Transgenic mice designed to express human α-1,2-fucosyltransferase in combination of human DAF and CD59 to avoid xenograft rejection

The expression of human α-1,2-fucosyltransferase (HT) or complement regulatory proteins has been proved as an strategy to overcome hypercute rejection in discordant xenogeneic organ transplantation. In this study, we examined whether peripheral blood mononuclear cells (PBMCs) from polytransgenic mice expressing the human HT, and complement regulatory proteins (DAF and CD59), can provide more effective protection against xenograft rejection. Transgenic mice were produced by co-injection of gene constructs for human HT, DAF and/or CD59. Flow Cytometry (FCM) was used to screen the positive transgenic mice. PBMCs from transgenic mice were incubated with 15% human serum to evaluate natural antibody binding, complement activation and expression of adhesion molecules. Three transgenes were strongly expressed in PBMCs of transgenic mice, and HT expression signifi-cantly reduced expression of the major xenoepitope galactose-α-1,3-galactose (α-Gal). Functional studies with PBMCs showed that co-expression of HT and DAF or CD59 markedly increased their re-sistance to human serum-mediated cytolysis when compared with single transgenic PBMCs. Moreover, the combined expression of triple transgenes in PBMCs led to the greatest protection against human serum-mediated cytolysis, avoided hyperacute rejection and reduced expression of adhesion mole-cules. Strong co-expression of triple transgenes was completely protected from xenograft hyperacute rejection and partially inhibited acute vascular rejection. The studies suggest that engineering mice to express triple molecules represents an critical step toward prolonging xenograft survival and might be more suitable for xenotransplantation.     

The human apoE7 and apoE4 transgenic mice models

To scrutinize the disorders caused by human mutant apoE7/apoE4, human apoE4 and E7 transgenic mice were established with microinjection technique to examine molecular genetic phenomena in vivo. The integration and expression of h-apoE mutant genes in transgenic mice were determined with Southern blot, Northern blot and ELISA. The current studies indicated that the transgenes and the phenotypes regarding expression of transgenes could be transmitted stably in transgenic lines. The levels of serum lipid in transgenic mice showed the characteristics of hyperlipidemia. Besides, behavior tests demonstrated the degeneration of learning and memory in transgenic mice. Short life span was observed in 2 transgenic lines. After fed with high lipid food high serum lipid was found both in normal and transgenic mice, but their mechanism regulating lipid metabolism was different. It was also verified that the human apoE mutants located at either N-terminal or C-terminal had the same pathogenesis regarding disorders of     

Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA/G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of du     

Tetracycline-inducible Expression Systems： New Strategies and Practices in the Transgenic Mouse Modeling

To accurately analyze the function of transgene(s)of interest in transgenic mice,and togenerate credible transgenic animal models for multifarious human diseases to precisely mimic human dis-ease states,it is critical to tightly regulate gene expression in the animals in a conditional manner.The abilityto turn gene expression on or off in the restricted cells or tissues at specific time permits unprecedentedflexibility in dissecting gene functions in health and disease.Pioneering studies in conditional transgene ex-pression have brought about the development of a wide variety of controlled gene expression systems,whichmeet this criterion.Among them,the tetracycline-controlled expression systems(e.g.Tet-off system andTet-on system)have been used extensively in vitro and in vivo.In recent years,some strategies derived fromtetracycline-inducible system alone,as well as the combined use of Tet-based systems and Cre/lox P switch-ing gene expression system,have been newly developed to allow more flexibility for exploring gene functionsin health and disease,and produce credible transgenic animal models for various human diseases.In thisreview these newly developed strategies are discussed.     

Can overexpression of TGF—β1 gene change the sex ratio in transgenic mice？

Mouse TGF-β1 gene was microinjected into male pronuclei of F2 hybrid fertilized eggs obtained by mating CSJLF1 and C57BL/6J inbred strains to generate transgenic mice with over-expressed TGF-β1 gene.The rate of founder production is 31% and Southern blot analysis of founder mice tail DNAs gave an integration efficiency of 33%.TGF-β1 gene could be stably integrated to the chromosomes of transgenic mice and transmitted to their progeny at a rate of 33% in the second generation.Dot blot analysis of tail RNA of some transgenic mice indicated a moderate expression of the transgene.The most interestin finding of the present work is the striking eviation from the normal male:female sex ratio in transgenic mice,with an average ratio of 6.7:1.The possible nature of the predominance of male sex in transgenic mice overexpressing TGF-β1 is discussed.     

