Publications by authors named "Qisheng Zuo"

60 Publications

miR-302d Competitively Binding with the lncRNA-341 Targets TLE4 in the Process of SSC Generation.

Stem Cells Int 2021 8;2021:5546936. Epub 2021 Jun 8.

College of Animal Science and Technology, Yangzhou University, Jiangsu Province Key Laboratory of Animal Breeding and Molecular Design, Yangzhou, 225009 Jiangsu, China.

MicroRNAs (miRNAs) are essential factors in the reproductive process of poultry. Here, we found miR-302d is a potential differentiation and negative factor of chicken embryonic stem cells (ESCs) into spermatogonia stem cells (SSCs). The competition mechanism was carried out for the preliminary exploration to determine the relationship among miR-302d, lncRNA-341(interacting with miR-302d), and target gene TLE4. The results showed that lncRNA-341 can competitively bind to miR-302d to decrease the targeted binding of miR-302d and TLE4 which promotes the differentiation of chicken SSCs. Moreover, it is suggested that miR-302d may participate in the Wnt signaling pathway through TLE4.
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http://dx.doi.org/10.1155/2021/5546936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8205581PMC
June 2021

H3K9me2 regulates early transcription factors to promote mesenchymal stem‑cell differentiation into cardiomyocytes.

Mol Med Rep 2021 Aug 29;24(2). Epub 2021 Jun 29.

Key Laboratory of Animal Breeding and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225001, P.R. China.

Studies have shown that histone H3 at lysine 9 (H3K9me2) is an important epigenetic modifier of embryonic development, cell reprogramming and cell differentiation, but its specific role in cardiomyocyte formation remains to be elucidated. The present study established a model of 5‑Azacytidine‑induced differentiation of rat bone mesenchymal stem cells (MSCs) into cardiomyocytes and, on this basis, investigated the dimethylation of H3K9me2 and its effect on cardiomyocyte formation by knockdown of H3K9me2 methylase, euchromatic histone‑lysine N‑methyltransferase 2 (G9a) and H3K9me2 lysine demethylase 3A (KDM3A). The results demonstrated that, in comparison with the normal induction process, the knockdown of G9a could significantly reduce the H3K9me2 level of the MSCs in the induced model. Reverse transcription‑quantitative (RT‑q) PCR demonstrated that the expression of cardiac troponin T(cTnT) was significantly increased. In addition, flow cytometry demonstrated that the proportion of cTnT‑positive cells was significantly increased on day 21. With the knockdown of KDM3A, the opposite occurred. In order to explore the specific way of H3K9me2 regulating cardiomyocyte formation, western blotting and RT‑qPCR were used to detect the expression of key transcription factors including GATA binding protein 4 (GATA‑4), NK2 Homeobox 5 (Nkx2.5) and myocyte enhancer factor 2c (MEF2c) during cardiomyocyte formation. The decrease of H3K9me2 increased the expression of transcription factors GATA‑4, Nkx2.5 and MEF2c in the early stage of myocardial development while the increase of H3K9me2 inhibited the expression of those transcription factors. Accordingly, it was concluded that H3K9me2 is a negative regulator of cardiomyocyte formation and can participate in cardiomyocyte formation by activating or inhibiting key transcription factors of cardiomyocytes, which will lay the foundation for the optimized induction efficiency of cardiomyocytes in and clinical applications.
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http://dx.doi.org/10.3892/mmr.2021.12255DOI Listing
August 2021

Role and function of the in early sex differentiation in chicken () embryo.

Anim Biotechnol 2021 Jun 21:1-11. Epub 2021 Jun 21.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.

Mono-Sex culturing is an important methodology for intensive livestock and poultry production. Here, was identified as a potential key gene in sex-determination process in chickens via RNA-seq. Then we developed an effective method to interfere or overexpress in chicken embryos through the intravascular injection. QRT-PCR, ELISA and H&E staining were used to detect the effects of on gonadal development of chicken embryos. Results showed that exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads. The qRT-PCR analysis showed that , in females were upregulated under the overexpression of , while and were downregulated . Overexpression of can promote the development of gonadal cortex, while interference with show the opposite result. Additionally, we found that overexpression of the in male chicken embryos could inhibit androgen levels and increase estrogen levels. On the other hand, interfering with in female chicken embryos decreased estrogen levels and increased androgen levels. In conclusion, this work sets the basis for the understanding of the molecular regulatory network for the sex-determination process in chicken embryos as well as providing the theoretical basis for mono-sex culturing of poultry.
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http://dx.doi.org/10.1080/10495398.2021.1935981DOI Listing
June 2021

Promotion of Differentiating Bone Marrow Mesenchymal Stromal Cells (BMSCs) into Cardiomyocytes via HCN2 and HCN4 Cotransfection.

Biomed Res Int 2021 13;2021:5529276. Epub 2021 May 13.

Medical College of Yangzhou University, Yangzhou, Jiangsu 225001, China.

Aim: Investigation of the influences HCN2 and HCN4 has on bone marrow mesenchymal stromal cells (BMSCs) on cardiomyocyte differentiation.

Methods: Miniature adult pigs were used for bone marrow extraction and isolation of BMSCs. The identification of these BMSCs was done by using flow cytometry for the detection of expressed surface antigens CD45, CD11B, CD44, and CD90. Using HCN2 and HCN4 genes cotransfected into BMSCs as group HCN2+HCN4 while myocardial induction solution was used to induced BMSC differentiation in the BMSC induction group. Myocardial marker proteins -actin and cTnT were detected by immunofluorescence staining, while -actin, cTnT, and Desmin myocardial marker proteins expressed were detected by Western blot. The whole-cell patch-clamp technique was used to identify and detect cellular HCN2 channels, HCN4 channel current activation curve, and the inhibitory effect of CsCl on heterologous expression currents.

