lncCPSET1 acts as a scaffold for MLL2/COMPASS to regulate Bmp4 and promote the formation of chicken primordial germ cells.

Primordial germ cells (PGCs) are the ancestors of female and male germ cells. Recent studies have shown that long non-coding RNA (lncRNA) and histone methylation are key epigenetic factors affecting PGC formation; however, their joint regulatory mechanisms have rarely been studied. Here, we explored the mechanism by which lncCPSET1 and H3K4me2 synergistically regulate the formation of chicken PGCs for the first time. Combined with chromatin immunoprecipitation (CHIP) sequencing and RNA-seq of PGCs transfected with the lncCPSET1 overexpression vector, GO annotation and KEGG enrichment analysis revealed that Wnt and TGF-ß signaling pathways were significantly enriched, and Fzd2, Id1, Id4, and Bmp4 were identified as candidate genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that ASH2L, DPY30, WDR5, and RBBP5 overexpression significantly increased the expression of Bmp4, which was up-regulated after lncCPSET1 overexpression as well. It indicated that Bmp4 is a target gene co-regulated by lncCPSET1 and MLL2/COMPASS. Interestingly, co-immunoprecipitation results showed that ASH2L, DPY30 and WDR5 combined and RBBP5 weakly combined with DPY30 and WDR5. lncCPSET1 overexpression significantly increased Dpy30 expression and co-immunoprecipitation showed that interference/overexpression of lncCPSET1 did not affect the binding between the proteins in the complexes, but interference with lncCPSET1 inhibited DPY30 expression, which was confirmed by RNA immunoprecipitation that lncCPSET1 binds to DPY30. Additionally, CHIP-qPCR results showed that DPY30 enriched in the Bmp4 promoter region promoted its transcription, thus promoting the formation of PGCs. This study demonstrated that lncCPSET1 and H3K4me2 synergistically promote PGC formation, providing a reference for the study of the regulatory mechanisms between lncRNA and histone methylation, as well as a molecular basis for elucidating the formation mechanism of PGCs in chickens.

Dual regulatory actions of LncBMP4 on BMP4 promote chicken primordial germ cell formation.

The unique characteristics of chicken primordial germ cells (PGCs) provide potential strategies for transgenic animal generation; however, insufficient PGC availability has limited their application. Regulation of bone morphogenic protein 4 (BMP4), a crucial factor for PGCs formation, may provide new strategies for PGC generation. We here identify a long noncoding RNA (lncRNA) that targets BMP4 (LncBMP4). LncBMP4 has similar functions as BMP4, in that it facilitates the formation and migration of PGCs. LncBMP4 promotes BMP4 expression by adsorbing the miRNA gga-mir-12211, thus reducing its inhibitory effect on BMP4 expression. In addition, the small peptide EPC5 encoded by LncBMP4 promotes the transcription of BMP4. The competing endogenous RNA (ceRNA) effect of LncBMP4 requires N6-methyladenosine (m6A) modification, in a dose-dependent manner, and high levels of m6A modification hinder EPC5 translation. Understanding the molecular mechanisms through which LncBMP4 promotes BMP4 expression during PGC formation may provide new avenues for efficient PGC generation.

Identification of key events and regulatory networks in the formation process of primordial germ cell based on proteomics.

Currently, studies have analyzed the formation mechanism of primordial germ cell (PGC) at the transcriptional level, but few at the protein level, which made the mechanism study of PGC formation not systematic. Here, we screened differential expression proteins (DEPs) regulated PGC formation by label-free proteomics with a novel sampling strategy of embryonic stem cells and PGC. Analysis of DEPs showed that multiple key events were involved, such as the transition from glycolysis to oxidative phosphorylation, activation of autophagy, low DNA methylation ensured the normal formation of PGC, beyond that, protein ubiquitination also played an important role in PGC formation. Importantly, the progression of such events was attributed to the inconsistency between transcription and translation. Interestingly, MAPK, PPAR, Wnt, and JAK signaling pathways not only interact with each other but also interact with different events to participate in the formation of PGC, which formed the PGC regulatory network. According to the regulatory network, the efficiency of PGC formation in induction system can be significantly improved. In conclusion, our results indicate that chicken PGC formation is a complex process involving multiple events and signals, which provide technical support for the specific application in PGC research.

