显微注射法制备转基因小鼠模型的研究

将外源基因ＭＴ０ＬＭＰ,ｐＺｉｐＮｅｏＳＶ（Ｘ１）－ＬＭＰ及ＨＬＡ－ＤＲα分别注入昆明小鼠受精卵雄原核中,结果得到有这三种外源基因整合的三个转基因小鼠系,导入基因的整合率分别为８％,８０％骸６６．７％,它们的子一代及子二代（Ｆ２）基因中也均有外源基因整合,并且导入ｐＺｉｐ－ＮｅｏＳＶ９Ｘ１）－ＬＭＰ基因后经反转录聚合酶链反应,检测ｍＲＮＡ发现有该基因的表达,说明本法制备转基因小鼠是可行的。应用荧光     

人DAF基因在转基因小鼠中的遗传与表达

为研究人ＤＡＦ基因在小鼠体内遗传与表达的规律,从质粒ｐＳＦＦＶ－ＤＡＦ分离出一段包含人ＤＡＦ基因的ＤＮＡ片段。采用受精卵显微注射法建立转人ＤＡＦ基因小鼠。提取出生小鼠的染色体ＤＮＡ,经Ｄｏｔ－ｂｌｏｔ与Ｓｏｕｔｈｅｒｎ－ｂｌｏｔ杂交相结合确定首建转基因小鼠,并经Ｄｏｔ－ｂｌｏｔ杂交研究人ＤＡＦ基因在转基因小鼠体内的遗传特征,Ｎｏｒｔｈｅｒｎ杂交确定其表达情况。小鼠受精卵经基因导入后,共生出２４只小鼠,其中４只被确定为首建转基因小鼠,整合率为１５％,在首建转基因小鼠两两交配生出的Ｆ１代小鼠中分别有７０％和７５％继续携带人ＤＡＦ基因。首建转基因小鼠中有１只小鼠在ＲＮＡ水平表达了人ＤＡＦ基因。可见,人ＤＡＦ基因整合入小鼠基因组中,并能够稳定遗传及表达。     

胚胎发育早期转基因的PCR检测研究

转基因在受精卵中的整合时间对于转基因动物的建立十分重要。采用ＷＡＰ基因调控序列指导的人Ｇ－ＣＳＦ基因为构件,对小鼠受精卵进行显微注射。对培养至１细胞期、２细胞期和８细胞期的胚胎进行ＰＣＲ检测。结果表明,三个时期转基因的检出率分别为１００％、７７．７７％和４４．４４％。说明随着培养时间的增加,转基因逐渐丢失。     

组织纤溶酶原激活剂突变体乳腺表达载体构建及在转基因鼠中表 …

通过ＰＣＲ方法从羊肝总ＤＮＡ中获得了羊－β乳球蛋白（ＢＬＧ）基因第一和第二内含子,以羊β－乳球蛋白基因（ＢＬＧ）５’区５ｋｂ为调控序列,构建了乳腺表达组织纤溶酶原激活剂突变体载体。对５４０枚小鼠受精卵进行显微注射,经ＰＣＲ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ检测,获得６只整合有人Ｌａ－ｔＰＡ的转基因小鼠,整合率为３２％。同时研究了转基因在小鼠体内的表达。Ｎｏｒｔｈｅｒｎ ｂｌｏｔ分析表明,在一些转基因鼠乳     

RT-PCR测定组织纤溶酶原激活剂突变体(La-tPA)在转基因鼠乳腺表达的研究

利用所构建的乳腺表达组织纤溶酶原激活剂突变体（Ｌａ－ｔＰＡ）载体．对５４０枚小鼠受精卵进行显微注射,经ＰＣＰ和Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,获得６只整合有人Ｌａ－ｔＰＡ转基因小鼠．为了精确研究转基因在小鼠体内的表达,采用ＲＴ－ＰＣＰ方法测定转基因鼠在泌乳期１～２０ｄＬａ－ｔＰＡ基因的转录,结果表明,在转基因鼠乳腺的Ｌａ－ｔＰＡｍＲＮＡ水平在１０～１５ｄ最高,在２０ｄ时降为最低．外源基因在转基因小鼠乳腺的表达规律研究为未来利用转基因动物生产Ｌａ－ｔＰＡ提供依据     

组织纤溶酶原激活剂突变体(La-tPA)转基因鼠的建立

用羊β－乳球蛋白基因（ＢＬＧ）５区５×１０３ｂ（１０３ｂ,旧称ｋｂ）为调控序列,构建了乳腺表达组织纤溶酶原激活剂突变体（Ｌａ－ｔＰＡ）载体．对５４０枚小鼠受精卵进行显微注射,经ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,获得６只整合有人Ｌａ－ｔＰＡ的转基因小鼠,整合率为３２％．这为未来利用转基因动物生产Ｌａ－ｔＰＡ提供依据     

