Rescue of a tk-plasmid from transgenic mice reveals its episomal transmission

Summary This communication demonstrates the usefulness of the plamid rescue procedure for recovery of plasmids from transgenic mice. We have microinjected the plasmid pSK1 harbouring the Herpes simplex virus thymidine kinase gene into fertilized mouse oocytes and succeeded in recovering plasmids from newborns by transformation of E. coli either with HindIII cut cellular DNA or with uncut DNA. The majority of the rescued plasmids were indistinguishable from pSK1 by restriction analysis. The rescued plasmids proved to be functionally active in a transient expression assay in mouse Ltk^- cells. The pSK1 DNA sequences were inherited by up to 90% of the second generation progeny mice, which is not in agreement with a Mendelian transmission of heterozygous markers integrated into a single site of the chromosome.These data support the assumption that germ line transmission of non-integrated episomal plasmids can occur.     

转基因小鼠和基因突变小鼠微卫星不稳定性分析

目的探讨微卫星在转基因和基因突变小鼠中的变化,为基因修饰和遗传突变动物的遗传检测和表型分析提供理论依据和技术手段。方法根据文献报道,从GenBank中选取198个等位基因数量多、富含多态性的微卫星位点,以野生型动物为对照,对6种近交系遗传背景的转基因小鼠和5种自然基因突变的近交系小鼠进行微卫星多态性检测,选用1.5%琼脂糖凝胶电泳和STR扫描技术,比较分析微卫星不稳定性。结果共有40个微卫星位点在转基因和基因突变小鼠中表现出多态性。在基因突变小鼠中,微卫星不稳定性有55.6%(10/18)是由纯合变为杂合(Ⅰ型),有3个位点(16.6%,3/18)是纯合突变(Ⅱ型),有5个位点同时存在2种类型的突变。但是在转基因动物中,大多数的微卫星多态性为Ⅰ型突变(87.5%,28/32),只有2个位点(6.2%,2/32)是Ⅱ型突变。另外有2个位点同时存在2种类型的突变。结论基因修饰或基因突变可引起小鼠相关微卫星发生不稳定性,而且某些微卫星位点对基因改变敏感性较高。     

Functional recombinant human anti-HAV antibody expressed in milk of transgenic mice

Hepatitis A virus (HAV) is a wide spread pathogenic agent and is the common cause of acute Hepatitis A worldwide. Passive immunization of HAV plays an extremely important role in post-exposure prophylaxis with clinical applications often requiring large amounts of antibody. As an alternative to the in vitro production of recombinant proteins, expression of monoclonal antibodies (mAbs) in the milk of transgenic animals is currently used being associated with low production costs and high activity. In this paper, eight founder lines of transgenic mice were generated by co-microinjection of the two cassettes encoding the heavy- and light-chains of a neutralizing anti-HAV antibody, respectively. The expressed heavy- and light-chains of the mAb were correctly assembled and modified in the mammary gland as detected by western blotting. High expression levels of the antibody were achieved during the lactation period and found to be independent of the copy numbers of integrated transgenes. The highest level was up to 32.2 mg/ml. The binding specificity and neutralizing activity of the expressed mAb were assayed by ELISA and neutralizing test, showing that it is capable to neutralize the JN strain of Hepatitis A virus efficiently. Therefore, our results suggest that a large-scale and efficient production of the anti-HAV mAb in the milk of transgenic farm animals would be feasible in the future.     

显微注射法制备转基因小鼠的技术研究

转基因技术是发生工程学或胚胎操作技术中的一部分。这一技术包括有受精卵的采集,受精卵的体外培养,胚胎移植等技术,转基因动物制作是其应用技术之一,它是将外源ＤＮＡ片段用显微注射法直接注入受精卵原核,使外源ＤＮＡ随机整合到寄主基因组中而形成转基因小鼠。我们建立完善这一技术的目的是在实验动物的生产中为转基因动物的生产提供稳定可靠的保证。我们将ｐＣＸＴＧＡＰ、ｐＣＳＬＮ等基因分别注入受精卵原核制备相应的转基因动物模型,并从中总结较佳的操作技术。结果经检测阳性转基因小鼠占出生幼鼠的１６３８％。我们认为不仅要有精细的微注射技术、动物的选用和饲育、严密的胚胎操作都是转基因动物制作成功的重要条件。     

