Genetic transformation of Drosophila with transposable element vectors

Exogenous DNA sequences were introduced into the Drosophila germ line. A rosy transposon (ry1), constructed by inserting a chromosomal DNA fragment containing the wild-type rosy gene into a P transposable element, transformed germ line cells in 20 to 50 percent of the injected rosy mutant embryos. Transformants contained one or two copies of chromosomally integrated, intact ry1 that were stably inherited in subsequent generations. These transformed flies had wild-type eye color indicating that the visible genetic defect in the host strain could be fully and permanently corrected by the transferred gene. To demonstrate the generality of this approach, a DNA segment that does not confer a recognizable phenotype on recipients was also transferred into germ line chromosomes.     

Transposition of cloned P elements into Drosophila germ line chromosomes

Recombinant DNA carrying the 3-kilobase transposable element was injected into Drosophila embryos of a strain that lacked such elements. Under optimum conditions, half of the surviving embryos showed evidence of P element-induced mutations in a fraction of their progeny. Direct analysis of the DNA of strains derived from such flies showed them to contain from one to five intact 3-kilobase P elements located at a wide variety of chromosomal sites. DNA sequences located outside the P element on the injected DNA were not transferred. Thus P elements can efficiently and selectively transpose from extrachromosomal DNA to the DNA of germ line chromosomes in Drosophila embryos. These observations provide the basis for efficient DNA-mediated gene transfer in Drosophila.     

Introduction of human DNA into mouse eggs by injection of dissected chromosome fragments

A procedure has been developed for introducing exogenous DNA into mouse eggs by injection of chromosome fragments. Chromosome fragments were dissected from human metaphase spreads and microinjected into the pronuclei of fertilized mouse eggs. Many of the injected eggs subsequently exhibited normal pre- and postimplantation development. Embryos obtained from eggs injected with centromeric fragments retained human centromeric DNA as demonstrated by in situ hybridization analysis. From eggs injected with noncentromeric fragments, a mouse was obtained whose tail tissue exhibited the presence of human DNA. This procedure should facilitate incorporation of very large (more than 10 megabases) DNA fragments into cells and embryos without the need for cloned sequences.     

Integration and stable germ line transmission of genes injected into mouse pronuclei

Genetic material has been successfully transferred into the genomes of newborn mice by injection of that material into pronuclei of fertilized eggs. Initial results indicated two patterns of processing the injected DNA: one in which the material was not integrated into the host genome, and another in which the injected genes became associated with high molecular weight DNA. These patterns are maintained through further development to adulthood. The evidence presented indicates the covalent association of injected DNA with host sequences, and transmission of such linked sequences in a Mendelian distribution to two succeeding generations of progeny.     

利用水平微注射技术制备转基因兔的研究（英文）

利用常规微注射系统进行原核胚转基因时,由于其透光性能较差,操作不便等原因,使外源基因导入效率很低,另外其开放构造也易造成污染。为了解决这些问题,本研究有针对性地设计了新型的水平微注射系统,利用此系统,可明显缩短常规注射原核胚的平均时间,注射后原核胚的分裂率、8～16细胞胚胎发育率也明显提高。此外,利用该系统还可在低倍镜下（×100）将DNA常规注射量减半注射,提高了原核胚注射后的成活率,其注射胚的外源基因整合率亦明显提高。采用水平微注射系统,以pMT-hIGF-1为目的基因,在移植产下的24只仔兔中,有5只经Southern blot检测,证明为整合外源基因的转基因兔。     

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.     