Results: Flow cytometry results showed that CD45 and CD11B were expressed negatively while CD90 and CD44 were positive. Post HCN2 and HCN4 gene transfection, immunofluorescence staining, and Western blot showed significantly increased HCN2, HCN4, -actin, and cTnT expressed in group HCN2+HCN4 were, which could be compared to the expression levels in the BMSC-induced group. The HCN2+HCN4 group was able to document cell membrane channel ion currents that were similar to If properties.

Conclusion: HCN2 and HCN4 overexpression can considerably enhance the MSC ability to differentiate into cardiomyocytes in vitro and restore the ionic current.
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http://dx.doi.org/10.1155/2021/5529276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140823PMC
May 2021

H3K4me2 Promotes the Activation of lncCPSET1 by Jun in the Chicken PGC Formation.

Animals (Basel) 2021 May 27;11(6). Epub 2021 May 27.

Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.

Primordial germ cells are the ancestors of female and male cells. Current research has shown that long non-coding RNA (lncRNA) and Histone methylation are the pivotal epigenetic factors in the PGC formation. However, there are few studies on the regulatory mechanism of lncRNA in the formation of PGC. Here, we define the lncRNA highly expressed in chicken PGC, lncCPSET1 (chicken-PGC-specifically-expressed transcript 1) This study found that compared with the interference of lncCPSET1/histone methylase Mll2 alone, the PGC formation was severely inhibited with the interference of lncCPSET1 and histone methylase Mll2 jointly in vivo and in vitro. Studies on the transcription level of lncCPSET1 found that H3K4me2 and transcription factor Jun have a positive effect on the activation of lncCPSET1; while DNA hypomethylation inhibits the expression of lncCPSET1. In terms of mechanism, compared with DNA methylation, H3K4me2 dominates lncCPSET1 activation. H3K4me2 can be enriched in the lncCPSET1 promoter, change its chromosome conformation, recruit the transcription factor Jun, and activate the expression of lncCPSET1. Taken together, we confirmed the model that H3K4me2 rather than DNA hypomethylation mediates Jun to regulate lncCPSET1 transcription, which broadens the study of lncCPSET1 pre-transcriptional mechanism.
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http://dx.doi.org/10.3390/ani11061572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8227976PMC
May 2021

Characterization of Alternative Splicing (AS) Events during Chicken () Male Germ-Line Stem Cell Differentiation with Single-Cell RNA-seq.

Animals (Basel) 2021 May 20;11(5). Epub 2021 May 20.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.

Alternative splicing (AS) is a ubiquitous, co-transcriptional, and post-transcriptional regulation mechanism during certain developmental processes, such as germ cell differentiation. A thorough understanding of germ cell differentiation will help us to open new avenues for avian reproduction, stem cell biology, and advances in medicines for human consumption. Here, based on single-cell RNA-seq, we characterized genome-wide AS events in manifold chicken male germ cells: embryonic stem cells (ESCs), gonad primordial germ cells (gPGCs), and spermatogonia stem cells (SSCs). A total of 38,494 AS events from 15,338 genes were detected in ESCs, with a total of 48,955 events from 14,783 genes and 49,900 events from 15,089 genes observed in gPGCs and SSCs, respectively. Moreover, this distribution of AS events suggests the diverse splicing feature of ESCs, gPGCs, and SSCs. Finally, several crucial stage-specific genes, such as NANOG, POU5F3, LIN28B, BMP4, STRA8, and LHX9, were identified in AS events that were transmitted in ESCs, gPGCs, and SSCs. The gene expression results of the RNA-seq data were validated by qRT-PCR. In summary, we provided a comprehensive atlas of the genome-wide scale of the AS event landscape in male chicken germ-line cells and presented its distribution for the first time. This research may someday improve treatment options for men suffering from male infertility.
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http://dx.doi.org/10.3390/ani11051469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160964PMC
May 2021

Production of viable chicken by allogeneic transplantation of primordial germ cells induced from somatic cells.

Nat Commun 2021 05 20;12(1):2989. Epub 2021 May 20.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.

The allogeneic transplantation of primordial germ cells (PGCs) derived from somatic cells overcomes the limitation of avian cloning. Here, we transdifferentiate chicken embryo fibroblasts (CEFs) from black feathered Langshan chickens to PGCs and transplant them into White Plymouth Rock chicken embryos to produce viable offspring with characteristics inherited from the donor. We express Oct4/Sox2/Nanog/Lin28A (OSNL) to reprogram CEFs to induced pluripotent stem cells (iPSCs), which are further induced to differentiate into PGCs by BMP4/BMP8b/EGF. DNA demethylation, histone acetylation and glycolytic activation elevate the iPSC induction efficiency, while histone acetylation and glycolytic inhibition facilitate PGCs formation. The induced PGCs (iPGCs) are transplanted into the recipients, which are self-crossed to produce 189/509 somatic cells derived chicken with the donor's characteristics. Microsatellite analysis and genome sequencing confirm the inheritance of genetic information from the donor. Thus, we demonstrate the feasibility of avian cloning from somatic cells.
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http://dx.doi.org/10.1038/s41467-021-23242-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138025PMC
May 2021

Spin1z induces the male pathway in the chicken by down-regulating Tcf4.