Expression of recombinant Hirudin in transgenic mice milk driven by the goat beta-casein promoter

Hirudin, isolated from the leech Hirudo medicinalis, inhibits thrombin directly and several expression systems have been used to produce recombinant Hirudin (rHirudin) for pharmaceutical purposes. A DNA fragment containing the Hirudin coding sequence and goat beta-casein secretion signal was chemically synthesized in this study. The synthetic DNA then was further constructed into a goat beta-casein expression vector for mouse transgenesis. Four lines of transgenic mice were successfully developed and one line showed a meaningful anti-thrombin activity of 40,000 anti-thrombin units (ATU)/mL in their milk. In this animal line, Hirudin mRNA was found in samples of uterus and kidney with insignificant anti-thrombin activity (相似文献   

Efficient generation of transgenic mice by direct intraovarian injection of plasmid DNA

We established a rapid procedure for obtaining transgenic mice by directly injecting an enhanced green fluorescent protein (EGFP)-expressing plasmid (pIRES-EGFP) into the ovaries of fertile mice. The frequency of transgenic mouse production was determined by pair-mating, and by polymerase chain reaction (PCR) and sequence analysis of DNA taken from the tails of the offspring. The mice that received the EGFP gene transmitted it to their offspring (F(1)). Genetic and PCR analyses of F(1) progeny confirmed that the inserted EGFP was stably inherited. Of six female F(1) mice, all were able to pass the foreign DNA on to the next generation (F(2)). In situ hybridization using paraffin-embedded sections of ovarian and testicular tissues from the F(1) and F(2) progeny showed that the introduced gene was expressed in the gonads of the animals. The chromosomal location of the injected DNA was determined by fluorescence in situ hybridization, and the frequency of multiple site versus single site insertions is 85.71% (18/21) analyzed by FISH. We anticipate great progress in murine genetic engineering using this technique.     

Expression of active recombinant human lactoferrin in the milk of transgenic goats

This report details the establishment of a transgenic goat model in order to produce human lactoferrin (hLf) in the mammary gland for large-scale application and research. Two transgenic male goats were generated by microinjecting sequence encoding hLf cDNA to the pronuclear. In the two lines, derived from the two founders, eight lactating female goats could secrete recombinant human lactoferrin (rhLf) at concentrations of up to 0.765 mg/ml. The method of purifying the rhLf from the milk was achieved using ion-exchange chromatography and resulted in 97% purity. Biochemical and physicochemical characteristics of rhLf were similar to native lactoferrin (nhLf); this included N-terminal sequence, isoelectric point, molecular mass, glycosylation, iron-binding/releasing ability, thermal stability, and proteolysis. The rhLf showed broad spectrum antibacterial activity inhibiting the growth of several pathogenic bacterial strains. Also investigated, although to a lesser degree, was a practicable pasteurization method for the downstream processing of rhLf and, further, a method for the oral administration of rhLf. On the basis of these results, our studies show an optimistic and promising approach for the large-scale production and therapeutic application of rhLf expressed in transgenic goats.     

Activity of DNA vaccines encoding self or heterologous Her-2/neu in Her-2 or neu transgenic mice

To assess the efficacy of self versus heterologous ErbB-2 vaccines, the reactivity to human and rat ErbB-2 (Her-2 and neu, respectively) DNA vaccines were tested in normal, Her-2 or neu transgenic mice. When immunized with either Her-2 or neu DNA, normal BALB/c and C57BL/6 mice produced cross-reactive T cells, but only antigen specific antibodies. In Her-2 Tg mice, weak to no anti-Her-2 response was induced by either self Her-2 or heterologous neu DNA, demonstrating profound tolerance to Her-2 and the inability to induce anti-Her-2 immunity with either vaccine. In NeuT mice, vaccination with self neu but not heterologous Her-2 DNA induced anti-neu antibodies and delayed spontaneous tumorigenesis. Both neu and Her-2 DNA induced anti-neu T cell response, but depletion of CD8 T cells did not change the delay in tumorigenesis. Therefore, in NeuT mice, both self and heterologous DNA activated anti-neu T cells, although T cell response did not reach sufficient level to suppress spontaneous tumorigenesis. Rather, induction of anti-neu antibodies by self neu DNA is associated with the delay in spontaneous tumor growth. Overall, NeuT mice were more responsive to DNA vaccination than Her-2 Tg mice and this may be associated with the continuous production of neu by the 10 mammary glands undergoing tumor progression.     

