NTS influence on transgene expression from mono- and bicistronic plasmid vectors after lipofection in a human fibroblast cell line

 We evaluated the survival, transgene production, and copy numbers of integrated plasmid units per host genome after lipofection with mono- and bicistronic plasmid vectors in different cell lines and under various conditions. The addition of an integration enhancing murine sequence nontranscribed spacer (NTS) to the plasmids increased transfection efficiency, survival, and transgene expression. However, in human fibroblast cells this sequence had only marginal effects on overall plasmid copy number in bulk cultures. Clones producing the highest amounts of the transgene contained only one or two copies of plasmid per genome, independent of cell type and plasmid design. Received: 1 October 1996 / Accepted: 27 November 1996     

神经组织特异表达绿色荧光蛋白转基因小鼠的脑组织形态观察

目的 建立神经组织特异表达绿色荧光蛋白的转基因小鼠,为神经系统的形态学观察提供可以荧光示踪的工具动物.方法 把增强型绿色荧光蛋白(EGFP) 因插入血小板源性生长因(PDGF)B-链启动子下游构建转基因载体,用显微注射的方法建立转基因C57BL/6J小鼠.PCR鉴定转基因小鼠的基因表型,对阳性转基因小鼠的脑组织进行矢状面冷冻切片,分别进行HE染色,显微镜观察组织结构,荧光体视镜及荧光显微镜观察绿色荧光蛋白(GFP)在神经组织的表达.结果 在8个首建品系中筛选出1个神经组织高表达绿色荧光蛋白的转基因小鼠系.观察到绿色荧光蛋白在大脑皮层、海马、丘脑、小脑及脑干等部位表达.结论 建立了稳定遗传的神经组织特异表达绿色荧光蛋白转基因小鼠品系,为神经系统的生理学及病理学研究提供了可以荧光示踪的模型动物.     

哺乳动物精原干细胞技术的研究进展

精原干细胞（spermatogonial stem cells SSCs）具有高度的自我更新能力和分化潜能。其体外培养以及近年来兴起的移植、基因转染的深入研究，为探讨精子的发生机理、重建不育个体的精子发生、生产转基因动物提供了新的途径。本文对SSCs技术的研究进展作一综述。     

Expression of a transgenic class II Ab gene confers susceptibility to collagen-induced arthritis

The major histocompatibility complex (MHC) class II region is assumed to influence autoimmune diseases such as rheumatoid arthritis. In the mouse, the H-2^q haplotype is associated with susceptibility to collagen-induced arthritis, while the H-2^p haplotype is not. The class II A molecules of these haplotypes differ by only four amino acids in the first domain of the β chain. To test if this difference accounts for the MHC influence on susceptibility to collagen-induced arthritis, H-2^p mice were made transgenic with an Ab^p gene altered to resemble the Ab^q gene. The transgenic A_β chain hybridized with the A_α^p chain and was shown to be physiologically expressed by testing antigen-presentation capacity to A^q-restricted T cell hybridomas and with FACS analyses. These transgenic mice developed an autoimmune response to type II collagen and also collagen-induced arthritis. The data unequivocally suggest the Ab gene as a major genetic susceptibility locus for autoimmune collagen-induced arthritis.     

丝裂原活化蛋白激酶和磷酸酯酶-1在心肌病和细胞凋亡中的作用

ERKJ、NK和p38等丝裂原活化蛋白激酶通过生长因子、激动剂或应激反应等介导生长、分化、凋亡以及细胞间相互作用等多种过程。ERKJ、NK和p38是参与心衰病理过程的主要信号元件,MKP-1是丝裂原活化蛋白激酶等的去磷酸化因子,是一种应激蛋白,在应激反应中可以抑制ERKJ、NK和p38的活性,并通过调节ERK、JNK和p38的活性,参与对心衰病理过程的调节。本文以转基因研究结果为主要线索,对丝裂原活化蛋白激酶和磷酸酯酶-1在心衰病理过程中的作用进行了综述。     

