Functional expression of a cloned I-A beta k gene in B-lymphoma cells

The immune response genes of the mouse encode two cell-surface glycoproteins, I-A and I-E, that play critical roles in determining the animal's immune responsiveness. The I-A antigen contains two chains, alpha and beta. A cloned beta-chain gene, I-A beta k, was introduced into B-lymphoma cells that express I-Ad. The transfected gene was successfully expressed on the cell surface of the recipient cells and was functional in stimulating allospecific T cells.     

Synthesis of functional human hemoglobin in transgenic mice

Human alpha- and beta-globin genes were separately fused downstream of two erythroid-specific deoxyribonuclease (DNase) I super-hypersensitive sites that are normally located 50 kilobases upstream of the human beta-globin gene. These two constructs were coinjected into fertilized mouse eggs, and expression was analyzed in transgenic animals that developed. Mice that had intact copies of the transgenes expressed high levels of correctly initiated human alpha- and beta-globin messenger RNA specifically in erythroid tissue. An authentic human hemoglobin was formed in adult erythrocytes that when purified had an oxygen equilibrium curve identical to the curve of native human hemoglobin A (Hb A). Thus, functional human hemoglobin can be synthesized in transgenic mice. This provides a foundation for production of mouse models of human hemoglobinopathies such as sickle cell disease.     

Altering the genome by homologous recombination

Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.     

Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor

The gene for the human platelet alpha 2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of alpha 2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human beta 2- and beta 1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional alpha 2-adrenergic receptor subtypes.     

Chromosomal location of human T-cell receptor gene Ti beta

A complementary DNA probe corresponding to the beta-chain gene of Ti, the human T lymphocyte receptor, has been molecularly cloned. The chromosomal origin of the Ti beta gene was determined with the complementary DNA by screening a series of 12 cell hybrid (mouse X human) DNA's containing overlapping subsets of human chromosomes. DNA hybridization (Southern) experiments showed that the human Ti beta gene resides on chromosome 7 and is thus not linked to the immunoglobulin loci or to the major histocompatibility locus in humans.     

XERICO和Tyr-rich HRGP双价基因植物表达载体的构建

以拟南芥基因组DNA为模板,通过特异性PCR扩增,克隆了逆境诱导表达启动子rd29A和XERICO基因、组织特异性启动子PAL2和Tyr-rich HRGP基因。构建了pBrd-XERICO-PAL2-tyr-rich HRGP-nos双价基因植物表达载体,并将其导入根癌农杆菌EHA105中。     

蝴蝶兰基因工程研究进展

蝴蝶兰作为热带和亚热带最重要的观赏花卉之一,在基因工程的改良上有着诱人的前景。综述了蝴蝶兰遗传转化的转基因受体材料、导入的外源基因和克隆的相关基因、抗病毒基因的研究现状,展望了蝴蝶兰转基因技术及其应用前景。     

Using a combination of SAR element and enhancer to increase the expression level of hybrid genes in transgenic animals

A regulatory element consisting of a MAR element sequence of Drosophila heat shock genes and enhancer from a long terminal repeat of the mouse mammary tumor virus is created for studying the possibility of increasing the expression level of hybrid genes in transgenic animals. Two lines of transgenic mice containing the described regulatory element and hybrid gene intended for expressing human beta interferon in milk of transgenic animals are obtained by the method of coinjection into the pronuclei of murine zygotes. It is shown that the use of the given regulatory element doesn’t lead to an increase of activity of human beta interferon in milk compared with transgenic rabbits containing only the hybrid gene for expressing beta interferon. Furthermore, a decrease of tissue-specific expression of beta interferon is observed with the use of the regulatory element.     

J genes for heavy chain immunoglobulins of mouse

A 15,8-kilobase pair fragment of BALB/c mouse liver DNA, cloned in the Charon 4A lambda phage vector system, was shown to contain the mu heavy chain constant region (CHmu) gene for the mouse immunoglobulin M. In addition, this fragment of DNA contains at least two J genes, used to code for the carboxyl terminal portion of heavy chain variable regions. These genes are located in genomic DNA about eight kilobase pairs to the 5' side of the CHmu gene. The complete nucleotide sequence of a 1120-base pair stretch of DNA that includes the two J genes has been determined.     

热纤维素梭菌内切葡聚糖苷酶基因在枯草芽孢杆菌的表达

对热纤维素梭菌Clostridium thermocellum内切葡聚糖苷酶基因CelG及其信号肽序列进行了克隆及表达研究.以热纤维素梭菌总DNA为模板,经PCR扩增获得目的,并连接到大肠杆菌-枯草杆菌穿梭质粒pSW1载体,获得质粒pSW1-CelG;用感受态转化法将获得质粒pSW1-CelG转化到枯草芽孢杆菌BGSC中进行分泌表达,获得了大小约56KD的蛋白片段,且具有生物活性,证明CelG基因克隆成功并正确表达.     