BMP4 activates the Wnt-Lin28A-Blimp1-Wnt pathway to promote primordial germ cell formation via altering H3K4me2.

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 Lin28A by inducing ß-catenin to compete with LSD1 for binding to TCF7L2, causing LSD1 to dissociate from the Lin28A promoter and enhancing H3K4me2 methylation in this region. Lin28A promotes PGC formation by inhibiting gga-let7a-3p maturation to initiate Blimp1 expression. Interestingly, expression of Blimp1 helped sustain Wnt5A expression by preventing LSD1 binding to the Wnt5A promoter. We thus elucidated a positive feedback pathway involving Wnt-Lin28A-Blimp1-Wnt that ensures PGC formation. In summary, our data provide new insight into the development of PGCs in chickens.

Disrupting methyl-CpG-binding protein 2 expression induces the transformation of induced pluripotent stem cell cardiomyocytes into pacemaker-like cells by insulin gene enhancer binding protein 1.

BACKGROUND: The experiment will explore whether interfering with the expression of methyl-CpG-binding protein 2 (MecP2) can enhance the ability of insulin gene enhancer binding protein 1 (ISL1) to induce iPSC-CMs to differentiate into pacemaker-like cells. METHODS: Differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMs) can be induced via the regulation of the Wnt signaling pathway. Real-time quantitative PCR (qPCR), western blotting, immunofluorescence staining, and patch-clamp technique were used to analyze the ability of ISL1 to induce the transformation of iPSC-CMs into pacemaker-like cells. Calcium spark, patch-clamp technique, and real-time qPCR were used to verify whether disrupting the expression of MeCP2 enhanced this ability of ISL1 to induce the differentiation of iPSC-CMs into pacemaker cells. Transplant pacemaker-like cardiomyocytes into the myocardium of mice to observe whether the pacemaker cells can survive in the tissue for a long time. RESULTS: RT-qPCR and patch-clamp analyses showed that overexpression of ISL1 induced the successful differentiation of iPSC-CMs into pacemaker cells. ISL1-overexpressing pacemaker-like cells possessed typical characteristics of pacemaker morphology, including action potential and If inward current. Chromatin immunoprecipitation results showed that MeCP2 bound to the promoter region of HCN4. Following disruption of MeCP2 expression, the gene expression of sinoatrial node-specific transcription factors, If inward current, and cardiac rhythm changes in iPSC-CMs resembled those of sinoatrial node pacemaker cells. Therefore, ISL1 induced the differentiation of iPSC-CMs into pacemaker-like cells, and knockdown of MeCP2 increased this effect. Frozen section results showed that surviving pacemaker-like cells could still be observed in myocardial tissue after 45 days. CONCLUSIONS: Experiments have found that interfering with the expression of MeCP2 can increase the ability of ISL1 to induce iPSC-CM cells to differentiate into pacemaker-like cells. And the pacemaker-like cells obtained in this experiment can survive in myocardial tissue for a long time.

Jun-mediated lncRNA-IMS promotes the meiosis of chicken spermatogonial stem cells via gga-miR-31-5p/stra8.

Meiosis, a key step in spermatogenesis, is affected by many factors. Current studies have shown that long noncoding RNAs (lncRNAs) are potential factors regulating meiosis, and their regulatory mechanisms have received much attention. However, little research has been done on its regulatory mechanism in the spermatogenesis of roosters. Here, we found that lncRNA involved in meiosis and spermatogenesis (lncRNA-IMS) was involved in the regulation of Stra8 by gga-miR-31-5p and hindered the inhibition of Stra8 by gga-miR-31-5p. The acquisition and loss of function experiments demonstrated that lncRNA-IMS was involved in meiosis and spermatogenesis. In addition, we predicted and determined the core promoter region of lncRNA-IMS. Prediction of transcription factors, deletion/overexpression of binding sites, knockdown/overexpression of Jun, and dual-luciferase reporter analysis confirmed that Jun positively activated transcription of lncRNA-IMS. Our findings further enrich the TF-lncRNA-miRNA-mRNA regulatory network during male meiosis and provide new ideas for studying the molecular mechanism of meiosis and spermatogenesis in chicken spermatogonial stem cells.