One year transgene expression with adeno-associated virus cardiac gene transfer

BACKGROUND: Adeno-associated virus (AAV) has shown promise as a vector for cardiac gene transfer given its ability to stably integrate into the host genome and its lack of immune reactivity. This study examined the feasibility of AAV-mediated myocardial gene transfer in mice, the animal which, because of transgenic technology, has become the disease model of choice for cardiovascular research. METHODS: AAV encoding the cytomegalovirus promoter driven LacZ reporter gene (10(7) LacZ-forming units per animal) or vehicle control was injected into the hearts of young adult C57Bl/6 mice by a transdiaphragmatic approach. At one, two, three, six, and twelve months post-injection, cardiac function was assessed by transthoracic echocardiography and hearts were assayed by X-gal histochemical staining. RESULTS: Echocardiography revealed normal left ventricular function in both AAV and control groups at all time points. X-gal staining of cryostat sections of hearts revealed uniform LacZ expression at all time points. There were minimal signs of immunologic infiltration by hematoxylin and eosin staining. CONCLUSIONS: AAV-mediated myocardial gene transfer by transdiaphragmatic injection can be conducted safely and results in long-term expression of the LacZ gene for at least one year without causing significant inflammatory response or adversely affecting LV systolic function.     

Neuropeptide alterations in the hippocampal formation and cortex of transgenic mice overexpressing beta-amyloid precursor protein (APP) with the Swedish double mutation (APP23)

The role of neuropeptides and the significance of peptidergic mechanisms in neurodegenerative diseases are still unclear. In the periphery, nerve injury results in dramatic changes in the expression of neuropeptides. An important question regards to what extent similar changes occur, and similar mechanisms operate, after lesions and/or degeneration in the brain. The purpose of this work is, therefore, to study neuropeptides with regard to their presence and distribution in the APP23 mouse (HuAPP(751) K670M/N671L under the murine Thy-1 promoter), a model for Alzheimer's disease, or cerebral amyloidosis, using the immunohistochemical technique. In addition, tyrosine hydroxylase and acetylcholinesterase were analyzed. This study shows marked neuropeptide changes in the hippocampal formation and the ventral cortex, whereas the dorsolateral neocortex was less affected. There was a considerable variation with regard to peptide expression among animals of the same age which was related to the variation in Abeta deposition. Dystrophic and varicose fibers containing galanin, neuropeptide Y, enkephalin, and especially cholecystokinin were commonly seen in close proximity to amyloid plaques. In addition, generalized changes were observed, such as increases of enkephalin and neuropeptide Y in stratum lacunosum moleculare and of neuropeptide Y, enkephalin, and dynorphin in mossy fibers. In contrast, cholecystokinin was decreased in mossy fibers. Comparatively small differences were observed between wild-type and transgenic mice with regard to tyrosine hydroxylase (noradrenergic but also dopaminergic fibers) and acetylcholine esterase (mainly cholinergic fibers). The increase of neuropeptides in dystrophic fibers in this model may represent a response to nerve injury caused by the amyloid accumulation and may reflect attempts to counteract degeneration by initiating protective and/or regenerative processes.     

外源基因在低温保存后同种带瓣大动脉中的表达

目的观察外源性基因在低温保存不同时间后的同种带瓣大动脉中的表达。方法将30只幼兔的同种带瓣大动脉分为5组,分别在4℃营养液中（M199）保存2、6、12、24、72h。保存后将每组中的5个同种带瓣大动脉浸入300μl含重组腺病毒(Ad5CMVLacZ)的DMEM液中,而另1个则浸入300μl不含重组腺病毒的DMEM液中,均在室温下放置30min,然后分别移植于15只成兔受体的两侧颈动脉。术后5d取出移植物,进行组织化学染色。结果所有转染重组腺病毒的同种带瓣大动脉均有不同程度的基因表达,Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组阳性细胞平均表达量分别42.6±8.5、38.2±12.7、34.5±10.2、31.6±9.6、36.4±13.2;组间转基因细胞表达量差异无显著性（P＞0.05）。对照组均为阴性。结论对低温保存不同时间后的同种带瓣大动脉进行转基因是可行的。     

TARGETED EXPRESSION OF TEMPERATURE-SENSITIVE DYNAMIN TO STUDY NEURAL MECHANISMS OF COMPLEX BEHAVIOR IN Drosophila

Sites in the brain that show functional, sexual dimorphism in courtship behavior have been mapped at high resolution in male/female mosaics of Drosophila melanogaster. The sex mosaics were produced by enhancer-trap expression of GALA driving the female-spliced form of the transformer gene (tra), revealing sites in the dorsal brain, lateral proto-cerebrum, suboesophageal, thoracic and abdominal ganglia, and suggesting the importance of cross-talk between these regions in the implementation of the courtship sequence.