Gene 2021 May 22;780:145521. Epub 2021 Feb 22.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China. Electronic address:

SPINDLIN1-Z (SPIN1Z), a member of the Spin/Ssty(Y-linked spermiogenesis specific transcript) protein family, participates in the early embryonic development process. Our previous RNA-seq analysis indicates that the level of Spin1z was abundantly expressed in male embryonic stem cells (ESCs) and primitive germ cells (PGCs), we speculate that Spin1z may play an important role in chicken male differentiation. Therefore, the loss- and gain-of-function experiments provide solid evidence that Spin1z is both necessary and sufficient to initiate male development in chicken. Furthermore, chromatin immunoprecipitation (ChIP) assay and the dual-luciferase assay was performed to further confirm that Spin1z contributed to chicken male differentiation by inhibiting the Tcf4 transcription. Our findings provide a novel insight into the molecular mechanism for chicken male differentiation.
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http://dx.doi.org/10.1016/j.gene.2021.145521DOI Listing
May 2021

Glycolysis Combined with Core Pluripotency Factors to Promote the Formation of Chicken Induced Pluripotent Stem Cells.

Animals (Basel) 2021 Feb 6;11(2). Epub 2021 Feb 6.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) in vitro. Previously, a lentivirus induction strategy of introducing Oct4, Sox2, Nanog and Lin28 (OSNL) into the iPSC process has been shown as a possible way to produce chicken iPSCs from chicken embryonic fibroblasts, but the induction efficiency of this method was found to be significantly limiting. In order to help resolve this efficiency obstacle, this study seeks to clarify the associated regulation mechanisms and optimizes the reprogramming strategy of chicken iPSCs. This study showed that glycolysis and the expression of glycolysis-related genes correlate with a more efficient reprogramming process. At the same time, the transcription factors Oct4, Sox2 and Nanog were found to activate the expression of glycolysis-related genes. In addition, we introduced two small-molecule inhibitors (2i-SP) as a "glycolysis activator" together with the OSNL cocktail, and found that this significantly improved the induction efficiency of the iPSC process. As such, the study identifies direct molecular connections between core pluripotency factors and glycolysis during the chicken iPSC induction process and, with its results, provides a theoretical basis and technical support for chicken somatic reprogramming.
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http://dx.doi.org/10.3390/ani11020425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915628PMC
February 2021

promotes chicken female differentiation by inhibiting .

Cytotechnology 2021 Feb 2;73(1):101-113. Epub 2021 Jan 2.

College of Animal Science and Technology, Jiangsu Province Key Laboratory of Animal Breeding and Molecular Design, Yangzhou University, 88 South University Ave, Yangzhou, 225009 Jiangsu China.

The sex determination and control of poultry is a key problem in production and scientific research despite few studies on regulatory factors, especially transcription factors in sex determination. In the early stage of this study, high-throughput sequencing was used to screen the differentially expressed gene in male and female embryonic stem cells (ESCs) and primordial germ cells (PGCs). The qRT-PCR discovered that the JUN gene significantly increased from embryonic days (E) 2.5 later in chicken embryo development, and the female gonad expression was much higher than that of the male after E14.5. Lentivirus shRNA, shRNA- interference, and OE- overexpression vectors were successfully constructed. After interfering with in vivo, male characteristics appeared in ZW embryonic gonads at E18.5. Meanwhile, the male-specific genes and were upregulated, the female-specific genes and were downregulated, and the estradiol in the gonads was significantly decreased. The situation was reversed after the overexpression of , ZZ chicken embryo developed into female sexual characteristics. The double luciferase report has found that the promoter activity was significantly upregulated after interference with , and significantly increased after the deletion of the binding site. After the injection of the -shRNA vector into the blood vessel in vivo, it was discovered that and of ZW embryos at E18.5 were downregulated, and were significantly upregulated, and the gonads show femininity. In conclusion, this study proves that is a key regulator in the process of chicken female sex differentiation, which can inhibit the transcription of and promote the synthesis of estradiol, and participate in the process of chicken sex differentiation. This study lays a foundation for the analysis of the molecular mechanism of chicken sex determination and the development of poultry sex control technology.
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http://dx.doi.org/10.1007/s10616-020-00447-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817717PMC
February 2021

Long Noncoding RNA Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens.

Animals (Basel) 2021 Jan 24;11(2). Epub 2021 Jan 24.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu, China.

Although lncRNAs have been identified as playing critical roles in the development of germ cells, their potential involvement in the development of PGCs in chickens remains poorly understood. Differentially expressed lncRNAs (DELs) from previous RNA-seq of embryonic stem cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) were analyzed by K-means clustering, from which a key candidate, lncRNA (lncRNA PGC regulator, ) was obtained. We confirmed that plays a positive role in the development of PGCs by increasing the expression of the PGC marker gene ( and ), while downregulating the pluripotency-associated gene () in vitro and in vivo. The activation and expression of are regulated by histone acetylation, and transcription factor TCF7L2. Mechanistically, a rescue assay was performed to further confirm that contributed to the development of PGCs by regulating the gga-miR-6577-5p/ signaling pathway. Adsorption of gga-miR-6577-5p activated the WNT signaling cascade by relieving the gga-miR-6577-5p-dependent inhibition of expression. Taken together, our study discovered the growth-expedited role of in PGCs development, showing the potential /miR-6577-5p/ pathway. The results and findings provide a novel insight into the development of PGCs.
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http://dx.doi.org/10.3390/ani11020292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912682PMC
January 2021

BMP4 activates the Wnt---Wnt pathway to promote primordial germ cell formation via altering H3K4me2.

