Li Bichun

Study on the role of JAK/STAT signaling pathway during chicken spermatogonial stem cells generation based on RNA-Seq

Abstract Spermatogonial stem cells (SSCs) form the foundation for spermatogenesis and sustain male fertility. To explore the regulatory mechanisms of chicken SSCs generation, we obtained highly purified chicken embryonic stem cells (ESCs), primordial germ cells (PGCs) and SSCs by fluorescence-activated cell sorting (FACS). High-throughput analysis methods (RNA-Seq) were used to sequence the transcriptome level of these cells. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment were used to analyze RNA-Seq results. BMP4 was used to induce chicken ESCs differentiation to SSCs-like cells in vitro. The quantitative real-time (qRT)-PCR was used to detect the expression changes of the key genes. The results showed that 22 relevant critical pathways were found by RNA-Seq, one of them was the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. Total of 103 related genes were detected in this pathway. Protein-protein interactions analysis found that 87 proteins were significantly related to 19 key proteins in this pathway. These 87 proteins were enriched in 21 biological processes and 18 signaling pathways. Moreover, during the differentiation of chicken ESCs to SSCs-like cells induced by BMP4 in vitro , JAK2 and STAT3 were activated. The qRT-PCR results showed that the expression trends of JAK2 and STAT3 were basically the same as in vivo. We concluded that JAK/STAT signaling pathway plays an important role in the process of chicken SSCs generation both in vivo and in vitro ; it may achieve its function through multiple biological processes and other related pathways.

Nanos2 promotes differentiation of chicken (Gallus gallus) embryonic stem cells to male germ cells

Nanos2 is an evolutionarily conserved RNA‐binding protein containing 2 CCHC‐type zinc finger motives. Here, we report that Nanos2 is strongly expressed in the testis compared to other tissues in chicken (Gallus gallus). Overexpression and knockout plasmid vectors were constructed, and in‐vitro Cas9/gRNA digestion and T7 endonuclease I (T7E1) assay indicated that Nanos2‐g1 possessed the highest knockout activity. In vitro and in vivo, Nanos2 overexpression accelerated the production of embryoid bodies (EBs) and SSC‐like cells and promoted cvh, c‐kit, and integrin α6 expression. Immunofluorescence staining, periodic acid schiff (PAS) and flow cytometry (FCM) assay showed that primordial germ cells (PGCs) and spermatogonial stem cells (SSCs) formation were significantly promoted. On the contrary, Nanos2 knockout delayed the production of EBs and SSC‐like cells and correspondingly reduced cvh, c‐kit, and integrin α6 expression. Simultaneously, the quantity of PGCs and SSCs was blocked. Collectively, these results uncovered a novel function of Nanos2 involved in chicken male germ cell differentiation, where it acts as a facilitator.

Research on the appropriate way to transfer exogenous substances into chicken embryos

Abstract In biological research, chicken embryos are a classic experimental model for the exploration of the embryonic development and cell differentiation. Transferring exogenous substances into chicken embryos for producing medical antibodies has been widely used in the production practice. However, there are few studies about the effect of the different injection site and dosage on chicken embryos. The aim of this study was to explore the effects of different injection sites and dosages on chicken embryo hatching rate and development, so as to provide a basis for further studies using the chicken embryo model. Freshly laid eggs (Rugao yellow chicken) were injected with different doses of saline at the tip, equatorial plane and the blunt end of the egg shell, respectively. Egg hatching rate was recorded and compared among injection sites and different doses. A trypan blue stain was also injected at the aforementioned sites and the growth of chicken embryos was observed. The SPSS (statistical package for the social science) software was used to analyze the relationship between the chicken eggs hatching rate and the different injection sites or the different dosages. The experimental results showed that there were significant differences on egg hatching rates among the different injection sites and doses (P

Genetic diversity of KAP6.1 gene in 6 rabbit populations.

Full-length CDS of KAP6.1 gene was obtained by sequence assembly with 2 specific primers.Then the sequences in 6 rabbit populations were scanned by PCR-SSCP.The results showed that 3 genotypes and 2 alleles were found at KAP6.1-1 loci,as well as the A 755 G mutation which was located at the enhancer region,while no mutation was found at KAP6.1-2 loci.The distribution of these genotypes in all populations was consistent with the Hardy-Weinberg equilibrium and presented either intermediate level or low level of diversity;and its difference was either significant(P0.05) or very significantly(P 0.01) from one another populations except between Fujian black rabbit and Jiuyishan rabbit,chinchilla and white rex rabbit,wan-line and su-line angora rabbit(P0.05).