CRISPR/Cas9-Mediated Suds3 Knockout Inhibited Mouse Embryonic Stem Cell Proliferation and Mesoderm/Endoderm Differentiation / 中国生物化学与分子生物学报

Embryonic stem cells (ESCs) have the ability to differentiate into various adult cells, and their fate in the process of development and differentiation is determined by the comprehensive regulation of multiple factors such as gene expression, epigenetics, and extracellular signals. Epigenetic regulation, such as DNA methylation, histone acetylation, and methylation, plays an important role in the maintenance of pluripotency and differentiation of ESCs. Suds3 (Sin3 histone deacetylase corepressor complex component SDS3) is one of the important components of Sin3 histone deacetylase complex. It played an important role in embryonic development, cell proliferation, chromosome separation and other biological processes. However, the functions of Suds3 in ESCs, such as its influence on the proliferation, maintenance of pluripotency and differentiation of ESCs, were rarely reported. In this study, we used CRISPR/Cas9 gene editing technology to construct a Suds3 knockout mouse embryonic stem cell line, and combined cell culture, in vitro embryoid body (EB) formation and in vivo teratoma formation, CCK-8 and cell counting experiments to study the function of Suds3 in ESCs. Western blotting results showed that the SUDS3 protein was not expressed, and the Suds3 gene was successfully knocked out. Through the observation of cell morphology and fluorescence quantitative PCR (QRT-PCR) to detect the expression of pluripotency genes, we found that the knockout of Suds3 had no significant effect on the maintenance of pluripotency of ESCs. Embryoid body (EB) formation experiments revealed that on the fourth and sixth days of EB formation, the pluripotency gene expression was not down-regulated as quickly as WT cells but increased in Suds3

Roles of TRIM28 in tumorigenesis and embryogenesis / 上海交通大学学报（医学版）

TRIM28, known as a kind of macro-molecules, consists of multiple domains and belongs to one of human tripartite motif-containing proteins families. This superfamily contains more than 60 proteins in total and is characterized by four conservative domains, also referred to the RBCC domain, which is a RING (really interesting new gene) finger, two B-boxes (type 1 and type 2) and a leucine zipper coiled-coil region. TRIM28 plays its pivotal roles in transcriptional co-activation or co-repression by interacting directly with transcription factors, within which is a Kruppel-associated box domain (KRAB domain). Moreover, it is critical during the development of embryos, the differentiation of cells and the regulation of cancers.

Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.

The expression of BCORL1 and E-cadherin in gastric carcinoma and their correlation analysis / 重庆医学

Objective To investigate the expression of BCORL1 and E-cadherin and their correlation analysis in gastric carcino-ma .Methods We freshly collected 58 samples of surgically resected paired gastric carcinoma and normal tumor-adjacent tissues and detected BCORL1 and E-cadherin expression in the samples using immunohistochemical staining .The correlation between BCORL1 and E-cadherin protein expression was analysed .Results The protein expression of BCORL1 in gastric carcinoma tissues was sig-nificantly upregulated compared to those of the normal tumor-adjacent tissues(60 .3% vs .17 .2% ,P=0 .000) ,but expression of E-cadherin in gastric carcinoma tissues was significantly lower than those in the normal tumor-adjacent tissues (27 .6% vs .63 .8% , P=0 .000) .Clinicopathological analysis suggested that EphA2 and E-cadherin protein expression were associated with histopatho-logical differentiation ,depth of invasion ,lymph node metastasis and TNM stage(P<0 .05) .BCORL1 was significantly negative cor-related with E-cadherin protein in gastric carcinoma(r= -0 .571 ,P=0 .002) .Conclusion The high-expression of BCORL1 is cor-related with malignant clinicopathological characteristics ,and BCORL1 is negative associated with E-cadherin ,suggesting that BCORL1 promotes tumor progression and metastasis through transcriptional regulating E-cadherin in gastric carcinoma .

The Hairless Gene: A Putative Navigator of Hair Follicle Development

The Hairless (HR) gene regulates the expression of several target genes as a transcriptional corepressor of nuclear receptors. The hair follicle (HF), a small independent organ of the skin, resides in the epidermis and undergoes regenerative cycling for normal hair formation. HF development requires many genes and signaling pathways to function properly in time and space, one of them being the HR gene. Various mutations of the HR gene have been reported to cause the hair loss phenotype in rodents and humans. In recent studies, it has been suggested that the HR gene is a critical player in the regulation of the hair cycle and, thus, HF development. Furthermore, the HR gene is associated with the Wnt signaling pathway, which regulates roliferation and differentiation of cells and plays an essential role in hair and skin development. In this review, we summarize the mutations responsible for human hair disorders and discuss the roles of the HR gene in HF development.

Proteomics analysis for the signal transduction under stimulation of prolactin on Jurkat cells / 中国免疫学杂志

Objective:The experiment was designed to apply proteomics to analyze the signal transduction molecules in T lymphocyte strain JurkatD1.1 cells with the stimulation of prolactin.Methods:JurkatD1.1 cells were cultured in the presence of recombinant human prolactin(rhPRL).Phosphated metal affinity chromography(PMAC) and immunoprecipitation(IP) assay were applied to enrich phosphoproteins from the total cell lystates which were resolved by one-dimensional electrophoresis(1 DE) or 2 DE.The different bands and different spots in the gels between control group and rhPRL-treated group were excised,digested,and analyzed by MALDI-TOF mass spectrometry.Results:Three major protein bands were observed specifically in the rhPRL-treated group and other two major protein bands were observed specifically in the control group.The bands were excised and analyzed by MALDI-TOF mass spectrometry.Protein band a in rhPRL-treated group was successfully identified as hot shock protein 90(hsp90).Nine different protein spots which were observed in the 2 DE gels between the two groups were excised and analyzed by MALDI-TOF mass spectrometry.In the rhPRL-treated group the protein spot 4 was identified as nuclear receptor co-repressor 2 variant(NcoR 2 variant);The protein spot 5 was identified as galactose-1-phosphate uridyl transferase;And the protein spot 6 was identified as zinc finger ZIM3.Conclusion:Hot shock protein 90 participates the signal transduction pathways of prolactin.Nuclear receptor co-repressor 2 variant and zinc finger ZIM3 may play a role in the process of prolactin modulating target genes' expression.