PiggyBac转座子介导的诱导细胞永生化转基因载体的构建及其整合效率的检测

【目的】构建在小鼠水平上实现多个目的基因的表达以及标记基因安全删除的转基因载体。【方法】以载体为模板,分别扩增SV40P neo、IRES、tk-PloyA,通过overlap PCR连接,并在两端添加方向相同的LoxP序列,构建转基因基本载体PB-NIT;然后通过overlap PCR扩增获得Tet-CMV-SV40 T-T2A-p 53-PolyA基因表达盒,将其插入PB-NIT中,构建载体PB-NIT-STP;最后将转录因子激活域 rtTA 通过同尾酶连接插入PB-NIT-STP,构建载体PB-rtTA-NIT-STP。【结果】经酶切和测序等鉴定,以上载体均正确;经转座活性鉴定,共转染转座子载体PB-rtTA-NIT-STP和转座酶载体比只转染转座子载体获得的阳性克隆数提高了20倍。【结论】得到了基于PiggyBac转座子的可用于正负向筛选和诱导目的基因表达的转基因载体。     

拟南芥2A6基因正义、反义植物表达载体的构建

利用PCR技术从番茄基因组中扩增了长约1.1kb的E8基因启动子,构建了中间表达载体pCAMBI-AE8;利用RT-PCR方法从拟南芥中扩增了长约1 197bp的2A6基因全长cDNA编码区,将其定向插入pCAMBI-AE8,构建了正义植物表达载体pCAMBIAE8-2A6;同时扩增了长约500bp的cDNA片段,定向插入pCAMBI-AE8,构建了反义植物表达载体pCAMBIAE8-2A6anti.以此为基础,可以进一步研究2A6基因的差异表达对转基因拟南芥角果发育的影响,达到揭示2A6基因功能的目的.     

鸭维甲酸诱导基因I克隆及其结构域功能分析

【目的】克隆鸭维甲酸诱导基因I（retinoic acid inducible gene I,RIG-I）,分析其不同结构域的功能。【方法】根据GenBank上公布的鸭RIG-I序列设计引物,利用RT-PCR克隆鸭RIG-I基因CDS（coding sequence）区,根据保守结构域预测结果,构建携带6*his组氨酸标签的不同结构域缺失突变体的真核表载体（RIG-I-Full、RIG-I-N和RIG-I-C）,转染鸡胚成纤维细胞DF1,经RT-PCR、间接免疫荧光方法鉴定重组质粒在细胞中转录与表达;同时,利用RT-qPCR检测RLR抗病毒信号通路中的IFN-β、Mx1和PKR等下游基因的表达变化。【结果】鸭RIG-I基因CDS区全长2 802 bp,共编码933个氨基酸;不同结构域缺失突变体的真核表载体转染DF1细胞后,重组蛋白均在DF1细胞中表达;RT-qPCR结果显示,N端能显著激活RLR通路上IFN-β、Mx1及PKR基因的表达上调。【结论】 duRIG-I及不同区段均能在DF1细胞中表达,其中N端在调节RLR抗病毒信号通路下游基因的表达过程中发挥了重要作用。     

Human beta-globin gene sequences injected into mouse eggs, retained in adults, and transmitted to progeny

Foreign gene sequences were retained in two adult mice (out of 62 analyzed) from fertilized eggs injected with a recombinant plasmid containing the human beta-globin genomic region and the herpes simplex viral thymidine kinase gene. The intact human and viral genes were found in DNA of one of the animals and, in the other, at least part of the human globin gene was present. The latter individual transmitted these sequences to its progeny in a Mendelian ration. Thus, human DNA may be incorporated into the germ line of mice for in vivo studies of regulation of gene expression in development, genetic diseases, and malignancy.     

牦牛2个α-珠蛋白基因在染色体上排列顺序的确定

本试验扩增并克隆测定了牦牛2个α-珠蛋白基因间区域的序列.根据所测结果及人、鼠、山羊、马和黄牛的α-珠蛋白基因序列比对中的保守区设计了2对特异性引物,扩增并克隆测定了牦牛α-珠蛋白的上、下游基因序列.将测定的牦牛α-珠蛋白基因间区域序列进行同源性搜索,发现没有任何一个基因与它同源.上、下游基因的测序结果表明,上游基因为牦牛α1-珠蛋白基因,下游基因为α2-珠蛋白基因.这表明牦牛的2个α-珠蛋白基因在染色体上紧密排列,其中α1-珠蛋白基因在前,α2-珠蛋白基因在后.     

番茄脯氨酸脱氢酶基因的克隆与RNAi植物表达载体的构建

[目的]克隆了脯氨酸脱氢酶ProDH基因的全长cDNA,构建了ProDH基因的RNA干扰植物表达载体,并转化到农杆菌.[方法]以“耐运2000番茄”幼苗为试材,根据GenBan中公布的番茄脯氨酸脱氢酶ProDH基因的序列信息设计了一对特异性引物,克隆了该基因的全长cDNA,分析基因序列选择正反向片段并扩增,并通过酶切、连接的方法构建了ProDH基因的RNA干扰植物表达载体PBI121-PDHi利用冻融法将表达载体转化到农杆菌EHA105中.[结果]所克隆到的ProDH基因片段长2 001 bp,其中CDS为1 491 bp,编码380个氨基酸.测序结果与公布序列同源性100％,因此可以用来构建干扰载体;通过酶切与测序鉴定,证明表达载体构建成功.[结论]经特异性引物扩增检测,证明表达载体已转入农杆菌,为进一步的研究该基因奠定了基础.