转大豆铁结合蛋白基因水稻的初步研究

采用冻融法将含有大豆铁结合基因的质粒pGTV-35S-fer转入农杆菌菌株LBA4404中,以宁夏4个水稻主栽品种为受体材料,通过农杆菌介导法转化水稻愈伤组织,经2轮次除草剂筛选获得了再生植株。经过对转化植株的PCR扩增检测,证明外源大豆铁结合基因已经转入宁夏水稻植株中。大豆铁结合蛋白基因在水稻种子中特异表达,转基因植株种子中的铁含量是未转化植株的1.7~2.2倍。     

Transferring a Gene Expression Cassette Lacking the Vector Backbone Sequences of the 1Ax1 High Molecular Weight Glutenin Subunit into Two Chinese Hexaploid Wheat Genotypes

1Ax1 high molecular weight glutenin subunit （HMW-GS） gene expression cassette （GEC） lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Een 1 and Emai 12 to test the feasibility and the efficiency of explant regeneration, transformation frequency and transgene expression comparing with whole vector transformation by the approaches of plasmid extraction and excision, immature embryo isolation, particle co-bombardment, tissue culture, DNA extraction, PCR amplification, southern hybridization, leaf-painting test and SDS-PAGE etc. No significant difference was shown in tissue culture response of the proportion of embryogenic calli, somatic embryogenesis and regeneration frequency between GEC and whole plasmid bombarded embryos, but both regenerated less well than non-bombarded control. Total 56 plantlets that survived PPT selection had insertion of at least the Bar gene, 18 were from the GEC treatment and 38 from the whole plasmid treatment, the escape ratio averaged 0.23. Six independent transplants f230 - f235 with GEC transformation from genotype Emai 12 presented clear PCR amplification bands of Bar and 1Ax1 gene. The transformation and co-transformation frequency were 3.51 and 100% respectively. PCR amplification using a primer-pair specific for ampicillin resistant gene indicated the existence of Amp^R gene in whole vectors but the removal in GECs and transplants. Southern blot of total DNA and PCR products from transgenic plants of 1Ax1 GEC confirmed the integration of the transgene 1Ax1 and the absence of the EcoR Ⅰ recognition site at both ends of the 1Ax1 GEC when integrated. SDS-PAGE showed the expression of 1Ax1 GEC and un-expression of whole plasmid. The length of integrated fragment, the proportion of the gene of interest （GOI） and the selectable marker （MG）, bombardment pressure and genotypes are vital for the expression of a transformed GEC.     

质粒拯救型T-DNA标签质粒的构建

[目的]为了高效率地获得转基因植物的旁邻序列。[方法]以pUC18及pWM101为出发质粒,通过用限制性内切酶EcoRⅠ和HindⅢ双酶切质粒pUC18,得到含大肠杆菌复制起点及氨苄青霉素抗性基因的pUC18大片断,并将这段DNA整合到pWM101的T-DNA之中构建重组质粒。[结果]该质粒利用根癌农杆菌转化植物后,可利用潮霉素筛选转化细胞,拯救质粒则可转化大肠杆菌后,以氨苄青霉素进行筛选。[结论]该研究为以后的用T-DNA标签研究植物功能奠定了基础。     

利用体细胞核移植技术生产表达绿色荧光蛋白转基因猪胚胎的研究

试验对脂质体转染猪胎儿成纤维细胞的技术程序进行了筛选,以绿色荧光蛋白基因转染后的阳性细胞作为体细胞核移植的核供体,以体外成熟卵母细胞为核受体构建了绿色荧光蛋白转基因克隆猪胚胎,并对重构胚在体外发育情况以及绿色荧光蛋白表达情况进行了跟踪研究。结果显示,采用4.0μg.mL-1脂质体转染试剂,1.6μg.mL-1质粒DNA,转染6 h可以获得最佳转染效果,转染效率达4.48%;GFP转基因体细胞重构胚体外囊胚发育率为10%,GFP阳性胚胎率为48%。结果表明,脂质体转染试剂可以高效转染猪胎儿成纤维细胞,获得的阳性细胞具有支持猪全程发育的潜能。     

鸡卵清蛋白基因调控序列指导人促红细胞生成素转基因鸡的制备

本研究将鸡卵清蛋白5端调控区调控人促红细胞生成素的pOV-hEPO特异表达载体用脂质体包埋，和供体细胞融合后，显微注射入450枚受体胚盘，采用代用蛋壳法培养制备转基因鸡，PCR和斑点杂交检测外源基因已整合入早期鸡胚胎生殖系统，为制备表达人促红细胞生成素的鸡输卵管生物反应器奠定了基础。     

