Tartaric acid induces toxicity in Madin-Darby canine kidney cells, but not human kidney-2 cells in vitro, and is prevented by organic anion transporter inhibition and human OAT-4 transfection.

OBJECTIVES: To evaluate the effect of tartaric acid (TTA) on Madin-Darby canine kidney (MDCK) cells compared to human kidney (HK)-2 cells. Secondarily, to evaluate the effects of probenecid, an organic anion transporter (OAT)-1 inhibitor, as well as human (h)OAT-4 transfection into MDCK cells to prevent TTA-induced cytotoxicity through decreasing accumulation via OAT-1 uptake inhibition or increasing OAT-4-mediated TTA efflux. DESIGN: Seventy-two-hour TTA concentration response and inhibitor studies in immortalized cell lines. SETTING: School of Pharmacy biomedical research laboratory and tissue culture facility. ANIMALS/SAMPLES: MDCK and HK-2 immortalized cell lines. INTERVENTIONS: Both cell lines were treated with increasing concentrations of TTA for 72 hours. Additionally, MDCK cells were co-incubated with TTA and increasing concentrations of probenecid or had been transfected with hOAT-4 and subsequently treated with TTA for 72 hours. MEASUREMENTS AND MAIN RESULTS: Media and samples were collected and lactate dehydrogenase (LDH) release was measured. LDH release was measured to assess TTA-induced cytotoxicity after 72 hours. LDH was not significantly increased in the HK-2 cells at any concentration but was significantly increased in the MDCK cells from 10 to 100 mM. LDH concentrations were significantly decreased (61%) in MDCK cells incubated with 50 mM TTA and probenecid when compared to TTA alone. hOAT-4 MDCK cell transfection also significantly reduced LDH release (57%) when comparing the transfected MDCK cells to the nontransfected MDCK cells treated with 50 mM TTA. CONCLUSIONS: TTA is a species-specific nephrotoxicant in dogs due to an interspecies difference in OAT-4 expression. Inhibiting TTA uptake in MDCK cells in vitro using the OAT-specific inhibitor, probenecid, prevents TTA-induced cytotoxicity.

Experimental evidence for occurrence of putative copy-choice recombination between two Senecavirus A genomes.

Senecavirus A (SVA), formerly known as Seneca Valley virus, belongs to the genus Senecavirus in the family Picornaviridae. SVA has a single-stranded, positive-sense RNA genome, which is actually an mRNA that initiates translation via its own internal ribosome entry site (IRES). The SVA IRES has been demonstrated to be the hepatitis C virus (HCV)-like IRES, containing eight stem-loop domains: domain (D)II, DIIIa, DIIIb, DIIIc, DIIId1, DIIId2, DIIIe and DIIIf. In this study, stem-forming motifs (SFMs) in the eight domains were independently subjected to site-directed mutagenesis (SDM) to construct eight SVA minigenomes for dual-luciferase reporter assay. The result suggested that except the DII, the other seven domains were closely evolved in the IRES activity. Subsequently, a full-length SVA cDNA clone tagged with a reporter gene was genetically modified to construct eight SFM-mutated ones, separately transfected into BSR-T7/5 cells in an attempt to rescue replication-competent SVAs. Nevertheless, no virus was successfully rescued from its own cDNA clone, implying each of the putative domains necessary in SVA IRES for viral replication. Further, we attempted to rescue replication-competent SVA via pairwise transfection of cDNA clones. Out of 28 combinations of co-transfection, four were demonstrated to be able to rescue replication-competent SVAs. Sanger sequencing showed that all four viruses had the wild-type IRES genotype, suggesting the occurrence of putative copy-choice recombination between two IRES-modifying genomes.

Gene editing in porcine embryos using a combination of electroporation and transfection methods.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation- and lipofection-mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)-intact and -free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP-intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9-based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP-intact or -free zygotes.

Application of transposon systems in the transgenesis of bovine somatic and germ cells.