J Cell Sci 2021 02 1;134(3). Epub 2021 Feb 1.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China

The unique developmental characteristics of chicken primordial germ cells (PGCs) enable them to be used in recovery of endangered bird species, gene editing and the generation of transgenic birds, but the limited number of PGCs greatly limits their application. Studies have shown that the formation of mammalian PGCs is induced by BMP4 signal, but the mechanism underlying chicken PGC formation has not been determined. Here, we confirmed that Wnt signaling activated via BMP4 activates transcription of by inducing β-catenin to compete with LSD1 for binding to TCF7L2, causing LSD1 to dissociate from the promoter and enhancing H3K4me2 methylation in this region. promotes PGC formation by inhibiting maturation to initiate expression. Interestingly, expression of helped sustain expression by preventing LSD1 binding to the promoter. We thus elucidated a positive feedback pathway involving Wnt---Wnt that ensures PGC formation. In summary, our data provide new insight into the development of PGCs in chickens
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http://dx.doi.org/10.1242/jcs.249375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875490PMC
February 2021

Study on the Function and Mechanism of Lin28B in the Formation of Chicken Primordial Germ Cells.

Animals (Basel) 2020 Dec 28;11(1). Epub 2020 Dec 28.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.

and are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of in the formation of primordial germ cells (PGCs) are very clear, but the related research on is rarely reported. Here, we found that the overexpression of can promote the formation of PGC in vivo. Furthermore, the overexpression of also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a significant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited expression showed completely opposite results, which were confirmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of can inhibit the maturation of , and the results of high-throughput sequencing indicated that was a negative regulator of the formation process of PGCs. Therefore, we conclude that our results determine that participates in the formation of PGCs through , which set a theoretical foundation for improving the function and mechanism of family in the formation of PGCs.
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http://dx.doi.org/10.3390/ani11010043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823903PMC
December 2020

Transcriptome Sequencing and Comparative Analysis of Amphoteric ESCs and PGCs in Chicken ().

Animals (Basel) 2020 Nov 27;10(12). Epub 2020 Nov 27.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.

Chicken () pluripotent embryonic stem cells (ESCs) and primordial germ cells (PGCs) can be broadly applied in the research of developmental and embryonic biology, but the difference between amphoteric ESCs and PGCs is still elusive. This study determined the sex of collected samples by identifying specific sex markers via polymerase chain reaction (PCR) and fluorescence activated cell sorter (FACS). RNA-seq was utilized to investigate the transcriptomic profile of amphoteric ESCs and PGCs in chicken. The results showed no significant differentially expressed genes (DEGs) in amphoteric ESCs and 227 DEGs exhibited in amphoteric PGCs. Moreover, those 227 DEGs were mainly enriched in 17 gene ontology (GO) terms and 27 pathways according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Furthermore, qRT-PCR was performed to verify RNA-seq results, and the results demonstrated that Notch1 was highly expressed in male PGCs. In summary, our results provided a knowledge base of chicken amphoteric ESCs and PGCs, which is helpful for future research in relevant biological processes.
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http://dx.doi.org/10.3390/ani10122228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760303PMC
November 2020

Analysis of lncRNA Expression Profile during the Formation of Male Germ Cells in Chickens.

Animals (Basel) 2020 Oct 11;10(10). Epub 2020 Oct 11.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.

Germ cells have an irreplaceable role in transmitting genetic information from one generation to the next, and also play an important role in sex differentiation in poultry, while little is known about epigenetic factors that regulate germ cell differentiation. In this study, RNA-seq was used to detect the expression profiles of long non-coding RNAs (lncRNAs) during the differentiation of chicken embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs). The results showed that a total of 296, 280 and 357 differentially expressed lncRNAs (DELs) were screened in ESCs vs. PGCs, ESCs vs. SSCs and PGCs vs. SSCs, respectively. Gene Ontology (GO) and KEGG enrichment analysis showed that DELs in the three cell groups were mainly enriched in autophagy, Wnt/β-catenin, TGF-β, Notch and ErbB and signaling pathways. The co-expression network of 37 candidate DELs and their target genes enriched in the biological function of germ cell development showed that XLOC_612026, XLOC_612029, XLOC_240662, XLOC_362463, XLOC_023952, XLOC_674549, XLOC_160716, ALDBGALG0000001810, ALDBGALG0000002986, XLOC_657380674549, XLOC_022100 and XLOC_657380 were the key lncRNAs in the process of male germ cell formation and, moreover, the function of these DELs may be related to the interaction of their target genes. Our findings preliminarily excavated the key lncRNAs and signaling pathways in the process of male chicken germ cell formation, which could be helpful to construct the gene regulatory network of germ cell development, and also provide new ideas for further optimizing the induction efficiency of germ cells in vitro.
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http://dx.doi.org/10.3390/ani10101850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599500PMC
October 2020

CYP19A1 (aromatase) dominates female gonadal differentiation in chicken (Gallus gallus) embryos sexual differentiation.

Biosci Rep 2020 10;40(10)

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China.

Cytochrome P450 Family 19 SubFamily A member 1 (CYP19A1) gene encodes an aromatase which regulates the sexual differentiation in vertebrates by initiating and maintaining 17β-Estradiol (E2) synthesis. Here, we described the spatiotemporal expression pattern of CYP19A1 and its functional role in the embryonic gonad development in amphoteric chickens (Gallus gallus). Results showed that CYP19A1 exhibited a sexually dimorphic expression pattern in female gonads early at embryonic day 5.5 (HH 28) and robustly expressed within the cytoplasm in ovarian medullas. Most importantly, we induced the gonadal sex reversal by ectopically delivering the aromatase inhibitor (AI) or estradiol (E2) into chicken embryos. To further explore the role of CYP19A1 in chicken embryonic sexual differentiation, we successfully developed an effective method to deliver lentiviral particles with CYP19A1 manipulation into chicken embryos via embryonic intravascular injection. The analysis of interference and overexpression of CYP19A1 provided solid evidences that CYP19A1 is both necessary and sufficient to initiate sex differentiation toward female in chicken embryos. Collectively, this work demonstrates that CYP19A1 is a crucial sex differentiation gene in the embryonic development, which provides a foundation for understanding the mechanism of sex determination and differentiation in chickens.
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http://dx.doi.org/10.1042/BSR20201576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560524PMC
October 2020

5-Azacytidine-Induced Cardiomyocyte Differentiation of Very Small Embryonic-Like Stem Cells.

