全鱼基因的构建及其在鲫鱼体内的整合与转录

利用PCR技术删除大麻哈鱼生长激素基因的启动序列,通过基因重组构建出全鱼基因（鲤鱼MT启动子－大麻哈鱼生长激素基因）;以融合全鱼基因为外源基因,通过显微注射方法将其线性片段导入鲫鱼受精卵内,研究其整合与转录效率。结果表明,全鱼基因在鲫鱼基因组中的整合率为36．4％（16／44）,对转基因阳性鱼的RNA样本进行Northern印迹杂交检测,转录率为25％（1／4）。因此,该全鱼基因可以作为转基因鱼研究和应用的外源基因。     

Transplantation of the human insulin gene into fertilized mouse eggs

A circular recombinant plasmid composed of a 12.5 kb fragment of human DNA including the entire insulin gene and the 4.3 kb bacterial plasmid pBR322 was microinjected into fertilized C57BL/6 mouse eggs. 753 eggs were injected with 30000 gene copies in a volume of 1-2 pl; 379 eggs survived micromanipulation and were subsequently cultured to the blastocyst stage. From 282 embryos that were transferred into the uteri of pseudopregnant ICR/Swiss foster females, 60 fetuses and corresponding placentas could be recovered at day 16-19 of pregnancy. High molecular weight DNA was extracted from these tissues and was screened with radioactively labelled hybridization probes for the presence of the injected DNA sequences. By restriction endonuclease analysis in conjunction with Southern blot hybridization, we found that in two normally developed fetuses at day 18, the fetal and placental tissues contained the human insulin gene including the flanking regions and bacterial plasmid sequences. Our results indicate that the injected DNA integrated into the mouse genome within its pBR322 region and properly replicated with the host DNA during development. The intensities of the hybridization bands suggest that at least one copy of foreign plasmid DNA was present per cell in the two fetuses and their placentas.     

外源猪生长激素基因在金鱼体内的整合与表达研究

自1982年Palmiter^[1]等人给小鼠受精卵雄原核注射大鼠生长激素基因培养成功“超级”鼠以来,由于人们认识到转基因技术导致动植物品种定向改良以及利用转基因动物作为生物反应器,大量合成和分泌贵重而安全的基因工程产品,因此转基因动物技术得到了迅速发展,相继开展了兔、鱼、猪、鸡、牛、羊等动物的转基因研究,并取得了一定的结果^[2,3,4]。但是在上述具有经济价值的高等脊椎动物中从事转基因研究,成本高、操作困难,而金鱼属于低等脊椎动物．具有产卵多、易获得同步卵、可控制体外受精和体外发育等特点,因而成为转基因动物研究的方便材料。生长激素是动物脑下垂体前叶分泌的单链多肽⑸,生理功能是促进碳水化合物代谢及核酸、蛋白质合成,整体效应表现为动物生长。本文以金鱼为实验材料,探讨猪生长激素基因导入其受精卵后整合、表达以及生物效应等问题,为进一步研究外源基因在高等动物内整合和表达调控机理提供参考。     

Stimulation and inhibition of the growth of mice carrying the human growth hormone gene

The human growth hormone gene, containing mouse metallothionein gene promotor, was injected into the male pronuclei of fertilized mouse ova. The progeny of transgenic mice included animals with both accelerated and inhibited growth. Radioimmunochemical analysis has revealed human growth hormone synthesis in both groups of transgenic mice. The molecular weight of the hormone synthesized in liver cells was 25,000 daltons. A possible mechanisms of foreign hormone effect on the growth of transgenic mice is discussed.     

Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (Oryzias latipes).

Gene constructs consisting of human growth hormone (hGH) gene driven by promoter/regulatory sequence of mouse metallothionein (mMT), viral thymidine kinase (vTK), rat cholecystokinin (rCCK), or chicken beta-actin (cBA) gene were injected into the cytoplasm of fertilized medaka eggs via the micropyle. More than 49% of the injected embryos survived at hatching. Up to 26% of the survivors showed integration of the introduced gene construct, as determined by polymerase chain reaction analysis and subsequent confirmation by Southern blot hybridization of the genomic DNA. A significant fraction of F1 progeny, derived from crosses between transgenic founders and the nontransgenic individuals, inherited the transgene. Expression of hGH gene was also observed in some of the P1 founders and F1 transgenic progeny carrying mMT-hCG or cBA-hGH gene. Furthermore, the growth performance of the P1 mMT-hGH and cBA-hGH transgenic founders and F1 cBA-hGH F1 transgenic progeny was significantly greater than their full sibling, nontransgenic individuals. In addition to the microinjection experiment, a gene construct containing the long-terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) and rainbow trout (rt) GH2 cDNA was introduced into embryos of medaka by electroporation using an exponential decay electroporator. Approximately 70% of the electroporated embryos survived at hatching, and 20% of the survived individuals integrated RSVLTR-rtGH2 cDNA into their genomes. These two techniques will greatly enhance the ability to study regulation of gene expression in transgenic animals during differentiation and development.     

New approach to cell lineage analysis in mammals using the Cre-loxP system

The Cre-loxP site-specific recombination system was used for cell lineage analysis in mammals. We constructed an expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a portion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E. coli beta-galactosidase (beta-gal). When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for beta-gal. When both circular plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity. This indicates that transient expression of the Cre recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing two-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four-cell stage. When the developing four-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for beta-gal activity was observed in most (77.8%, 7/9) embryos. These findings were further confirmed using two-cell embryos derived from a transgenic mouse line carrying CETZ-17 transgene. Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals.     