BACKGROUND: Several DNA transposons including PiggyBac (PB), Sleeping Beauty (SB), and Tol2 have been applied as effective means for of transgenesis in many species. Cattle are not typically experimental animals, and relatively little verification has been presented on this species. Thus, the goal here was to determine the applicability of three transposon systems in somatic and embryo cells in cattle, while also investigating which of the three systems is appropriate for each cell type. Green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, the GFP-positive cells or blastocysts were observed through fluorescence, while the transfection efficiency was calculated by FACS. RESULTS: In bovine somatic cells, the PB (63.97 ± 11.56) showed the highest efficiency of the three systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Conversely, Tol2 (75.00%) and SB (70.00%) presented a higher tendency in the embryonic cells compared to PB (42.86%). CONCLUSIONS: These results demonstrate that these three transposon systems can be used in bovine somatic cells and embryos as gene engineering experimental methods. Moreover, they demonstrate which type of transposon system to apply depending on the cell type.

Evaluation of a D-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines.

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.

Generation of mutant pigs by lipofection-mediated genome editing in embryos.

The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.

INHBA transfection regulates proliferation, apoptosis and hormone synthesis in sheep granulosa cells.

Inhibin subunit beta A (INHBA) participates in the synthesis of inhibin A, activin A and activin AB. Here we investigated the effect and molecular mechanism of INHBA on proliferation, apoptosis and hormone synthesis in sheep granulosa cells (GCs) using in vitro transfection. We first noticed that INHBA expression increased with follicle diameter and was widely distributed in ovarian tissue. The proliferation rate of GCs was significantly increased and decreased with overexpression and silence of INHBA, respectively, compared with the negative controls. INHBA transfection affected GC proliferation and apoptosis, regulating the expression of many cell cycle-related and apoptosis-related genes. INHBA overexpression significantly decreased activin and estradiol secretion while increasing inhibin and progesterone secretion. The expression of follicle-stimulating hormone beta subunit was significantly decreased and increased with INHBA overexpression and knockdown, respectively. Notably, silence of INHBA inhibited the expression of many transforming growth factor beta-related genes. Overall, the functional molecule of INHBA gene may be associated with follicular development via regulating proliferation, apoptosis and folliculogenesis-related hormone secretion of sheep GCs. In addition, our findings may contribute to a better understanding of the law of follicular development and thus improve the reproductive performance of female animals.

Safe and effective aerosolization of in vitro transcribed mRNA to the respiratory tract epithelium of horses without a transfection agent.

Vaccines and therapeutics using in vitro transcribed mRNA hold enormous potential for human and veterinary medicine. Transfection agents are widely considered to be necessary to protect mRNA and enhance transfection, but they add expense and raise concerns regarding quality control and safety. We found that such complex mRNA delivery systems can be avoided when transfecting epithelial cells by aerosolizing the mRNA into micron-sized droplets. In an equine in vivo model, we demonstrated that the translation of mRNA into a functional protein did not depend on the addition of a polyethylenimine (PEI)-derived transfection agent. We were able to safely and effectively transfect the bronchial epithelium of foals using naked mRNA (i.e., mRNA formulated in a sodium citrate buffer without a delivery vehicle). Endoscopic examination of the bronchial tree and histology of mucosal biopsies indicated no gross or microscopic adverse effects of the transfection. Our data suggest that mRNA administered by an atomization device eliminates the need for chemical transfection agents, which can reduce the cost and the safety risks of delivering mRNA to the respiratory tract of animals and humans.

Transfection of exogenous DNA complexed to cationic dendrimer induces alterations of bovine sperm microRNAome.

MicroRNAs have been hypothesized to be involved in the regulation of male fertility potential. The primary aim of our study was to demonstrate the effects of transfection with dendrimer nanostructure on the parameters of bovine sperm quality and to investigate whether the microRNA profile could be disturbed after cationic dendrimer-mediated exogenous DNA transfection of bovine spermatozoa. The binding of exogenous DNA was significantly increased when dendrimer-based transfection was implemented. However, cationic dendrimer transfection induced detrimental changes in the kinetics and sperm quality parameters, such as membrane integrity, acrosome reaction, and mitochondrial membrane potential, when compared to the control group. Sperm microRNA sequencing revealed 218 known and 106 novel microRNAs in the sperm samples, among which nine were dysregulated after transfection (one was upregulated and eight were downregulated), in comparison to the non-transfected sperm. All the dysregulated microRNAs were related to sperm quality and embryonic development. These results suggest that the transfection process using the dendrimer nanostructure has an impact on the quality and microRNA profile of bovine sperm.

Introduction of a plasmid and a protein into bovine and swine cells by water-in-oil droplet electroporation.

