Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs

Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs

Atorvastatin suppresses lipopolysaccharideinduced inflammation in human coronary artery endothelial cells

he present study was designed to examine the effects of atorvastatin on vascular inflammatory responses in human coronary artery endothelial cells(HCAECs), when challenged by lipopolysaccharide (LPS), a Toll-like receptor-4 (TLR4) ligand. HCAECs were pretreated with atorvastatin and induced by LPS. The expression of TLR4, interleukin -6(IL-6), monocyte chemoattractant protein 1(MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecular-1(ICAM-1), nuclear factor-κB (NF-κB) and p38 mitogen activated protein kinase(p38 MAPK) were evaluated using Real-time polymerase chain reaction, cytokine ELISA assay and Western blotting. The results showed that pretreatment with atorvastatin down-regulated the expression of TLR4 in LPS-activated HCAECs. Atorvastatin also attenuated the LPS-induced expression of interleukin IL-6 and MCP-1, at both the transcription and translation level in HCAECs. LPS-induced endothelial cell adhesion molecules, ICAM-1 and VCAM-1 expression were also reduced by pretreatment with atorvastatin. Furthermore, atorvastatin efficiently suppressed LPS-induced phosphorylation of NF-κB and p38 MAPK in HCAECs. These findings show that atorvastatin suppresses endothelial cell inflammation, suggesting that atorvastatin may be suitable for development as a therapeutic agent for inflammatory cardiovascular disease

Effect of IGFBP2 Overexpression on the Expression of Fatty Acid Synthesis Genes in Primary Cultured Chicken Hepatocytes

The effects of insulin-like growth factor binding protein 2 (IGFBP2) on the expression of fatty acid synthesis regulators and triglyceride production were investigated in primary cultured chicken hepatocytes. The full-length chicken IGFBP2 coding region was synthesized by overlap extension PCR and cloned into the pcDNA3.1 vector. An in situ digestion method was used to prepare the chicken hepatocytes. Primary chicken hepatocytes were maintained in monolayer culture. Real-time PCR was used to detect changes in the expression of IGFBP2, PPARG, IGF1, IGF1R, APOAI, and LFABP, after the overexpression of IGFBP2 in chicken hepatocytes. Triglyceride production and glucose content were also evaluated using triglyceride and glucose analysis methods. The expression level of IGFBP2 increased after transfection of the IGFBP2-containing vector. The expression levels of PPARG, IGF1, and IGF1R also increased in cultured chicken hepatocytes after the overexpression of IGFBP2, whereas the expression of LFABP and APOAI decreased. Triglyceride production in primary cultured chicken hepatocytes increased after the overexpression of IGFBP2. These results suggest that IGFBP2 is involved in lipogenesis, increasing both the expression of fatty acid synthesis regulators, and triglyceride production in primary cultured chicken hepatocytes

CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts

The multi-gene editing porcine cell model can analyze the genetic mechanisms of multiple genes, which is beneficial for accelerating genetic breeding. However, there has been a lack of an effective strategy to simultaneously perform precise multi-gene editing in porcine cells. In this study, we aimed to improve the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells. CRISPR RNP, including Cas9 protein, sgRNA, and ssODN, was used to generate precise nucleotide substitutions by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by PCR for each target site. To enhance HDR efficacy, small-molecule M3814 and phosphorothioate-modified ssODN were employed. All target DNA samples were sequenced and analyzed, and the efficiencies of different combinations of the CRISPR RNP system in target sites were compared. The results showed that when 2 μM M3814, a small molecule which inhibits NHEJ-mediated repair by blocking DNA-PKs activity, was used, there was no toxicity to PFFs. The CRISPR RNP-mediated HDR efficiency increased 3.62-fold. The combination of CRISPR RNP with 2 μM M3814 and PS-ssODNs achieved an HDR-mediated precision gene modification efficiency of approximately 42.81% in mutated cells, a 6.38-fold increase compared to the control group. Then, we used the optimized CRISPR RNP system to perform simultaneous editing of two and three loci at the INS and RLN3 genes. The results showed that the CRISPR RNP system could simultaneously edit two and three loci. The efficiency of simultaneous editing of two loci was not significantly different from that of single-gene editing compared to the efficiency of single-locus editing. The efficiency of simultaneous precise editing of INS, RLN3 exon 1, and RLN3 exon 2 was 0.29%, 0.24%, and 1.05%, respectively. This study demonstrated that a 2 μM M3814 combination with PS-ssODNs improves the efficacy of CRISPR RNP-mediated precise gene editing and allows for precise editing of up to three genes simultaneously in porcine cells