Stem Cells Int 2020 8;2020:5162350. Epub 2020 Sep 8.

Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou 225001, China.

The use of stem cells in generating cell-based pacemaker therapies for bradyarrhythmia is currently being considered. Due to the propensity of stem cells to form tumors, as well as ethical issues surrounding their use, the seed cells used in cardiac biological pacemakers have limitations. Very small embryonic-like stem cells (VSELs) are a unique and rare adult stem cell population, which have the same structural, genetic, biochemical, and functional characteristics as embryonic stem cells without the ethical controversy. In this study, we investigated the ability of rat bone marrow- (BM-) derived VSELs to differentiate into cardiomyocytes by 5-Azacytidine (5-AzaC) treatment. The morphology of VSELs treated with 10 M 5-AzaC increased in volume and gradually changed to cardiomyocyte-like morphology without massive cell death. Additionally, mRNA expression of the cardiomyocyte markers cardiac troponin-T (cTnT) and -sarcomeric actin (-actin) was significantly upregulated after 5-AzaC treatment. Conversely, stem cell markers such as Nanog, Oct-4, and Sox2 were continuously downregulated posttreatment. On day 14 post-5-AzaC treatment, the positive expression rates of cTnT and -actin were 18.41 ± 1.51% and 19.43 ± 0.51%, respectively. Taken together, our results showed that rat BM-VSELs have the ability to differentiate into cardiomyocytes . These findings suggest that VSELs would be useful as seed cells in exploring the mechanism of biological pacemaker activity.
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http://dx.doi.org/10.1155/2020/5162350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495233PMC
September 2020

Comparison of the effects of three cryoprotectants on the cryopreservation of mouse subcutaneous tissue under different conditions.

Exp Ther Med 2020 Oct 29;20(4):3285-3289. Epub 2020 Jul 29.

College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, P.R. China.

The subcutaneous tissue of animals contains different cell types, and different cells have different requirements for cryopreservation. This establishes obstacles that need to be overcome in the clinical application of tissue preservation. In the present study, the effects of different freezing rates and various concentrations of cryoprotectants on the cryopreservation of subcutaneous tissue of mice were compared, and these results provided basic research data that can be used to explore the optimal cryopreservation method for tissue. The effects of three cryoprotectants, dimethyl sulfoxide, glycerinum and 1,2-propanediol, and their concentrations on the cryopreservation of subcutaneous tissue of mice were compared with slow and rapid freezing rates. The results revealed that under various cryopreservation conditions, the percentage of fibroblasts that grow from the tissue following slow cryopreservation (19.8%) was significantly higher than that following rapid freezing (6.7%) at osmotic equilibrium for 10-20 min (P<0.05). After 19 days of culture, under the conditions of slow freezing, with 10, 20 and 30% glycerinum as a cryoprotectant, respectively, fibroblasts grew from 26.0, 16.7 and 16.7% of the tissues, respectively. No fibroblasts were indicated in the tissue mass cultured in any other tissue blocks treated with cryopreservation solutions. Under the condition of rapid freezing, fibroblasts grew from 6.7 and 6.7% tissue blocks of 20% DMSO and 10% glycerinum, respectively, following 19 days of culture. No fibroblasts were identified in the tissue mass cultured in the other tissue blocks treated with cryopreservation solutions, and no fibroblasts were identified in the tissue blocks without osmotic balance before freezing.
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http://dx.doi.org/10.3892/etm.2020.9076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444325PMC
October 2020

Regulatory functions of gga-miR-218 in spermatogonial stem cells meiosis by targeting Stra8.

Mech Dev 2020 12 13;164:103636. Epub 2020 Aug 13.

College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu province, PR China; Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou 225009, Jiangsu province, PR China. Electronic address:

MicroRNAs play a crucial role in sperm formation, but its specific function remains unknown. Here, we found that gga-miR-218 regulates chicken sperm formation through in/ex vivo experiments. We constructed over-expression/interference carrier to overexpress and inhibit gga-miR-218 in chicken spermatogonial stem cells, separately, the detection of haploid and QRT-PCR of meiosis related genes revealed that gga-miR-218 inhibits meiosis. After injection of miR-218 in vivo, semen concentration and HE (Hematoxylin and Eosin staining) revealed that gga-miR-218 inhibits meiosis. Meanwhile, we discovered that gga-miR-218 could target Stra8 by prediction software which can inhibit the wild-type fluorescence activity by co-transfection of gga-miR-218 with the Stra8 3' untranslated regions fluorescent reporter vector (wild-type/mutant), QRT-PCR and Western blot showed that gga-miR-218 inhibits the expression level of Stra8 by targeting its 3' untranslated regions directly. Finally, we suggest that gga-miR-218 could target to srta8 directly and inhibit spermatogenesis.
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http://dx.doi.org/10.1016/j.mod.2020.103636DOI Listing
December 2020

Narrow H3K4me2 is required for chicken PGC formation.

J Cell Physiol 2021 Feb 4;236(2):1391-1400. Epub 2020 Aug 4.

Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC-related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor-β). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.
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http://dx.doi.org/10.1002/jcp.29945DOI Listing
February 2021

P53 and H3K4me2 activate N6-methylated LncPGCAT-1 to regulate primordial germ cell formation via MAPK signaling.

J Cell Physiol 2020 12 27;235(12):9895-9909. Epub 2020 May 27.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, China.

Long noncoding RNAs (lncRNAs) participate in the formation of primordial germ cells (PGCs); however, the identity of the key lncRNAs and the molecular mechanisms responsible for the formation of PGCs remain unknown. Here, we identify a key candidate lncRNA (lncRNA PGC transcript-1, LncPGCAT-1) via RNA sequencing of embryonic stem cells, PGCs, and Spermatogonial stem cells (SSCs). Functional experiments confirmed that LncPGCAT-1 positively regulated the formation of PGCs by elevating the expression of Cvh and C-kit while downregulating the pluripotency(Nanog) in vitro and in vivo; PAS staining of genital ridges in vivo also showed that interference with LncPGCAT-1 can significantly reduce the number of PGCs in genital ridges, while overexpression of LncPGCAT-1 had the opposite result. The result of luciferase reporter assay combined with CHIP-qPCR showed that the expression of LncPGCAT-1 was promoted by the transcription factor P53 and high levels of H3K4me2. Mechanistically, the luciferase reporter assay confirmed that mitogen-activated protein kinase 1 (MAPK1) was the target gene of LncPGCAT-1 and gga-mir-1591. In the ceRNA system, high levels of N methylation of LncPGCAT-1 enhanced the adsorption capacity of LncPGCAT-1 for gga-mir-1591. Adsorption of gga-mir-1591 activated the MAPK1/ERK signaling cascade by relieving the gga-mir-1591-dependent inhibition of MAPK1 expression. Moreover, LncPGCAT-1 interacted with interleukin enhancer binding factor 3 (ILF3) to regulate the ubiquitination of P53 and phosphorylation of JNK. Interaction with ILF3 resulted in positive self-feedback regulation of LncPGCAT-1 and activation of JNK signaling, ultimately promoting PGC formation. Altogether, the study expands our knowledge of the function and molecular mechanisms of lncRNAs in PGC development.
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http://dx.doi.org/10.1002/jcp.29805DOI Listing
December 2020

Distinct roles of retinoic acid and BMP4 pathways in the formation of chicken primordial germ cells and spermatogonial stem cells.

Food Funct 2019 Nov 9;10(11):7152-7163. Epub 2019 Oct 9.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China. and Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu 225009, China.

This study demonstrated different effects of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) signaling on the induction of germ cell formation in chickens. In vitro, BMP4 significantly promoted primordial germ cell (PGC) formation, while RA promoted spermatogonial stem cell (SSC) formation. Hematoxylin-Eosin (HE) staining of reproductive ridge and testicular slices showed that BMP4 signaling was activated during PGC formation but was inhibited during PGC differentiation into SSC. In contrast, RA signaling was significantly activated during PGC differentiation to SSC. Mechanistically, elevated expression of phosphorylated mothers against decapentaplegic homolog 5 (p-Smad5) activated BMP4 signaling, while inhibition of p-Smad5 significantly reduced the PGC formation. Additionally, BMP4 regulated the PGC formation through histone acetylation and DNA methylation in deleted in azoospermia-like (DAZL) gene. Luciferase report showed RA binding to RARα regulated stimulated by RA 8 (Stra8) promoter activity during SSC formation, while mutations in RAR binding sites inhibited the Stra8 expression and SSC formation. Further, both HAT and HDAC regulated the RARα isoform, and HAT binding to RARα activated the Stra8 transcription. RNA-seq of embryonic stem cells (ESC), PGC, and SSC showed inverse expression of genes related to the BMP4 and RA pathways during PGC and SSC formation. Additionally, Smad5 and Smurf were critical for the interactions between the two pathways. Specifically, through Smurf promotion of Smad5 ubiquitination, RA could inhibit the BMP4 signal transduction. In conclusion, the BMP4 and RA signaling pathways play opposing roles in germ cell formation, driven by epigenetic processes such as phosphorylation, ubiquitination, and histone acetylation.
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http://dx.doi.org/10.1039/c9fo01485cDOI Listing
November 2019

Comparison between curcumin and all-trans retinoic acid in the osteogenic differentiation of mouse bone marrow mesenchymal stem cells.

Exp Ther Med 2019 May 20;17(5):4154-4166. Epub 2019 Mar 20.

Key Laboratory of Animal Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, P.R. China.

The use of bone marrow mesenchymal stem cells (BMSCs) has great potential in cell therapy, particularly in the orthopedic field. BMSCs represent a valuable renewable cell source that have been successfully utilized to treat damaged skeletal tissue and bone defects. BMSCs can be induced to differentiate into osteogenic lineages via the addition of inducers to the growth medium. The present study examined the effects of all-trans retinoic acid (ATRA) and curcumin on the osteogenic differentiation of mouse BMSCs. Morphological changes, the expression levels of the bone-associated gene markers bone morphogenetic protein 2, runt-related transcription factor and osterix during differentiation, an mineralization assay, and changes in osteocalcin expression revealed that curcumin supplementation promoted the osteogenic differentiation of BMSCs. By contrast, the application of ATRA increased osteogenic differentiation during the early stages, but during the later stages, it decreased the mineralization of differentiated cells. In addition, to the best of our knowledge, the present study is the first to examine the effect of curcumin on the osteogenic potency of mouse embryonic fibroblasts (MEFs) after reprogramming with human lim mineralization protein (hLMP-3), which is a positive osteogenic regulator. The results revealed that curcumin-supplemented culture medium increased hLMP-3 osteogenic potency compared with that of MEFs cultured in the non-supplemented medium. The present results demonstrate that enrichment of the osteogenic culture medium with curcumin, a natural osteogenic inducer, increased the osteogenic differentiation capacity of BMSCs as well as that of MEFs reprogrammed with hLMP-3.
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http://dx.doi.org/10.3892/etm.2019.7414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447915PMC
May 2019

The Lbc gene promotes differentiation of chicken embryo stem cell into spermatogonial stem cells via the regulation of transcriptional factor Hoxa5.