Analysis of transfer of microinjected zygotes in production of transgenic mice

The data on transfer of mouse eggs microinjected with DNA during production of transgenic mice were analyzed. The transfer of mouse eggs into both oviducts did not lead to a reliably higher birth rate. It did not affect the frequency of recipients’ pregnancy and, although somewhat increased the frequency of multiple birth, led, finally, to unjustified loss of the major part of viable DNA-injected eggs. We recommend transferring no less than 15 microinjected eggs only in one oviduct of each recipient. The transfer into another oviduct is acceptable if the transfer into the first oviduct failed or its outcome is doubtful.     

Prnp knockdown in transgenic mice using RNA interference

RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance.     

Advances in transgenic rat production

Predictable and reproducible production of transgenic rats from a standardized input of egg donors and egg recipients is essential for routine rat model production. In the course of establishing a transgenic rat service, transgenic founders were produced from three transgenes in outbred Sprague-Dawley (SD) rats and four transgenes in inbred Fischer 344 (F344) rats. Key parameters that affect transgenesis efficiency were assessed, including superovulation treatments, methods to prepare pseudopregnant recipients, and microinjection technique. Five superovulation regimens were compared and treatment with 20 IU PMSG and 30 IU HCG was selected for routine use. Four methods to prepare pseudopregnant egg recipients were compared and estrus synchronization with LHRHa and mating to vasectomized males was selected as most effective. More than 80% of eggs survived microinjection when modified pronuclear microinjection needles and DNA buffers were used. The efficiencies of transgenic production in rats and C57BL/6J (B6J) mice were compared to provide a context for assessing the difficulty of transgenic rat production. Compared to B6J mice, SD rat transgenesis required fewer egg donors per founder, fewer pseudopregnant egg recipients per founder, and produced more founders per eggs microinjected. Similar numbers of injection days were required to produce founders. These results suggest that SD rat transgenesis can be more efficient than B6J mouse transgenesis with the appropriate technical refinements. Advances in transgenic rat production have the potential to increase access to rat models.     

Rearrangements of large-insert T-DNAs in transgenic rice

Introduction of large-DNA fragments into cereals by Agrobacterium-mediated transformation is a useful technique for map-based cloning and molecular breeding. However, little is known about the organization and stability of large fragments of foreign DNA introduced into plant genomes. In this study, we produced transgenic rice plants by Agrobacterium-mediated transformation with a large-insert T-DNA containing a 92-kb region of the wheat genome. The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers (fiber FISH). By using this cytogenetic technique, we showed that rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. Deletion of long stretches of the large-insert DNA was also observed in Agrobacterium.     

Heterologous CpG island becomes extensively methylated in the genome of transgenic mice

It is demonstrated that a heterologous (chicken) CpG island containing five Sp1 canonical recognition sequences becomes highly methylated in the genome of transgenic mice bearing one or several copies of the transgene. Similar levels of methylation of the chicken CpG island were observed in different tissues of transgenic mice except the brain where the level of methylation of this chicken CpG-rich fragment was significantly lower than in other tissues. Analysis of susceptibility of the "transgenic" CpG island to Hpa II and Msp I restriction nucleases revealed an unusual methylation pattern interfering with the action of both of these enzymes. A conclusion has been drawn that heterologous CpG island per se does not contain all necessary signals permitting to maintain its own non-methylated status in the genome of transgenic animals.     