组织纤溶酶原激活剂突变体（La—tPA）转基因鼠的建立

用羊β－乳球蛋白基因（ＢＬＧ）５’区５＊１０＾３ｂ（１０＾３ｂ,旧称ｋｂ）为调序列,构建了乳腺表达组织纤溶酶原激活剂突变体载体。对５４０枚小鼠受精卵进行显微注射,经ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,获得６只整合有人Ｌａ－ｔＰＡ的转基因小鼠,整合率为３２％。     

猪生长激素基因在金鱼中的整合、表达与生物活性(英文)

自Ｐａｌｍｉｔｅｒ于１９８２年首次将大鼠生长激素基因导入小鼠受精卵,培育出“超级鼠”以来,转基因动物技术获得迅速发展。本文以低等脊锥动物为实验动物,探讨猪生长激素基因导入金鱼受精卵后的整合与表达、生物学效应及后代遗传等问题,为进一步研究外源基因在高等脊椎动物（包括猪）内整合与表达的调控机理提供方法上的参考。实验结果表明：采用显微注射方法,将羊金属硫蛋白基因启动子与猪生长激素基因重组的线型ＤＮＡ（Ｆｉｇ．１）片段导入金鱼受精卵中,获得成活实验鱼,经斑点（Ｆｉｇ．２）,Ｓｏｕｔｈｅｒｎ杂交（Ｆｉｇ．３）,筛选ＰＧＨ阳性的转基因鱼作为亲本交配,分别得到Ｆ１代和Ｆ２代。经斑点、ＰＣＲ－Ｓｏｕｔｈｅｒｎ分析（Ｆｉｇｓ．４,５＆６）及放射免疫检测（Ｔａｂ．１）,表明外源基因在部分受体鱼中得到整合和表达,并能通过有性繁殖传递给后代,且仍具生长效应（Ｆｉｇ．７;Ｔａｂ．２）。本实验获得的转基因阳性金鱼数量有限,且只传了两代,似乎不足以说明转基因金鱼后代表观特征的遗传稳定,但转基因金鱼Ｆ１代中存在外源基因整合位点纯合的个体是可能的。这为建立转基因动物纯系奠定了基础。     

庚型肝炎病毒转基因小鼠的建立

利用受精卵原核显微注射的方法,产生了含有ＨＧＶ结构蛋白Ｃ、Ｅ１、Ｅ２及部分非结构蛋白ＮＳ２、ＮＳ３的转基因小鼠。得到１０只ｆｏｕｍｄｅｒ小鼠,其中有３只ｆｏｕｎｄｅｒ小鼠与正常小鼠交配后得到了整合有外源基因的Ｆ１代阳性小鼠。ＲＴ－ＰＣＲ的结果显示,外源基因可在ｆｏｕｎｄｅｒ小鼠及Ｆ１代小鼠的血液有核细胞及肝细胞内转录;组织病理学检查显示,某些转基因小鼠的肝细胞出现了水样变、脂肪变性及轻微炎性反应等     

小鼠细小病毒非结构基因转基因小鼠的建立

为探讨小鼠细小病毒（ＭＶＭ）非结构蛋白在ＭＶＭ感染中的抗肿瘤作用,酶切表达质粒ｐＵＬＢ３２３８获取该非结构基因,通过显微注射法接种入小鼠受精卵内制备转基因鼠。共注射受精卵７２０枚,选取存活受精卵２２５枚植入假孕小鼠输卵管,产仔１４只。转基因小鼠尾部组织ＰＣＲ法ＤＮＡ检测证明,其中４只整合靶基因。整合转基因的４只Ｇ０代小鼠与正常Ｃ５７ＢＬ／５Ｊ小鼠配种均可生产整合靶基因的小鼠。ＲＴ－ＰＣＲ法ｍＲＮＡ     

Exploring transgene transfer from the transgenic chicken model to its offspring through a nonviral vector