J Cell Biochem 2019 Jan 17. Epub 2019 Jan 17.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.

Recently, the surface marker genes of spermatogonial stem cells (SSCs) were increasingly excavated and verified. However, few studies focused on the key genes involved in the regulation of SSCs differentiation. Our laboratory has screened the Lbc gene (GenBank accession number: XM_429585.3), which is specifically expressed on the SSCs. The aim of this study is to investigate the function of Lbc and its regulatory mechanism for SSCs. The indirect immunofluorescence assay (IFA) showed that Lbc was located in both nucleus and cytoplasm. Lbc was also overexpressed and knocked out both in vitro and in vivo to verify its function in SSCs, respectively. As a result, the overexpressed Lbc could promote the formation of spermatogonial stem cells like cells (SSCs-like), while the deficiency of Lbc blocked the formation of SSCs-like. We also identified the core region of Lbc promoter that located into the upstream of the transcription initiation site -247 to -2bp. Moreover, the activity of Lbc promoter could be increased by histone acetylation which is leading to the higher expression of Lbc. When we mutated the transcription factor HOXA5 and SOX10 that bound to the core region of Lbc promoter, HOXA5 could reduce the transcription activity of Lbc whereas the SOX10 was not. Currently, we found Lbc is a new specific marker of SSCs. This gene can be modified by histone acetylated and promote the formation of chicken SSCs via the transcription factor HOXA5. The present research will lay the foundation for further study on the regulatory mechanism of SSCs.
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http://dx.doi.org/10.1002/jcb.27760DOI Listing
January 2019

Acetyl-coenzyme A acyltransferase 2 promote the differentiation of sheep precursor adipocytes into adipocytes.

J Cell Biochem 2018 Nov 28. Epub 2018 Nov 28.

College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, China.

The acetyl CoA acyltransferase 2 (ACAA2) is a key enzyme of the fatty acid oxidation pathway, catalyzing the last step of the mitochondrial beta oxidation, thus playing an important role in the fatty acid metabolism. The purpose of this study was to investigate the effect of knocking out ACAA2 on the expression of genes lipoprteinlipase (LPL), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase, fat mass and obesity-associated gene, adipocyte fatty acid-binding protein (AP2) in precursor adipocytes and their differentiation into adipocytes. The knockout vector was constructed using CRISPR-Cas RNA-guided nuclease technology with an efficiency of 23.80%, and the vector was transfected into precursor adipocyte cells, while an overexpression vector of the ACAA2 gene was also transfected in another group of preadipocytes. Quantitative polymerase chain reaction showed that the expression of the PPAR-γ, LPL, and AP2 was significantly lower in the knockout compared with the overexpression group, while there was no difference in cell growth. After induction of adipocyte precursor cells into adipocytes using dexamethasone, insulin, and IBMX, oil red staining showed a significantly different number of lipid droplets in the knockout group. These results provide a preliminary indication for a possible involvement of the ACAA2 gene in adipocyte differentiation in vitro.
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http://dx.doi.org/10.1002/jcb.28080DOI Listing
November 2018

Direct conversion of mouse embryonic fibroblast to osteoblast cells using hLMP-3 with Yamanaka factors.

Int J Biochem Cell Biol 2019 01 16;106:84-95. Epub 2018 Nov 16.

Key Laboratory of Animal Breeding, Reproduction and Molecular Design for Jiangsu Provience, College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China. Electronic address:

Large bone defects and bone loss after fractures remain significant challenges for orthopedic surgeons. Our study aims to find an available, applicable and biological treatment for bone regeneration overcoming the limitations in ESC/iPSC technology. We directly reprogrammed the mouse embryonic fibroblast (MEF) into osteoblast cells using different combinations of Yamanaka factors with human lim mineralization protein-3 (hLMP-3). LMP is an intracellular LIM-domain protein acting as an effective positive regulator of the osteoblast differentiation. After transduction, cells were cultured in osteogenic medium, and then examined for osteoblast formation. The expression of osteogenic markers (BMP2, Runx2 and Osterix) during reprogramming and in vitro mineralization assay revealed that the best reprogramming cocktail was (c-Myc - Oct4) with hLMP-3. In addition, both immunofluorescent staining and western blot analysis confirmed that osteocalcin (OCN) expression increased in the cells treated with the c-Myc/Oct4/hLMP3 cocktail than using hLMP-3 alone. Furthermore, this reprogramming cocktail showed efficient healing in an induced femoral bone defect in rat animal model one month after transplantation. In the present study, we reported for the first time the effect of combining Yamanaka factors with hLMP-3 to induce osteoblast cells from MEF both in vitro and in vivo.
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http://dx.doi.org/10.1016/j.biocel.2018.11.008DOI Listing
January 2019

Study on immortal conditions of chicken embryonic stem cells.

J Cell Biochem 2018 Nov 16. Epub 2018 Nov 16.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.