鸡IL-18真核表达质粒与新城疫病毒F基因疫苗联合免疫探讨

为探讨鸡IL-18基因真核表达质粒pcDNA-chIL-18在新城疫病毒(NDV)F基因疫苗免疫中的作用,克隆了新城疫病毒标准株F48E9的F基因,构建F基因重组真核表达质粒pcDNA-F;将11日龄试验鸡分为4组,每组10只,14日龄一免,28日龄二免,分别肌注pcDNA-F、pcDNA-F+pcDNA-ckIL-18、新城疫传统疫苗(14日龄NDV弱毒疫苗首免,28日龄NDV油苗二免)、生理盐水,一免疫后第7、14、21、28 d均采血进行ND抗体(ELISA)检测和T淋巴细胞转化(MTT)试验;第29 d均肌注50LD50NDV强毒F48E9株.结果显示:免疫保护效率,生理盐水组0/10,pcDNA-F免疫组3/10,pcDNA-F+pcDNA-chIL-18免疫组5/10,新城疫传统疫苗免疫组10/10;ND抗体水平,pcDNA-F免疫组与pcDNA.F+pcDNA-chIL-18免疫组无差异(P>0.05),均显著低于传统疫苗免疫组(P<0.05);T细胞转化水平,pcDNA.F+pcDNA-cML-18免疫组明显高于pcDNA-F免疫组(P<0.05),pcDNA-F免疫组与传统疫苗免疫组无差异(P>0.05).试验结果综合显示pcDNA-F免疫试验鸡可产生一定程度的免疫保护;pcDNA-chIL-18对pcD-NA-F具有一定的免疫增强作用.     

导入外源GRF基因质粒对犊牛生长性能的影响

为了研究导入GRF基因质粒对犊牛生长性能的影响,选取36头体重为约102kg西门塔尔杂交牛,随机分成四组每组9头,分别注射含有0mg,3.37mg,6.74mg,10.11mg质粒的5ml生理盐水。在试验过程中,定期进行增重,饲料采食量和生长激素水平的测定。试验结果表明,各处理组均在一定程度上提高了动物的日增重和饲料转化率,其中6.74mg处理组效果最好,日增重比对照组高出17.33%(P<0.01),饲料转化率提高18.05%(P<0.01)。屠宰后进行组织质粒残留检测,未发现有残留。     

鸭多杀性巴氏杆菌外膜蛋白A基因表达及抗原性鉴定

根据已发表的Pm70株的序列(GenBank登录号:AE004439)设计了两对引物,用PCR方法扩增了鸭多杀性巴氏杆菌标准株C48-102的外膜蛋白基因A(ompA),扩增的片段为1062bp。将测序结果与GenBank中多杀性巴氏杆菌P52、PM70、T931317、T94289的ompA序列比对结果表明,核苷酸水平上同源性为89.0%～98.9%;在氨基酸水平上同源性为90.7%～99.2%。全ompA序列在大肠杆菌中干扰蛋白的正常表达,因此截断ompA蛋白的信号肽序列,将去信号肽的ompA基因插入到pPRO-EX-Htb载体上,构建了原核表达载体Htb-ompA,转化BL21,并诱导表达,SDS-PAGE结果表明,表达蛋白约为35kDa,与预期的分子量大小相符。Western-blot结果表明,表达的蛋白具有良好的抗原活性。本研究重组蛋白ompA的获得,为敲除ompA基因多杀性巴氏杆菌突变株的血清学检测奠定基础。     

电穿孔法用于大肠杆菌和苏云金杆菌的转化及质粒消除

利用电脉冲将质粒pHT3101和pHE－4D分别转入了大肠杆菌和苏云金杆菌,结果显示大肠杆菌DNA的转化率（转化子）为104～105·μg－1,苏云金杆菌的DNA的转化率（转化子）为102～103·μg－1．同时利用该法消除了Escherichiacoli菌株TG1和Bt菌株ISP78／11中的pHT3101质粒,消除率分别为88．6％和66．4％．比较了转化和质粒消除的条件并构建了一个工程菌．     

