Uses of knockout,knockdown, and transgenic models in the studies of glucose transporter 4

Currently, glucose transporter 4 (GLUT4) has been considered as the key player for the insulin-stimulated glucose transport in the muscle and adipose tissues. The development of recombinant DNA techniques allows the creations of genetically knockout, knockdown and transgenic animals and cells for the study of GLUT4’s physiological functions. Here, we have used key words to search the PubMed and summarized the methods used in Slc2a4 gene knockout, GLUT4 knockdown and overexpression in the whole body and tissue specific manner. The whole body GLUT4-null mice have growth retardation, but normal glucose tolerance and basal glucose turnover rates. Compared with whole body Slc2a4 knockout mice, adipose and muscle double knockout mice have impaired insulin tolerance and glucose intolerance. The results of GLUT4 knockdown in 3T3-L1 adipocytes have shown that its expression is needed for lipogenesis after, but not during, differentiation. Transgenic mice with the whole body GLUT4 overexpression have normal body weight and lowered blood glucose level. The adipose tissue specific overexpression of GLUT4 leads to increases in mouse body weight and adipose tissue weight. The insulin-stimulated GLUT4 translocation in the skeletal muscle contributes to the regulation of glucose homeostasis. Data from both transgenic overexpression and tissue specific Slc2a4 knockout indicate that GLUT4 probably plays a role in the glucose uptake in the fasting state. More studies are warranted to use advanced molecular biology tools to decipher the roles of GLUT4 in the control of glucose homeostasis.     

The renin-angiotensin system in transgenic rats

Transgenic animals are used to study the function, regulation and in vivo expression of genes. The effects of the genes of the renin-angiotension system on blood pressure regulation and hypertension were investigated in transgenic rats. The role of the renin-angiotensin system in the development of cardiovascular hypertrophy of hypertensive renal damage was analysed, as well as its interaction with other hormonal systems, i.e. adrenal steroids. The development of a transgenic rat strain carrying the mouse REN-2 gene has provided a new model of hypertension with systolic blood pressure values of 200 mmHg. This model is characterised by low active plasma renin, hyperproreninaemia and high expression of renin in the adrenal gland and other extrarenal tissues. Transgenic rats with thehuman components of the renin-angiotensin system expressed the human renin and angiotensinogen proteins which interacted species-specifically in transgenic rats. These transgenic models demonstrate the feasibility of studying the function of candidate hypertension genes in transgenic animals. In the future, further refinements in transgene construction, mutation, and modification can be tested in such transgenic animal models.     

转基因食品的安全性评价初探

综述了转基因食品的概念、分类、转基因食品的现状与未来发展趋势,旨在消除人们对转基因食品的疑虑,对转基因技术及食品安全性方面有一总体认识,使现代生物技术能更好地造福人类。     

异种移植模型转人a1,2岩藻糖基转移酶基因小鼠显微注射DNA片段的制备

目的制备转人琢1,2岩藻糖基转移酶(HT)基因小鼠显微注射DNA片段。方法引物两端设计酶切识别位点,聚合酶链反应(PCR)扩增HTcDNA全长序列,两端含有EcoRⅠ和BamHⅠ酶切识序列;回收HTcDNA片段并与PMD18-T载体连接,EcoRⅠ和BamHⅠ双酶切纯化质粒鉴定;EcoRⅠ和BamHⅠ双酶切pMD18-HTcDNA重组质粒和pCMV-MCS质粒,回收HTcDNA片段和pCMV-MCS质粒片段,进而连接,转化感受态细菌,纯化质粒对其进行酶切、PCR和测序鉴定;PvuⅠ和NotⅠ依次单酶切重组质粒pCMV-MCS-HTcDNA,回收大小约2.85kb片段,溶于适量显微注射用缓冲液。结果成功构建了重组质粒pCMV-MCS-HTcDNA,酶切回收了2.85kb的显微注射DNA片段。结论通过基因工程技术可以获得转人HT基因小鼠显微注射DNA片段,包含基因表达元件,可以用于显微注射法建立转人HT基因小鼠。     

