provide diverse health benefits including antimicrobial, antiviral, immune-modulating, and antihypertensive effects. Thus, it is worth investigating the effects on offspring development of increasing HLA in milk. In this study, we found that recombinant human α-lactalbumin (rHLA) exhibits efficient inhibition of dipeptidyl peptidase-IV (DPP-IV) activity in an in vitro simulated gastrointestinal digestion system. Using a BAC clone containing the complete HLA gene as a candidate vector, we generated two lines of transgenic cloned sows via somatic cell nuclear transfer that over-expressed rHLA. The average concentrations of rHLA in milk from the two lines of transgenic cloned sows were 2.24 ± 0.71 mg/ml and 2.67 ± 1.29 mg/ml. The feeding experiments revealed that rHLA represses dipeptidyl peptidase-IV (DPP-IV) activity in vivo . Furthermore, the piglets reared by rHLA transgenic cloned sows exhibit better performance in gain of body weight and intestine growth than the control piglets reared by non-transgenic sows. Therefore, these findings indicate that rHLA could serve as a natural precursor for a DPP-IV inhibitor, and the transgenic technology that produced the over-expression of rHLA could be a useful method for pig breeders to improve lactation performance. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P!0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression. [ABSTRACT FROM AUTHOR]

Neuropeptide W (NPW), a novel hypothalamic peptide, is an endogenous ligand for the orphan G protein-coupled receptors GPR7 (NPBWR1) and GPR8 (NPBWR2). Although several studies have implicated NPW in the regulation of feeding and energy metabolism in many species, the precise physiological function of NPW in pigs remains unclear. In this study, we cloned and sequenced NPW, GPR7, and GPR8 cDNA from pigs. NPW, GPR7, and GPR8 mRNA expression was quantified in the pig brain and peripheral tissues by semiquantitative reverse transcriptase polymerase chain reaction. Immunohistochemistry showed that NPW protein expression was limited in the brain and abundant in peripheral tissues. These results suggest that NPW is involved in the regulation of various physiological functions in pigs. The molecular and morphological data from this study provide a basis for further research on the functions of NPW in pigs. [ABSTRACT FROM AUTHOR]

Incomplete reprogramming of pluripotent genes in cloned embryos is associated with low cloning efficiency. Epigenetic modification agents have been shown to enhance the developmental competence of cloned embryos; however, the effect of the epigenetic modification agents on pluripotent gene reprogramming remains unclear. Here, we investigated Nanog reprogramming and the expression patterns of pluripotent transcription factors during early embryo development in pigs. We found that compared with fertilized embryos, cloned embryos displayed higher methylation in the promoter and 5′-untranslated region and lower methylation in the first exon of Nanog. When 5-aza-2′-deoxycytidine (5-aza-dC) or trichostatin A (TSA) enhanced the development of porcine cloned embryos, Nanog methylation reprogramming was also improved, similar to that detected in fertilized counterparts. Furthermore, our results showed that the epigenetic modification agents improved the expression levels of Oct4 and Sox2 and effectively promoted Nanog transcription in cloned embryos. In conclusion, our results demonstrated that the epigenetic modification agent 5-aza-dC or TSA improved Nanog methylation reprogramming and the expression patterns of pluripotent transcription factors, thereby resulting in the enhanced expression of Nanog and high development of porcine cloned embryos. This work has important implications in the improvement of cloning efficiency. [ABSTRACT FROM AUTHOR]

Incomplete DNA methylation reprogramming in cloned embryos leads to poor cloning efficiency. Epigenetic modification agents can improve genomic methylation reprogramming and the development of cloned embryos, however, the effect of epigenetic modification agents on gene-specific methylation reprogramming remains poorly studied. Here, we investigated DNA methylation reprogramming of pluripotency (Oct4) and tissue specific (Thy1) genes during early embryo development in pigs. In this study, we found that compared with in vitro fertilized counterparts, cloned embryos displayed the disrupted patterns of Oct4 demethylation and Thy1 remethylation. When 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) enhanced the development of cloned embryos, the transcripts of DNA methyltransferases (Dnmt1 and Dnmt3a), histone acetyltransferase 1 (Hat1) and histone deacetylase 1 (Hdac1) and the methylation and expression patterns of Oct4 and Thy1 became similar to those detected in in vitro fertilized counterparts. Further studies showed that Dnmt1 knockdown in cloned embryos enhanced the methylation reprogramming of Oct4 and Thy1 and promoted the activation of Oct4 and the silence of Thy1. In conclusion, our results demonstrated that cloned embryos displayed incomplete gene-specific methylation reprogramming and disrupted expression patterns of pluripotency and tissue specific genes, and epigenetic modification agents improved gene-specific methylation reprogramming and expression pattern by regulating epigenetic modification related genes. This work would have important implications in improving cloning efficiency. [ABSTRACT FROM AUTHOR]

Most animals produced by somatic cell nuclear transfer (SCNT) are heteroplasmic for mitochondrial DNA (mtDNA). Oxidative phosphorylation (OXPHOS) in clones therefore requires the coordinated expression of genes encoded by the nuclear DNA and the two sources of mitochondria. Such interaction is rarely studied because most clones are generated using slaughterhouse oocytes of unrecorded origin. Here we traced the maternal lineages of seven diseased and five one-month-old live cloned piglets by sequencing their mtDNA. Additionally by using a 13K oligonucleotide microarray, we compared the expression profiles of nuclear and mtDNA-encoded genes that are involved in mitochondrial functions and regulation between the cloned groups and their age-matched controls (n=5 per group). We found that the oocytes used to generate the cloned piglets were of either the Large White or Duroc background, and oocyte genetic background was not related to the clones’ survival. Expression profiles of mtDNA-encoded genes in clones and controls showed intermixed clustering patterns without treatment or maternal lineage-dependency. In contrast, clones and controls clustered separately for their global and nuclear DNA-encoded mitochondrial genes in the lungs for both the deceased and live groups. Functional annotation of differentially expressed genes encoded by both nuclear and mtDNA revealed abnormal gene expression in the mitochondrial OXPHOS pathway in deceased clones. Among the nine differentially expressed genes of the OXPHOS pathway, seven were down-regulated in deceased clones compared to controls, suggesting deficiencies in mitochondrial functions. Together, these data demonstrate that the coordination of expression of mitochondrial genes encoded by nuclear and mtDNA is disrupted in the lung of diseased clones. [ABSTRACT FROM AUTHOR]

Cloned pigs produced by somatic cell nuclear transfer (SCNT) are important as a potential alternative source of organs. Although SCNT has created new possibilities for targeted gene modification, the successful cloning of pigs is rare. Here, we successfully conducted serial SCNT for three generations. We determined that the piglet genome was inherited from donor cell nuclei using microsatellite analysis of each generation. The methylation of differentially methylated regions (DMRs) inH19was gradually reduced over the three generations of serial SCNT. By contrast, methylation of the insulin-like growth factor 2 (Igf2) DMR increased in the F1 generation, compared to the F0, and remained at the higher level in the F2 and F3 generations. The methylation patterns of housekeeping genes such asGAPDHandβ-actinwere unchanged in the serially cloned pigs. In addition, expression levels ofH19andIgf2were variable for each generation of serial SCNT piglets, but there was no clear relationship between the methylation and gene expression patterns. Our study conclusively demonstrates that the methylation patterns of DMRs inH19andIgf2were altered, compare to the F0 donor, during serial SCNT, but housekeeping genes were unaffected. [ABSTRACT FROM PUBLISHER]