弓形虫自然弱毒株棒状蛋白2基因克隆及序列分析

应用PCR法从弓形虫QHO株速殖子基因组DNA中扩增ROP2的基因片断,连接到pMD18-T载体中,转化大肠杆菌JM109,经酶切及PCR鉴定后进行测序,并进行序列分析.结果表明,自然弱毒株的开放阅读框架(ORF)与ROP2基因已知序列(Z36906)的ORF具有97%的同源性,氨基酸序列具有95%的同源性.     

In vivo imaging of quantum dots encapsulated in phospholipid micelles

Fluorescent semiconductor nanocrystals (quantum dots) have the potential to revolutionize biological imaging, but their use has been limited by difficulties in obtaining nanocrystals that are biocompatible. To address this problem, we encapsulated individual nanocrystals in phospholipid block-copolymer micelles and demonstrated both in vitro and in vivo imaging. When conjugated to DNA, the nanocrystal-micelles acted as in vitro fluorescent probes to hybridize to specific complementary sequences. Moreover, when injected into Xenopus embryos, the nanocrystal-micelles were stable, nontoxic (<5 x 10(9) nanocrystals per cell), cell autonomous, and slow to photobleach. Nanocrystal fluorescence could be followed to the tadpole stage, allowing lineage-tracing experiments in embryogenesis.     

伪狂犬病病毒gG基因缺失通用转移载体的构建

对克隆有伪狂犬病病毒Ea株基因组DNA SphI 15kb片段的质粒pBSA用SphI和KpnI消化，将含gG全基因及其上游PK基因，下游gD基因部分编码区的约2．6kb的片段亚克隆到消除了EcoRI位点的载体pUC19中，获得重组质粒pUSK(E)。以pEGFP-N1为模板，通过PCR扩增约0．3kb的SV40Poly(A)片段，并将其克隆到杆状病毒转座载体pFastBac1的NotI和PstI位点，利用pFastBac1的BamHI和PstI将含有9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区约9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区的400bp的缺失，构建了由gG启动子驱动gG部分编码区缺失的通用转移载体pgG-Uni。序列分析进一步证实：有7个单一酶切位点可供外源基因直接插入。上述结果为构建以伪狂犬病病毒作载体的多价基因工程疫苗以及利用标记蛋白探讨伪狂犬病病毒在体内的增殖与分布奠定了基础。     

基于酿酒酵母的多片段质粒的构建

为避免现有的多片段质粒构建技术的弊端,提高多片段质粒构建效率,以构建树干毕赤酵母Agglutinin-like基因的敲除载体为例,利用酿酒酵母活体细胞重组系统,一次性将多个外源DNA片段和线性化质粒重组,形成环状质粒。通过引物设计在相互连接的DNA片段之间引入50bp重叠序列,用PCR扩增DNA片段后,与线性化质粒一起转化酿酒酵母,再用PCR和测序等方法鉴定阳性酵母菌落中质粒的正确性。通过本方法构建的多片段质粒正确率高、耗时短。     

Immune Responses in Mice Injected with gD Plasmid DNA of Infectious Bovine Rhinotracheitis Virus

The gene encoding gD of isolate Luojing of infectious bovine rhinotracheitis virus (IBRV)was amplified,sequenced, and cloned into plasmid pcDNA3.1, resulting in a recombinant pcDNA-gD. Groups of BALB/c mice were injected with 100μg of plasmid only or together with liposome. After immunization, serum samples were collected from mice every 2 weeks for a 10-week period and tested for protein-specific antibody with enzyme-linked immunosorbent assay (ELISA). It was showed that the plasmid encoding IBRV glycopretein D developed gene-specific antibody. This report indicates the potential of DNA injection as a method of vaccination.