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

Mono-Sex culturing is an important methodology for intensive livestock and poultry production. Here, Hintw 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 Hintw in chicken embryos through the intravascular injection. QRT-PCR, ELISA and H&E staining were used to detect the effects of Hintw on gonadal development of chicken embryos. Results showed that Hintw exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads. The qRT-PCR analysis showed that Foxl2, Cyp19a1 in females were upregulated under the overexpression of Hintw, while Sox9 and Dmrt1 were downregulated Hintw. Overexpression of Hintw can promote the development of gonadal cortex, while interference with Hintw show the opposite result. Additionally, we found that overexpression of the Hintw in male chicken embryos could inhibit androgen levels and increase estrogen levels. On the other hand, interfering with Hintw 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.

Analysis of the Promoter Regions of gga-miR-31 and Its Regulation by RA and C-jun in Chicken.

The role of gga-miR-31 in chicken germ cell differentiation and spermatogenesis is of significant importance. The transcriptional properties of gga-miR-31 are crucial in establishing the foundation for the formation of chicken spermatogonia stem cells and spermatogenesis. In this study, a series of recombinant vectors including varying lengths of the gga-miR-31 promoter were predicted and constructed. Through the utilization of the dual luciferase reporting system, the upstream -2180~0 bp region of gga-miR-31 was identified as its promoter region. Furthermore, it was predicted and confirmed that the activity of the gga-miR-31 promoter is increased by retinoic acid (RA). The binding of RA to the gga-miR-31 and Stra8 promoter regions was found to be competitive. Through the deletion of C-jun binding sites and the manipulation of C-jun expression levels, it was determined that C-jun inhibits the activity of the gga-miR-31 promoter. Furthermore, the combined treatment of C-jun and RA demonstrated that the positive regulatory effect of RA on the gga-miR-31 promoter is attenuated in the presence of high levels of C-jun. Overall, this study establishes a foundation for further investigation into the regulatory mechanisms of gga-miR-31 action, and provides a new avenue for inducing chicken embryonic stem cells (ESC) to differentiate into spermatogonial stem cells (SSC), and sperm formation.

DNA hypomethylation activation Wnt/TCF7L2/TDRD1 pathway promotes spermatogonial stem cell formation.

Detailed analysis of the regulatory mechanism of spermatogonia stem cell (SSCs) genesis can provide a novel strategy for the application of SSCs in the fields of transgenic animal production and regenerative medicine. Previous studies in this study showed that WNT signaling can positively regulate the formation of SSCs, but the exact regulatory mechanism is not clear. Here, we predicted the target gene of the Wnt/TCF7L2 pathway, namely TDRD1, by bioinformatics analysis. Functional studies revealed that overexpression of TDRD1 during RA-induced SSCs formation in vitro significantly upregulated the expression of reproductive marker genes (Integrinß1 and Integrinα6), and further flow cytometric analysis also confirmed that the formation efficiency of SSCs was significantly increased after overexpression of TDRD1; while interference with TDRD1 showed the exact opposite result. The in vivo experiments were consistent with the results of the in vitro experiments. Interestingly, although Wnt/TCF7L2 can promote the formation of SSCs, its function must be dependent on the expression of TDRD1, which was also repeatedly demonstrated as a target gene of the Wnt/TCF7L2 signaling pathway. Mechanistically, we found a large number of CpG sites in the TDRD1 promoter, and BSP analysis also confirmed that DNA methylation modifications in the TDRD1 promoter were significantly higher in embryonic stem cells than in SSCs, and further dual-luciferase reporter system assays revealed that low DNA methylation modification levels could enhance TDRD1 promoter activity; although previous studies demonstrated that TCF7L2 could enrich in the TDRD1 promoter region, the binding of the two was dependent on low DNA methylation modification. Taken together, we confirmed that low DNA methylation mediates Wnt/TCF7L2 regulation of TDRD1 to promote the formation of SSCs, providing a basis for SSCs in improving animal productivity.

Characteristics of the TDRD1 gene promoter in chickens.