In recent years, considerable attention has been paid to chicken embryonic stem cells (ESCs) studies in relation to extensive applications in gene therapy and regenerative medicine. However, the approaches used are still immature. In this study, we showed that the chicken ESCs clones with a clear border can express alkaline phosphatase and marker proteins such as SSEA-1, SOX2, and OCT4 stably. In addition, culture medium containing 10 μmol/L of vitamin C (VC) could significantly promote the proliferation of ESCs cells. Moreover, ESCs transfected with p:enhanced green fluorescent protein (pEGFP)-hTERT could be subcultured more than tenth generations in culture medium containing exogenous factors (mLIF + bFGF + hSCF) and VC, and these ESCs clone could still be regenerated following cryopreservation. Quantitative real-time polymerase chain reaction results showed that there was no significant difference between SSEA-1, SOX2, and OCT4 expression during ESCs immortalization and that the tenth generation of ESCs was still able to express marker proteins SSEA-1, SOX2, and OCT4. Our results showed that an immobilized system for ESCs was established, and the ESCs were cultured in vitro maintaining their pluripotency.
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http://dx.doi.org/10.1002/jcb.27173DOI Listing
November 2018

Functional characterization of the Sox2, c-Myc, and Oct4 promoters.

J Cell Biochem 2019 01 11;120(1):332-342. Epub 2018 Sep 11.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.

To better understand the mechanisms in transcriptional regulation, we analyzed the promoters of the reprogramming key genes Sox2, c-Myc, and Oct4. Here, we cloned different 5' deletions of the goat Sox2, c-Myc, and Oct4 promoters, and evaluated their functions by green fluorescent protein reporter system and dual-luciferase reporter system. Site-directed mugagenesis and epigenetic modifiers were used to explore the influence of transcription binding sites and epigenetic status on the promoters. The results suggested that the basal promoters were located in the - 109 to 49, - 147 to 1, and - 96 to 30 bp regions of the Sox2, c-Myc, and Oct4 promoters. The transcription factors that identified to influence the Sox2, c-Myc, and Oct4 promoter activities were Elf-1 and activating protein 2 (AP-2), C/EBP and Sp1, and Mzf1 and Sp1, respectively. The epigenetic alternation of the Sox2, c-Myc, and Oct4 promoters by 5-aza-2'-deoxycytidine or/and trichostatin A significantly increased the promoter activities. In conclusion, the result determined the core promoter areas of the Sox2, c-Myc, and Oct4 genes, and identified the transcription factors that influence their promoter activities. We also verified that the Sox2, c-Myc, and Oct4 promoters were hypermethylated and hypoacetylated.
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http://dx.doi.org/10.1002/jcb.27374DOI Listing
January 2019

CRISPR/Cas9-mediated sheep MSTN gene knockout and promote sSMSCs differentiation.

J Cell Biochem 2018 Sep 22. Epub 2018 Sep 22.

Institutes of Agricultural Science and Technology Development, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.

Myostatin (MSTN) is an important gene involved in the regulation of embryonic muscle cells and adult muscle development; it has a good application prospect in transgenic animal production by improving the yield of muscle. The purpose of this study is to construct MSTN gene knockout vector using clustered regularly interspaced short palindromic repeats ( CRISPR)/CRISPR-associated protein 9 ( Cas9). The knockout efficiency was evaluated in sheep ear fibroblasts (SEFs) by cleavage activity of transcription of guide RNA ( gRNA), luciferase-single-strand annealing assay, T7 endonuclease I assay (T7E1), and TA clone sequence (10/38); and above all, detection showed that the cleavage activity of CRISPR/Cas9-mediated MSTN reached 29%. MSTN-Cas9/gRNA4 was transfected into sheep skeletal muscle satellite cell (sSMSC) to confirm the function of MSTN in myotomes formation induced by starvation in low-serum medium. The results showed that myotubes formation efficiency were 11.2 ± 1.3% and 19.5 ± 2.1% in the control group and knockout group, respectively. The average length of myotomes was 22 ± 5.3 and 47 ± 3.6 μm, displaying that MSTN knockout can promote sSMSC differentiation in number and length. The unlabeled MSTN-Cas9/gRNA4 was transfected into SEFs and monoclonal positive cells was obtained after 48 hours transfection. The MSTN-positive cells were used as donor cells to perform somatic cell nuclear transplantation to produce transgenic sheep. A total of 20 embryos were transplanted into surrogate mothers, four of them normally produce offspring. The genomic DNA of surviving lambs were used as a template, three positive individuals were identified by T7E1 digestion. All the results demonstrated that the CRISPR/Cas9 system has the potential to become an important and applicable gene engineering tool in animal breeding.
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http://dx.doi.org/10.1002/jcb.27474DOI Listing
September 2018

The establishment of clonally derived chicken embryonic fibroblast cell line (CSC) with high transfection efficiency and ability as a feeder cell.

J Cell Biochem 2018 11 4;119(11):8841-8850. Epub 2018 Aug 4.

Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.

This study established a single cloned chicken embryonic fibroblast (CEF) cell line. It solves the main problem of the instability of a cultured primary cell and its impact on the experiment. In this study, CEF pass through this crisis and formed a continuous cell line after subculture. We isolated single postcrisis CEF by a mouth pipette under a convert microscope then established a single cloned cell line named CSC-1-5 which passaged continuously from 96-well plates to 60 mm culture plates. CSC has a normal chicken diploid karyotype, no tumorigenicity, and a high G2/M phase cell ratio. We found that Fugene could mediate the transfection of CSCs efficiently; it was significantly improved compared with the primary cells. It could also promote the proliferation of chicken embryonic stem cell as a feeder layer.
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http://dx.doi.org/10.1002/jcb.27137DOI Listing
November 2018
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