超声成像技术分析糜酶转基因小鼠心脏功能

目的应用小动物高频超声技术对糜酶转基因小鼠的心功能进行分析,以探索糜酶基因对心脏结构和功能的影响。方法通过VisualSonics Vevo770高分辨率小动物超声系统检测不同年龄的转基因小鼠及对照小鼠包括心壁厚度、主动脉血流速度、左心室内径、左心室容积、每搏输出量、射血分数、短轴缩短率和心输出量等的心脏功能指标的改变进行比较分析。结果随着小鼠年龄的增大,转基因阳性小鼠心壁厚度和主动脉血流速度逐渐增加,至6月龄时,转基因小鼠的左心室前壁收缩期厚度增加37%,增长率是对照小鼠的1.2倍(P<0.05);主动脉血流速度比对照小鼠高29%(P<0.05);转基因阳性小鼠的左心室内径、左心室容积、每搏输出量、射血分数、短轴缩短率和心输出量等的心脏功能指标表现出和以上变化相一致的表型。结论转基因小鼠糜酶表达水平升高,引起心脏AngⅡ形成增多,可使心肌细胞的收缩力提高;心肌细胞肥大,心壁增厚;且有随年龄增加的趋势,可研究建立慢性、老年性肥厚型心脏病模型。     

Effects of trigeminal ganglion stimulation on the central auditory system

It is very important to determine if recombinant adenoviral vector (Ad) can damage the auditory hair cells in guinea pig cochlea after transgene expression. In this study, the scanning electron microscope was used to determine if there was loss of the auditory hair cells after Ad.LacZ (Ad5 containing Escherichia coli galactosidase) was inoculated into the cochlea through the round window membrane. Seven days later all inner and outer hair cells were found to express the LacZ gene. Except for the sparse loss of outer hair cells in the basal turn and the second turn, there was insignificant loss in the other turns. All inner hair cells were present. The damage to auditory hair cells resulting from intracochlear inoculation of Ad is limited, and this vector can be used as one of the ideal delivery tools in gene therapy of the cochlea.     

新生大鼠耳蜗Corti器体外培养及其转基因表达

目的建立新生大鼠耳蜗Corti器体外培养模型及观察外源基因表达。方法采用出生后1~3天龄大鼠,分离出的耳蜗Corti器组织置入表面覆盖有胶原凝胶的培养皿中,在含有10%DMEM液体培养基中培养;加入携带报告基因的腺病毒悬液,观察外源基因的表达。结果耳蜗Corti器体外培养8小时后,培养组织生长良好,形态结构清晰可辨,在该实验条件下,Corti器形态结构可以维持5天以上,最长达7天,随着培养时间的延长,由于周围组织生长的蔓延,Corti器的结构分界不清楚。腺病毒加入Corti器后12小时即有基因表达,24小时达到高峰,表达时间可持续1周。结论新生大鼠离体内耳组织转基因培养法是内耳Corti器体外培养实验研究的一种可行方法。     

T cell tolerance and activation to a transgene-encoded tumor antigen

Much has been learned in recent years concerning the nature of tumor antigens recognized by T cells. To apply this knowledge clinically, the nature of the host response to individual and multiple tumor antigens has to be characterized. This will help to define the efficacy of immune surveillance and the immune status of the host following exposure to tumor antigens expressed on pre-neoplastic tissue. To approach these questions, we have developed a transgenic mouse which expresses the tumor-specific antigen P91A. The single amino acid substitution in P91A results in the expression of a new MHC class I (H-2L^d)-binding peptide. In transgenic tissue, the H-2L^d/P91A complex is expressed in isolation from other tumor-associated antigens, allowing definition of the immune response to a single defined tumor antigen, a situation closely analogous to events during tumorigenesis. We show that CD8⁺ T cell immune surveillance of P91A is ineffective without the introduction of a helper determinant operating through stimulation of CD4⁺ T cells. Recognition of the isolated P91A tumor antigen on normal tissue by CD8⁺ T cells is a tolerogenic process. Induction of T cell tolerance suggests tumor antigen-T cell interactions occurring during tumorigenesis may elicit T cell tolerance and hence confound some immunotherapeutic approaches.     

EB病毒BNLF—1基因转基因小鼠整合位点FISH检测的研究

本文应用荧光原位杂交技术研究了ＥＢ病毒潜伏膜蛋白基因（ＢＮＬＦ１） 在转基因小鼠染色体上的整合。结果：在转基因小鼠的单条染色体上检测到了杂交信号,检出率为３８ ．３ ％ ,表明该转基因已以单位点、多拷贝的形式稳定整合于转基因小鼠的染色体上。