There is much interest in using chickens as “bioreactors” to produce large quantities of biopharmaceuticals. However, transient expression of foreign genes have been known to cause low efficiency of obtaining transgenic offspring, especially when using nonviral vectors. In present study, a transgenic chicken model was investigated to determine whether an exogenous gene can be expressed stably and transferred to its offspring through a matrix attachment region (MAR)-mediated non-viral vector using the eGFP marker gene. The eukaryotic expression vector pEGFP-N1-MAR, which contains the eGFP gene and MAR, was constructed and transfected into a chicken stage-X blastoderm to produce a G0 generation of transgenic chickens. The hatchabilities of different injection regions were tested; 18 of the 40 eggs injected with pEGFPN1- MAR in the area opaca hatched after 21 days of incubation, and had a hatchability rate of 45%. By contrast, eggs injected at the area pellucida did not hatch. Results from the fluorescence signal detection and polymerase chain reaction (PCR) verified that four hatched chicks from the G0 generation expressed the eGFP gene. Furthermore, fluorescence signal detection results indicated that 2 of the 65 chicks from the G1 generation expressed the eGFP gene. We conclude that MAR facilitates the production of transgenic chickens; pEGFP-N1-MAR application is a novel approach that can produce transgenic chicken offspring.     

复制型HBV转基因小鼠遗传稳定性研究

目的：提高复制型HBV转基因小鼠的遗传稳定性。方法：应用回交传代及双杂交育种法,经荧光定量PCR、ELISA和化学发光法研究HBV基因在小鼠体内的复制与表达。结果：HBV转基因小鼠已稳定传至第23代,血清HBsAg达4122.31±2044.74IU/ml,93.93%的转基因小鼠血清HBV DNA达104-106copies/ml,表达复制水平较早期有显著提高并稳定传代;雌雄小鼠之间表达水平无显著性差异。结论：该转基因小鼠经过培育传代,已成为一个高表达且遗传稳定的复制型HBV小鼠模型。     

HBsAg/GM-CSF融合蛋白在毕赤酵母中的表达及其免疫原性研究

为探索一种提高乙肝病毒表面抗原免疫原性的新方法,用PCR和基因重组技术构建HBsAg与GM-CSF的融合基因,并在毕赤酵母中分泌表达HBsAg/GM-CSF(S-GM)融合蛋白。表达产物用SDS-PAGE检测,W estern b lot分析,离子交换柱纯化后免疫昆明鼠,ELISA检测免疫小鼠血清中抗HBsAg的抗体水平。结果显示S-GM融合蛋白在毕赤酵母中获得了表达,离子交换柱一步纯化即可得到纯度达90%以上的S-GM。W estern b lot分析S-GM可分别与抗HBsAg及抗GM-CSF的抗体特异结合。ELISA检测发现第一次免疫后4w出现抗HBsAg的抗体,加强免疫后融合蛋白组几乎全部阳转,且抗体水平较HBsAg组(P=0.009<0.05)及HBsAg和GM-CSF的混合物组(P=0.032<0.05)高。HBsAg/GM-CSF融合蛋白能够在毕赤酵母中表达,且可增强HBsAg的免疫原性,为提高乙肝疫苗的免疫效果提供了新的思路与方法。     

Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19–20 h after hCG injection. About 5 μg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3′ flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 μg/mL rhFVIII in milk, with an activity of 0.594 lU/mL. The DM founder female produced 118 μg/mL rhFVIII, with activity values of 18 lU/mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.     

Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen

5S is a mouse monoclonal IgG1 that binds to the ‘a’ epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K_D = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.     

High Efficiency Production of germ-line Transgenic Japanese Medaka (Oryzias latipes) by Electroporation with Direct Current-shifted Radio Frequency Pulses

Although there have been several studies showing the production of transgenic fish through electroporation techniques, success rates have been low and few studies show germ-line integration and expression. When electroporation has been successful, the device used is no longer commercially available. The goal of this experiment was to find an alternative efficient method of generating transgenic Japanese medaka (Oryzias latipes) using a commercially available electroporation device. The Gene Pulser II and RF module (Bio-Rad Laboratories, USA), along with two reporter gene constructs, were used. In contrast to other electroporation devices, which are based on a single pulse with exponential decay or square wave technology, the Gene Pulser II incorporates a direct current (DC)-shifted radio frequency (RF) signal. With this technique, over 1000 embryos can be electroporated in less than 30 min. The plasmid pCMV-SPORT--gal (Invitrogen, USA) was used in the supercoiled form to optimize parameters for gene transfer into single-celled embryos, and resulted in up to 100% somatic gene transfer. Similar conditions were used to generate fish transgenic for both the pCMV-EGFP plasmid (Clontech, USA) and a cytomegalovirus (CMV) driven phytase-EGFP construct. The conditions used were a voltage of 25 V, a percent modulation of 100%, a radio frequency of 35 kHz, a burst duration of 10 ms, 3 bursts, and a burst interval of 1.0 s. Seventy percent of the embryos electroporated with the pCMV-EGFP construct survived to sexual maturity, and of those, 85% were capable of passing the transgene on to their offspring. Transgenic second generation back-crossed (BC₂) fry were subjected to Southern blot analysis, which confirmed germ-line integration, and observation for green fluorescence protein, which confirmed protein expression. DC-shifted RF pulses are effective and efficient in the production of transgenic medaka, and germ-line integration and expression can be achieved without linearization of the transgene vector.     

