外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

Characterization of a S-layer protein from Lactobacillus crispatus K313 and the domains responsible for binding to cell wall and adherence to collagen

It was previously shown that the surface (S)-layer proteins covering the cell surface of Lactobacillus crispatus K313 were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-layer protein genes (slpA, slpB, and slpC) of L. crispatus K313 were amplified by PCR, sequenced, and characterized in detail. Quantitative real-time PCR analysis reveals that slpA was silent under the tested conditions; whereas slpB and slpC, the putative amino acid sequences which exhibited minor similarities to the previously reported S-layer proteins in L. crispatus, were actively expressed. slpB, which was predominantly expressed in L. crispatus K313, was further investigated for its functional domains. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-layer protein expression. By producing a set of N- and C-terminally truncated recombinant SlpB proteins in Escherichia coli, the cell wall-binding region was mapped to the C terminus, where rSlpB_380–501 was sufficient for binding to isolated cell wall fragments. Moreover, the binding ability of the C terminus was variable among the Lactobacillus species (S-layer- and non-S-layer-producing strains), and teichoic acid may be acting as the receptor of SlpB. To determine the adhesion region of SlpB to extracellular matrix proteins, ELISA was performed. Binding to immobilized types I and IV collagen was observed with the His-SlpB_1–379 peptides, suggesting that the extracellular matrix protein-binding domain was located in the N terminus.     

Impact of warm ischemia on gene expression analysis in surgically removed biosamples

Surgically removed samples of high quality provide more accurate and reliable results in downstream molecular assays. Some factors, including the type of anesthesia, surgical manipulation, transport time and mode, preservation method, storage length, and number of freeze-thaw cycles, can affect biosample quality and the subsequent gene expression analysis. Warm ischemia resulting from these factors has a substantial effect on biosample quality and is the focus of this mini review. We classified the effects of warm ischemia on gene expression as (i) warm ischemia-induced metabolic activity (WIMA) in living cells and (ii) warm ischemia-induced RNA degradation (WIRD). The differential effects of WIMA and WIRD on gene expression analysis appear to depend on the period after surgical removal. WIMA predominantly affects gene expression during the early stage after surgery, whereas WIRD has a more significant effect after tissue thawing. By a literature review, we also found that RNA isolated from surgically removed biopsies is stable, and high-quality RNA can be obtained for most nonfixed human tissue maintained at room temperature during the early period after surgery. Understanding these characteristics of gene expression variation should help biomedical researchers to avoid misleading gene expression results.     

Molecular characterization of a tandem-repeat galectin-9 (RuGlec9) from Korean rose bitterling (Rhodeus uyekii)

Galectin-9 is a b-galactoside-binding lectin that regulates many cellular functions, ranging from cell adhesion to pathogen recognition. We isolated and characterized the cDNA of tandem-repeat galectin-9 (RuGlec9) from the Korean rose bitterling (Rhodeus uyekii), an endemic Korean fish belonging to the Acheilognathinae subfamily of the Cyprinidae family. RuGlec9 cDNA is 1486 bp long and encodes a polypeptide of 323 amino acids containing two carbohydrate-recognition domains connected by a linker peptide. The deduced amino acid sequence of RuGlec9 shows 45-84% amino acid sequence identity to other galectin-9 sequences, including those from mammals and fish. RuGlec9 appeared in a large cluster with other galectin-9 sequences from fish and is more closely related to galectin-9 from Danio rerio than to those of other fish and mammals. RuGlec9 mRNA was expressed highly in the testis, spleen, intestine, stomach, and liver, and moderately in the brain, kidney, ovary, and gills of normal Korean rose bitterling. RuGlec9 mRNA expression in the spleen was increased by lipopolysaccharide. These results suggest that RuGlec9 plays a role in innate immunity in Korean rose bitterling.