Instrument cost is a major problem for the transduction of DNA fragments and proteins into cells. Water-in-oil droplet electroporation (droplet-EP) was recently invented as a low-cost and effective method for the transfection of plasmids into cultured human cells. We here applied droplet-EP to livestock animal cells. Although it is difficult to transfect plasmids into bovine fibroblasts using conventional lipofection methods, droplet-EP enabled us to introduce an enhanced green fluorescent protein (EGFP)-expressing plasmid into bovine earlobe fibroblasts. The optimal transfection condition was 3.0 kV, which allowed 19.1% of the cells to be transfected. For swine earlobe fibroblasts, the maximum transfection efficacy was 14.0% at 4.0 kV. After transfection with droplet-EP, 69.1% of bovine and 76.5% of swine cells were viable. Furthermore, droplet-EP successfully transduced Escherichia coli recombinant EGFP into frozen-thawed bovine sperm at 1.5 kV. Flow cytometry analysis revealed that 71.5% of spermatozoa exhibited green fluorescence after transfection. Overall, droplet-EP is suitable for the transfection of plasmids and proteins into cultured livestock animal cells.

Successful transplantation of transfected enriched buffalo (Bubalus bubalis) spermatogonial stem cells to homologous recipients.

Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ±â€¯4.56%, P < 0.05) for enriched SSCs in comparison to fugene HD (6.7 ±â€¯0.25%) and lipofectamine 3000 (15.57 ±â€¯0.74%). The transfected enriched SSCs were transplanted into buffalo males under the ultrasound guidance and testis was removed by castration after 7-8 weeks of transplantation. Persistence and localization of donor cells within recipient seminiferous tubules was confirmed using fluorescent microscopy. Further confirmation was done by flow cytometric evaluation of GFP expressing cells among those isolated from two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated for the first time, generation of buffalo transfected enriched SSCs and their successful homologous transplantation in buffaloes. This study represents the first step towards genetic modifications in buffaloes using SSC transplantation technique.

Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3) contains two major open reading frames (ORFs) and the ORF2 gene encodes the major structural capsid protein. In this study, nuclear localization of ORF2 was demonstrated by fluorescence observation and subcellular fractionation assays in ORF2-transfected PK-15 cells. The subcellular localization of truncated ORF2 indicated that the 38 N-terminal amino acids were responsible for the nuclear localization of ORF2. The truncated and site-directed mutagenesis of this domain were constructed, and the results demonstrated that the basic amino acid residues at positions 8-32 were essential for the strict nuclear localization. The basic motifs 8RRR-R-RRR16 and 16RRRHRRR22 were further shown to be the key functional nucleolar localization signals that guide PCV3 ORF2 into nucleoli. Furthermore, sequence analysis showed that the amino acids of PCV3 nuclear localization signals were highly conserved. Overall, this study provides insight into the biological and functional characteristics of the PCV3 ORF2 protein.

Krüppel-like factor KLF9 inhibits chicken intramuscular preadipocyte differentiation.

1. Poultry meat quality is affected by many factors, among which intramuscular fat (IMF) is predominant. IMF content affects tenderness, juiciness and flavour of meat. Krüppel-like transcriptional factors (KLFs) are important regulators of adipocyte differentiation. However, little is known about the KLF9 gene associated with poultry IMF deposition, especially intramuscular adipocyte differentiation.2. Previous work has shown that chicken KLF9 was differentially expressed during adipogenesis of intramuscular preadipocytes differentiation. In this study, the function of KLF9 in chicken intramuscular preadipocytes differentiation was investigated.3. In the chicken preadipocyte differentiation model, KLF9 expression showed a major increase with adipogenic induction. Overexpression of KLF9 down-regulated the expression of the adipogenic marker gene AP2, and impaired triglyceride accumulation. Knockdown of KLF9 in chicken intramuscular preadipocytes increased the expression of PPARG, CEBPA and AP2. In addition, it was proposed that KLF9 may regulate adipogenesis via lncRNAs NONGGAT002209.2, NONGGAT003346.2, NONGGAT000436.2 and NONGGAT006302.2 in chicken.4. The data supported a novel role of KLF9 in regulating chicken intramuscular preadipocyte differentiation. Such findings may contribute to a more thorough understanding of chicken IMF deposition and the improvement of poultry meat quality.

Immune-related genes response to stimulation of miR-155 overexpression in CIK (ctenopharyngodon idella kidney) cells and zebrafish.