Tudor domain containing 1 (TDRD1) is a member of the TDRD family and plays an important role in embryogenesis and gametogenesis. A detailed study of the characteristics of chicken TDRD1 can lay a foundation for the study of chicken spermatogonia stem cell formation and spermatogenesis. We cloned 2117 bp upstream fragment of TDRD1 promoter and constructed a series of recombinant vectors with different length deletions. The dual-luciferase experiments reveal that the upstream region of - 161 to 0 bp was its core transcription promoter region. Bioinformatics analysis predicted the possible binding of Transcription Factor 7 Like 2 (TCF7L2) and Zinc Finger E-Box-Binding Homeobox 1(ZEB1) transcription factors in the core region. The transcriptional activity of TDRD1 was significantly decreased after mutation of TCF7L2-binding site, while that of TDRD1 was significantly increased after mutation of ZEB1-binding site. Further, ChIP experiments verified that TCF7L2 enriched in the TDRD1 core transcriptional initiation region, suggesting that TCF7L2 and ZEB1 play an important role in the regulation of TDRD1. In summary, the region from - 161 to 0 bp is the core promoter region of TDRD1; ZEB1 and TCF7L2 bind to the TDRD1 promoter region and TCF7L2 activates the transcription of TDRD1 gene.

A Dead Reckoning Calibration Scheme Based on Optimization with an Adaptive Quantum-Inspired Evolutionary Algorithm for Vehicle Self-Localization.

Parameter calibration is critical for self-localization based on dead reckoning in the control of intelligent vehicles such as autonomous driving. Most traditional calibration methods for robotics control based on dead reckoning rely on data collection with specially designed paths. For the calibration of parameters in the control of intelligent vehicles, the design of such paths is considered impossible due to the complexity of road conditions. To solve this problem, an optimization-based dead reckoning calibration scheme is introduced in this research using the differential global positioning system to obtain the actual positions of the intelligent vehicle. In this scheme, the difference between the positions obtained through dead reckoning and the positions obtained through the differential global positioning system is selected as the optimization objective function to be minimized. An adaptive quantum-inspired evolutionary algorithm is developed to improve the quality and efficiency of optimization. Experiments with an intelligent vehicle were also conducted to demonstrate the effectiveness of the developed calibration scheme. In addition, the newly introduced adaptive quantum-inspired evolutionary algorithm is compared with the classic genetic algorithm and the classic quantum-inspired evolutionary algorithm using eight benchmark test functions considering computation quality and efficiency.

Narrow H3K4me2 is required for chicken PGC formation.

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-ß). 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.

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

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 N6 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.

Nanos2 promotes differentiation of male germ cells basing on the negative regulation of Foxd3 and the treatment of 5-Azadc and TSA.

The transcription factor positioning in promoter regions relate to gene regulation, and the level of DNA methylation and histone acetylation also impact the promoter activity. In this study, we tested and verified the core promoter region and key transcription factor of Nanos2 which is a male-critical gene in the differentiation of embryonic stem cells to male germ cells, meanwhile, epigenetic effects by mean of 5-Aza-2'-deoxycytidine (5-Azadc) and Trichostin A (TSA) on the activity of Nanos2 promoter were detected. The results reveal that key transcription factor Foxd3 is a negative regulator of Nanos2, which suggests that loss-of-function of Foxd3 causes strong expression of Nanos2 responsive to large amounts of primordial germ cells and spermatogonial stem cells,whereas its overexpression causes the opposite effect. Furthermore, both 5-Azadc and TSA can provoke responses of Nanos2, but the combination effect of the two is better.

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

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.

Study on immortal conditions of chicken embryonic stem cells.

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.

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

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.

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

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.

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

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.

[Design and practice of the case study methodology throughout the "Animal Genetic Principles and Breeding Methods" course].

"Animal Genetics Principles and Breeding Methods" is a main course for Master students majoring in Agriculture (Livestock) and involves a combination of theory and practice. The traditional teaching method is difficult not only to meet the requirements of modern professional degree teaching, but also for the students to master the theory and practice of genetic breeding. We have employed the case study methodology during the entire course. This paper analyzes the connotation and characteristics of the method and expounds the design and discussion of the cases. Besides, the teaching evaluation is also included. It provides a reference for the application and promotion of the case teaching method in training graduate students majoring in agriculture.