Simultaneous synthesis of human-, mouse- and chimeric epidermal growth factor genes via ''hybrid gene synthesis'' approach.

Simultaneous synthesis of two DNA duplexes encoding human and mouse epidermal growth factors (EGF) was accomplished in a single step. A 174 b.p. DNA heteroduplex, with 16 single and double base pair mismatches, was designed. One strand encoded the human EGF, and the opposite strand indirectly encoded the mouse EGF. The heteroduplex DNA was synthesized by ligation of seven overlapping oligodeoxyribonucleotides with a linearized plasmid. After transformation in E. coli HB101 (recA 13), the resulting heteroduplex plasmid served as the template in plasmid replication. Two different plasmid progenies bearing either the human or mouse EGF-coding sequence were identified by colony hybridization using the appropriate probes. However, in E. coli JM103, the same process yielded plasmid progenies encoding different chimeric EGF molecules, presumably due to crossover of human and mouse EGF gene sequences.     

High level expression of human tissue-type plasminogen activator gene in mouse C127 cell.

We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.     

Transgenic mice containing growth hormone fusion genes

New genes composed of the mouse metallothionein I promoter-regulator and the rat or human growth hormone structural gene have been introduced into mice. A large proportion of mice containing these genes express them, and some animals grow to be up to twice as large as controls. The technique has been used in part to correct the genetic defect that results in the small stature of the mutant little mouse.     

猪生长激素基因表达质粒的构建及其转基因动物研究

将猪生长激素基因（ＰＧＨ）克隆到质粒ｐＵＣ１９上,经酶切分析,确定其酶切图谱．把ＰＧＨ转录起始位点以前的序列切掉,换上羊ＭＴ－１ａ基因的启动子,构建成可以调控的表达载体ｐＳＭＴＰＧＨ,用于转基因动物的研究．采用微注射法将线状ｐＳＭＴＰＧＨ导入猪、鼠和金鱼的受精卵中,得到了相应的转基因动物．对这些动物鉴定分析表明,外源基因整合率因动物不同而异,但该基因在这３种动物中的整合率均在９％以上,其生长速度都高于对照组．     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

Electroporation as a new technique for producing transgenic fish

A recombinant plasmid, pMV-GH, containing rainbow trout growth hormone cDNA fused to mouse metallothionein I promoter, was introduced into medaka (Oryzias latipes) by electroporation. Of 3109 fertilized eggs treated with electric pulses (750 V/cm, 50 μs, 5 times), 783 (25%) hatched out. Four percent of the hatchlings were transgenic. To obtain transgenic lines, 180 hatchlings were maintained and 35 of them grew into adult fish. Two of these fish were transgenic. When one transgenic fish was mated with a normal female, the transgene was found in all the F1 offspring assayed. In F2 offspring obtained by mating transgenic F1 fish, 88% were transgenic.     

Cloning of bovine growth hormone gene and its expression in bacteria.

A hybrid plasmid was constructed containing beta-lactamase gene of plasmid pBR322 and cloned coding sequences of bovine growth hormone (BGH). The constructed plasmid contains all DNA sequences required to encode BGH, and when used as a hybridization probe it detects one growth hormone gene in the bovine genome. The cloned DNA sequences are inserted into the beta-lactamase gene in the correct reading frame for BGH synthesis. The hybrid gene is expressed in bacteria and the product, a fused beta-lactamase-bovine growth hormone protein, is specifically immunoprecipitated with anti-serum to BGH. Unlike beta-lactamase, very little growth hormone containing sequences can be detected in the periplasmic space.     

Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO4-polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, we developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. In this case, a DNA fragment coding for the natural signal sequence, the hTF/2N lobe, and the two stop codons was cloned into the expression vector pNUT, such that the expression of hTF/2N was controlled by the mouse metallothionein promoter and the human growth hormone termination sequences. Baby hamster kidney cells containing this hTF/2N-pNUT plasmid secreted up to 20 mg of recombinant hTF/2N per liter of tissue culture medium. Recombinant hTF/2N was purified from the medium by successive chromatography steps on DEAE-Sephacel, Sephadex G-75, and FPLC on Polyanion SI. The purified protein was characterized by NaDodSO4-PAGE, urea-PAGE, amino-terminal sequence analysis, UV-visible spectroscopy, iron-binding titration, and proton NMR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice

Mature mouse sperm cells incubated in an isotonic buffer with cloned DNA capture DNA molecules over a 15 min period. Spermatozoa incubated with pSV2CAT plasmid in either circular or linear form were used to fertilize mouse eggs in vitro. Sequences complementary to pSV2CAT were identified in approximately 30% of 250 progeny by Southern blotting. A genomic library was constructed from the DNA of a positive mouse. Three positive clones were identified and two adjacent HincII restriction fragments of 240 and 370 bp showed identical sequences to the corresponding fragments of the pSV2CAT plasmid. F1 progeny showed paternal and maternal transmission of the transgenes from founders. CAT gene expression was detected on tissues of adult F1 individuals, preferentially on tails and muscle. We conclude that transgenic mice can be obtained using sperm cells as foreign DNA vectors.