MiR-155 regulates the development of germinal-center and the generation of immunoglobulin class-switched plasma cells. However, whether miR-155 is involved in immune response in fish is still unclear. Here, CIK cells transfected with miR-155 overexpressed plasmid inhibited mRNA expression of mIg and Rag2 (P < 0.05). Interestingly, mIg was predicted as a potential target gene of miR-155 by RNAhybrid, with a putative binding site in its CDS. Further, mIg luciferase reporter vectors with successive deletions of mIg cDNA sequence were constructed and dual luciferase reporter assay showed that vectors containing the sequence from 318 to 347 in CDS exhibited lower relative luciferase activity than others without predicted binding region (P < 0.05), which indicated mIg is the target gene of miR-155 and reveal bona fide targeted binding site of mIg for miR-155 in fish. In vivo, the zebrafish were respectively injected with miR-155 overexpressed and empty vector, and showed that miR-155 efficiently expressed in zebrafish (P < 0.01), which consistently decreased mRNA level of immune-related genes, including mIg (P < 0.01), sIg (P < 0.05), AID (P < 0.01), PU.1 (P < 0.05) and Rag2 (P < 0.05) at d 3 and d 6 post injection, comparing to control. Collectively, this work indicates that overexpression of miR-155 suppresses the mRNA level of immune-related genes in CIK cells and zebrafish, and mIg is a novel target gene of miR-155 in fish. These findings provide an insight into the miR-155 modulating adaptive immunity in grass carp and zebrafish.

Transient transfection of Babesia ovis using heterologous promoters.

Babesia species, etiological agents of babesiosis, a recognized emerging tick-borne disease, are a significant animal and human health concern with a worldwide socio-economic impact. The development of genetic manipulation techniques, such as transfection technology, is pivotal to improve knowledge regarding the biology of these poorly studied parasites towards better disease control strategies. For Babesia ovis, responsible for ovine babesiosis, a tick-borne disease of small ruminants, these tools are not yet available. The present study was based on the existence of interchangeable cross-species functional promoters between Babesia species. Herein, we describe for the first time B. ovis transient transfection using two heterologous promoters, the ef-1α-B intergenic regions from B. bovis and B. ovata. Their ability to drive expression of a reporter luciferase in B. ovis supports their cross-species functionality. Also, the ef-1α-B promoter region from B. ovata resulted in statistically significantly higher luminescence values in comparison to the control, thus a possibly suitable promoter for stable gene expression. Evaluation of transfection efficiency using qPCR demonstrated that higher luminescence levels were due to promoter strength rather than a higher transfection efficiency. These findings represent a step forward in the development of methods for B. ovis genetic manipulation, an undoubtedly necessary tool to study this parasite basic biology, including its life cycle, the parasite interactions with host cells and virulence factors.

Matrine inhibits IL-1ß secretion in primary porcine alveolar macrophages through the MyD88/NF-κB pathway and NLRP3 inflammasome.

Our previous studies demonstrated that matrine directly acts on the replication process of porcine reproductive and respiratory syndrome virus (PRRSV). Matrine inhibits viral replication and is also associated with the NF-κB signalling pathway. These results suggest that matrine has antiviral and anti-inflammatory effects. However, the specific anti-inflammatory mechanism of matrine is still unclear. In this study, we investigated the anti-IL-1ß mechanism of matrine, as IL-1ß is a major inflammatory cytokine, in porcine alveolar macrophages (PAMs) stimulated with 4 µg PRRSV 5'-untranslated region (UTR) RNA and 1 µg/mL LPS. After 5'UTR RNA and LPS co-stimulation of PAMs for 12 h, the expression of IL-1ß, IL-6, IL-8 and TNF-α was significantly increased. The results also showed that co-stimulation induced the expression of MyD88, and activated the NF-κB signalling pathway and NLRP3 inflammasome. Furthermore, matrine treatment downregulated MyD88, NLRP3 and caspase-1 expression, inhibited ASC speck formation, suppressed IκBα phosphorylation, and interfered with the translocation of NF-κB from the cytoplasm to the nucleus. These results suggest that matrine plays an important role in PAMs co-stimulated with PRRSV 5'UTR RNA and LPS via its effect on NF-κB and the NLRP3 inflammasome. These findings lay the foundation for the exploration of the clinical application of matrine in PRRSV disease.

ZB transposon and chicken vasa homologue (Cvh) promoter interact to increase transfection efficiency of primordial germ cells in vivo.