Improving the Efficiency of CRISPR Ribonucleoprotein-Mediated Precise Gene Editing by Small Molecules in Porcine Fibroblasts

The aim of this study was to verify whether small molecules can improve the efficiency of precision gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoprotein (RNP) in porcine cells. CRISPR associated 9 (Cas9) protein, small guide RNA (sgRNA), phosphorothioate-modified single-stranded oligonucleotides (ssODN), and different small molecules were used to generate precise nucleotide substitutions at the insulin (INS) gene by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by polymerase chain reaction (PCR) for the target site. All samples were sequenced and analyzed, and the efficiencies of different small molecules at the target site were compared. The results showed that the optimal concentrations of the small molecules, including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A, for in vitro-cultured PFFs’ viability were determined. Compared with the control group, the single small molecules including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A increased the efficiency of HDR-mediated precise gene editing from 1.71-fold to 2.28-fold, respectively. There are no benefits in using the combination of two small molecules, since none of the combinations improved the precise gene editing efficiency compared to single small molecules. In conclusion, these results suggested that a single small molecule can increase the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells

Trichostatin A Rescues the Disrupted Imprinting Induced by Somatic Cell Nuclear Transfer in Pigs

<div><p>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 <i>H19/Igf2</i> methylation and transcription in porcine cloned embryos treated with trichostatin A (TSA), and examined <i>H19/Igf2</i> imprinting patterns in cloned fetuses and piglets. Our results showed that compared with the maintenance of <i>H19/Igf2</i> methylation in fertilized embryos, cloned embryos displayed aberrant <i>H19/Igf2</i> methylation and lower <i>H19/Igf2</i> transcripts. When TSA enhanced the development of cloned embryos, the disrupted <i>H19/Igf2</i> 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 <i>H19/Igf2</i> 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.</p></div

<i>In vitro</i> development of cloned embryos treated with TSA.

<p>40 nM TSA enhanced the development of cloned embryos.</p><p>^a-bValues in the same column with different superscripts differ significantly (P<0.05).</p><p><i>In vitro</i> development of cloned embryos treated with TSA.</p

Methylation statuses of H19/Igf2 in 35-day fetuses.

<p>A, the morphologies of 35-day fetuses, B and C, the corresponding methylation statuses of H19/Igf2 in 35-day fetuses. IV, the in vivo produced fetuses, IVF, the IVF fetuses, NT-CON-N, the normal cloned fetuses in the NT-CON group, NT-CON-A, the abnormal cloned fetuses in the NT-CON group, NT-TSA-N, the normal cloned fetuses in the NT-TSA group, and NT-TSA-A, the abnormal cloned fetuses in the NT-TSA group. TSA prevented the H19/Igf2 imprinting disruption and morphological abnormality of cloned fetuses. Black or white circles indicate methylated or unmethylated CpG sites. The data were expressed as mean ± SEM. ^a-cValues with different superscripts differ significantly (P<0.05).</p

Relative H19/Igf2 transcripts in early embryos.

<p>A, relative Igf2 transcripts at 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages of IVF, NT-CON and NT-TSA embryos, B, H19 transcripts in IVF embryos, and B', H19 transcripts in blastocysts of IVF, NT-CON and NT-TSA embryos. TSA improved the expression levels of H19/Igf2 in cloned embryos. The transcript abundance in MII stage oocytes (A and B) or IVF blastocysts (B') was considered to be the control. The data were expressed as mean ± SEM. ^a-eValues with different superscripts differ significantly (P<0.05).</p

H19/Igf2 methylation statuses in early embryos.

<p>The methylation statuses of H19/Igf2 at 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages of IVF, NT-CON and NT-TSA embryos were examined. Cloned embryos displayed aberrant imprinting of H19/Igf2, while TSA rescued the disrupted imprinting in cloned embryos. Black or white circles indicate methylated or unmethylated CpG sites, respectively, and gray circles represent mutated and/or single nucleotide polymorphism (SNP) variation at certain CpG sites.</p