角质细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有角质细胞特异性角质素5启动子、Cre重组酶基因和人生长激素基因plyA的转基因载体pK5-Cre-hGH。以显微注射的方法将4．2kb的转基因片段K5-Cre-hGH引入小鼠基因组,共注射720枚受精卵,其中龄5枚移植至29只假孕母鼠的输卵管中发育,获得子代小鼠48只,经基因型鉴定有12只小鼠在其基因组上整合有Cre基因,整合率为25％。将带有cre重组酶基因的小鼠与基因组上携带loxP位点的smad4条件基因打靶小鼠杂交以检测Cre重组酶组织特异性表达情况以及介导重组的功能。结果表明,K5-Cre转基因小鼠只在皮肤组织中表达Cre重组酶并能在体内成功地介导loxP位点的重组。     

成骨细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有骨钙素基因启动子、Cre重组酶基因和人生长激素基因polyA的转基因载体pOC-Cre, 以显微注射的方法将4.6 kb的转基因片段OC-Cre导入小鼠受精卵。16只子代小鼠中经PCR和Southern杂交鉴定, 有2只小鼠携带外源基因, 整合率为12.5%。为了检测OC-Cre在转基因小鼠中表达的组织特异性, 将转基因首建者小鼠与基因组上携带有LoxP位点的条件性Smad4基因敲除小鼠交配, PCR结果显示, 仅在子代纯合型小鼠骨组织基因组中扩增出了Cre介导重组后的片段。将OC-Cre转基因小鼠与ROSA26报告小鼠交配, 利用LacZ染色对双转基因阳性子代小鼠进行检测, 结果显示Cre重组酶在成骨细胞中特异性表达并介导ROSA基因座LoxP位点间的重组。所有这些结果说明：所建立的OC-Cre转基因小鼠在成骨细胞中特异性表达Cre重组酶, 并能在体内介导成骨细胞基因组上LoxP位点间的重组, 是一种理想的研制成骨细胞特异性基因敲除小鼠的工具小鼠。     

Concomitant cellular expression of heat shock regulated genes of heapatitis B virus surface antigen and of human growth hormone by a NIH-3T3 cell line

A plasmid carrying a DNA fragment of hepatitis B virus, coding for the pre-S2 and the entire S region of the surface antigen (HBsAg), placed under the control of the promoter of the human 70 kDa heat shock protein gene (hsp70) was introduced into Line 6, a recombinant cell line that was selectedfromNIH-3T3 cellspreviously transfected with a similar construct coding for the human growth hormone cDNA gene (chGH) and with the plasmid pEJ carrying the Ha-ras^EJ activated cellular oncogene. The resulting cell line, EMS8, expressed: (1) hsp70/HBsAg and hsp70/hGH hybrid genes, (2) the human Ha-ras^EJ oncogene, and (3) the neomycin resistance gene, the two last plasmid markers being used for cell selection. EMS8 cells were able to carry outpost-translational modifications of the middle M and the major S envelope proteins of HBV, such as assembly and glycosylation. Accordingly, the cells synthesized and secreted both free and glycosylated M and S viralproteins, and the human growth hormone protein. In addition concomitant expression of HBsAg and hGH proteins as well as their mRNA were detected in EMS8 cells at least up to 72 hr after heat induction instead of 24 hr in the case of hGH in line 6 cells.     

Production of transgenic mice expressing the goat H-FABP gene by intratesticular injection

The objective of this study was to explore the possibility of obtaining stable transgenic animals by intratesticular injection. The recombinant vector pEGFP-H-FABP expressing the goat heart-type fatty acid binding protein and green fluorescent protein was mixed with liposome complexes and randomly injected into the testes of mice. Testicular section, fluorescence, and DNA detection assays of mouse sperm were performed to determine the integration of foreign DNA. The results showed that foreign DNA was successfully expressed in the treated mice. Furthermore, the expression and function of the foreign gene were analyzed in F₁ generation and F₂ generation mice at different levels, with the positive rates of foreign gene transfer into the F₁ and F₂ generations being 4.0 and 30.23 %, respectively. These results strongly support testicular injection as an effective method of producing transgenic animals and indicate that foreign genes can be stably passed on to the offspring. This research has theoretical and practical implications for the improvement in the quality of laboratory animals and for gene therapy.