1. In order to increase the efficiency of generating transgenic chicken, this trial focused on two points: primordial germ cells (PGCs）transfection in vivo and a germline-specific promoter.2. In order to transfect PGCs in vivo, two plasmids (pZB-CAG-GFP, pCMV-ZB）were co-injected into chicken embryos via the subgerminal cavity at Hamburger and Hamilton (HH) stage 2-3 or via blood vessel at HH stage 13-14. Results showed that the percentage of GFP+ embryos, viability and hatching rate of embryos injected at HH stage 13-14 were significantly higher than that at HH stage 2-3.3. Two plasmid transposon systems were used for chicken embryo micro-injections. The donor plasmid, with a green fluorescent protein (GFP) reporter gene, was mediated by the ZB transposon. The helper plasmid was a transposase expression vector driven by the promoter of the chicken vasa homologue (Cvh) gene or Human cytomegalovirus (CMV) promoter. Results showed that 60.98% of gonads in Cvh group expressed GFP, which was 52.50% higher than seen in the CMV group. Only gonad tissue from the Cvh group showed any GFP signal, whereas both gonads and other tissues in the CMV group showed green fluorescence.4. The data suggested that ZB transposon-mediated gene transfer was efficient for transfecting PGCs in vivo; the Cvh promoter drove the transposase gene specifically in the germline and increased the efficiency of germline transmission. Blood vessels injection at HH stage 13-14 may be a more efficient route for PGCs transfection in vivo.

In vivo miRNA delivery in whitefish: Synthetic MiR92b-3p uptake and the efficacy of gene expression silencing.

IMPACT STATEMENT: The delivery of short snippets of RNA, such as synthetic miRNA agents, is an essential step for achieving RNA-mediated knockdown, which has not been studied in sufficient detail in fish. Our results indicate that a MiR92b-3p mimic may be effectively delivered via intraperitoneal injection to the spleen and the liver of whitefish, and that it likely achieves functionality without causing any apparent toxic effects in the challenged animals. We report the novel finding that the MiR92b-3p mimic reduced the in vivo liver mRNA expression levels of its putative pro-apoptotic targets (p53, cdkn1a, and pcna), and important metabolic genes, e.g. cdo1. This shows that this methodology of MiR92b-3p mimic transfection in vivo may be a useful tool for studies that investigate the molecular pathways that confer pro-proliferative and anti-apoptotic phenotypes or those that regulate intracellular metabolism in fish and other vertebrates.

Generation of porcine induced-pluripotent stem cells from Sertoli cells.

Induced pluripotent stem cells (iPSCs) are generated by reprogramming of somatic cells using four transcription factors: OCT4, SOX2, KLF-4, and c-MYC (OSKM). However, reprogramming efficiency of iPSCs is currently poor. In this study, we used the Sertoli line as a novel cell source for somatic cell reprogramming. Neonatal testes were collected from 1-week-old piglets. The testes were digested by a two-step enzymatic method to isolate Sertoli cells. The latter were transfected with retroviral vectors expressing OSKM. The Sertoli iPSC-like colonies were subjected to morphological analysis, alkaline phosphatase staining, RT-PCR, G-banding karyotyping, in vitro differentiation, and in vivo differentiation. Primary Sertoli cells had polygon-shaped morphology and manifested phagocytic activity as determined by a fluorescent bead assay. Sertoli cells also expressed the anti-Müllerian hormone protein in the cytoplasm. According to RT-PCR results, these cells expressed Sertoli cell markers (FSHR, KRT18, and GATA6) and endogenous transcription factors genes (KLF4 and c-MYC). A total of 240 colonies (0.3% efficiency) were detected by day 7 after viral transduction of 72500 cells. The Sertoli iPSC-like colonies contained small cells with a high nucleus-to-cytoplasm ratio. These colonies tested positive for alkaline phosphatase staining, expressed endogenous pluripotency genes, and had a normal karyotype. All these cell lines could form in vitro three-dimensional aggregates that represented three germ layers of embryonic-like cells. A total of two cell lines used for in vivo differentiation produced high-efficiency teratoma. In conclusion, Sertoli cells can efficiently serve as a novel cell source for iPSC reprogramming.

Production of transgenic broilers by non-viral vectors via optimizing egg windowing and screening transgenic roosters.

The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)-transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP-positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence-activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene-positive roosters can improve the production of germline-transmitted transgenic birds.