Altered renal morphology in transgenic mice with cholecystokinin overexpression

Although cholecystokinin is a regulatory peptide with a predominant role in the brain and the gastrointestinal tract, there is an increasing evidence for its role in the kidney. The aim of this study was to reveal morphological changes in the structure of kidney of mice with cholecystokinin overexpression by means of light, transmission and scanning electron microscope, and atomic force microscopy. Using immunohistochemistry the expression of important basement membrane proteins collagen IV, laminin and fibronectin, as well the distribution of cholecystokinin-8 in the renal structures was evaluated. The altered morphology of kidneys of mice with cholecystokinin overexpression was seen by all microscopic techniques used. The renal corpuscles were relatively small with narrow capsular lumen. The basement membranes of renal tubules were thickened and the epithelial cells were damaged, which was more pronounced for distal tubules. Characteristic feature was the increased number of vesicles seen throughout the epithelial cells of proximal and especially in distal tubules reflecting to the enhanced cellular degeneration. The relative expression of laminin but not collagen IV in the glomerular basement membrane was higher than in the tubular basement membranes. The content of fibronectin, in opposite, was higher in tubular membranes. Cholecystokinin-8 was clearly expressed in the glomeruli, in Bowman’s capsule, in proximal and distal tubules, and in collecting ducts. Ultrastructural studies showed irregularly thickened glomerular basement membranes to which elongated cytopodia of differently shaped podocytes were attached. As foot processes were often fused the number of filtration pores was decreased. In conclusion, cholecystokinin plays important role in renal structural formation and in functioning as different aspects of urine production in mice with cholecystokinin overexpression are affected-the uneven glomerular basement membrane thickening, structural changes in podocytes and in filtration slits affect glomerular filtration, while damaged tubular epithelial cells and changed composition of thickened tubular basement membranes affect reabsorption.     

Production of transgenic mice carrying the Thanatin gene by intratesticular injection

Transgenic animals have potential applications in medicine, life sciences, and biopharmacy. In this study, we developed a convenient, economic, and efficient method for gene transfer by transfection of male spermatogonial stem cells. Three fragments of the Thanatin gene, encoding an antibacterial peptide, were synthesized and amplified by overlap extension polymerase chain reaction (PCR). They were inserted into vector pIRES2-EGFP. The pIRES2-EGFP-Thanatin plasmid was mixed with liposomes and injected into the testes of male mice by a minimally invasive operation. Six weeks after injection, male mice were mated with normal female mice to produce an F1 generation. PCR and Southern blotting were performed to analyze F1 mice. Among those 52 F1 mice produced, 38.46% were found to be positive for the Thanatin gene by PCR and 30.77% by Southern blotting. Six positive mice were selected from the F1 generation and mated with normal females to an F2 generation, in which 36.36% were found to be positive for the Thanatin gene. Expression of the green fluorescence protein in the transgenic mice was confirmed by fluorescence imaging. These results showed that the Thanatin gene was integrated into the mouse genome. The study provides a useful method for the future development of disease-resistant animals and production of antibacterial peptides through transgenic animals.     

Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

PiggyBac转座酶转基因小鼠模型的建立

目的 建立表达PiggyBac转座酶转基因小鼠模型,为研究PiggyBac转座子介导基因修饰在小鼠中的应用提供工具.方法 利用Cytomegalovirus( CMV)启动子驱动PiggyBac转座酶基因的表达,经显微注射法建立C57BL/6J表达PiggyBac转座酶的转基因小鼠.PCR鉴定转基因小鼠的基因型,RT-PCR检测PiggyBac转座酶在小鼠生殖系睾丸中的表达情况.PiggyBac转座酶转基因小鼠活性的检测,是通过与转座子供体转基因小鼠杂交检测供体位置变化来确定的.结果 显微注射产生7只转基因小鼠并能传代,经RT-PCR筛选出一株在睾丸中相对高表达PiggyBac转座酶的转基因小鼠.随后与转座子供体转基因小鼠杂交,子代双阳小鼠与野生型小鼠杂交基因型分离,产生的子代转座子供体单阳性小鼠中具有转座子供体片段的转座反应.结论 成功建立了表达PiggyBac转座酶转基因小鼠动物模型,该模型为PiggyBac转座子技术在小鼠中的应用提供了有价值的工具动物.     

Expression of functional recombinant human factor IX in milk of mice

Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed to express the FIX gene in the mammalian glands under control of milk β-casein promoter. The founding females secreted the FIX in their milk (3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the mammary gland of transgenic mice.