A New β-Estradiol-lnducible Vector Set that Facilitates Easy Construction and Efficient Expression of Transgenes Reveals CBL3-Dependent Cytoplasm to Tonoplast Translocation of CIPK5

Transient and stable expression of transgenes is central to many investigations in plant biology research. Chemical regulation of expression can circumvent problems of plant lethality caused by constitutive overexpression or allow inducible knock （out/down） approaches. Several chemically inducible or repressible systems have been described and successfully applied. However, cloning and application-specific modification of most available inducible expression systems have been limited and remained complicated due to restricted cloning options. Here we describe a new set of 57 vectors that enable transgene expression in transiently or stably transformed cells. All vectors harbor a synthetically optimized XVE expression cassette, allowing I~-estradiol mediated protein expression. Plasmids are equipped with the reporter genes GUS, GFP, mCherry, or with HA and Strepll epitope tags and harbor an optimized multiple cloning site for flexible and simple clon- ing strategies. Moreover, the vector design allows simple substitution of the driving promoter to achieve tissue-specificity or to modulate expression ranges of inducible transgene expression. We report details of the kinetics and dose-dependence of expression induction in Arabidopsis leaf mesophyll protoplasts, transiently transformed Nicotiana benthamiana leaves, and stably transformed Arabidopsis plants. Using these vectors, we investigated the influence of CBL （Calcineurin B-like） protein expression on the subcellular localization of CIPKs （Calcineurin B-like interacting protein kinases）. These analyses uncovered that induced co-expression of CBL3 is fully sufficient for dynamic translocation of CIPK5 from the cytoplasm to the tonoplast. Thus, the vector system presented here facilitates a broad range of research applications.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the hα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

Inheritance and Expression of Copies of Transgenes 1Dx5 and 1Ax1 in Elite Wheat （Triticum aestivum L.） Varieties Transferred from Transgenic Wheat through Conventional Crossing

To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Email 8 were carried out. Chuan89-107×B72-8-11b, Chuan89-107×B102-1-2 and Email 8×B72-8-11b, and F_1 plants were selfed or backcrossed to obtain different generation populations. Protein analysis in grains of F_1 and F_2 and backcross progenies of BC_1F_1, BC_1F_2, BC_1F_3, BC_2F_1, BC_2F_2 and BC_2F_3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes lDx5 and lAx1 were expressed and segregated in the target wheat according to Mendelian laws. A range of lDx5 expression levels were observed in the progenies of Chuan89-107×B72-8-11b and Emai 18×B72-8-11b, but the expression levels of lAx1 in progenies of Chuan89-107×B102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign lDx5 gene of 5-10 copies had the more complicated expression mechanism than the lAx1 gene of 4-5 copies.     

Characterization of transgene integration pattern in F4 hGH-transgenic common carp （Cyprinus carpio L.）

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.     

Secretion of Biologically Active Human Granulocyte Colony-Stimulating Factor (G-CSF) in Milk of Transgenic Mice

Two constructs were devised, containing the full-length gene of the human granulocyte colony-stimulating factor (G-CSF) fused with the 5′ and 3′ flanking promoter sequences of bovine alpha-S₁-casein gene (CSN1S1). Both constructs contained a 1518-bp fragment that included exons 18 and 19 and 320 bp of the 3′ flanking region of bovine gene CSN1S1, but differed in size of the 5′ flanking sequences, which were of 721 bp, and exon 1 in construct pGCm1 and 2001 bp and exon 1 and intron 1 in construct pGCm2. With both constructs, transgenic mice were produced. The transgene expression was assessed using RT-PCR and immunochemically from the production of human G-CSF in milk of lactating females. Secretion of human G-CSF into the milk varied in a wide range, from 0.8 μg/ml to over 1 mg/ml, in mice with construct pGCm1 and was low (up to 60 μg/ml) or absent in mice with construct pGCm2. G-CSF glycosylation was incomplete in mice with transgene pGCm1 and complete in mice with pGCm2. G-CSF of transgenic mouse milk was shown to stimulate the formation and growth of granulocyte-containing colonies in human umbilical blood cell culture and be close or identical in physiological activity to the natural human G-CSF. __________ Translated from Genetika, Vol. 41, No. 10, 2005, pp. 1331–1337. Original Russian Text Copyright ? 2005 by Dvoryanchikov, Serova, Andreeva, Dias, Azevedo, Serov.     

猪异种器官移植的人源化修饰

利用猪的器官来解决当前人源器官严重短缺,为解决移植器官短缺的可行的途径。用定向基因转移(gene targeting)手段,直接并准确地对α-1,3半乳糖苷转移酶（α-1,3GT）基因进行同源重组,使α-1,3GT失活,再结合猪体细胞克隆技术,对其进行人源化改造,减弱或消除排异反应。除对2-1.3GT进行基因定向修饰外,阻断由异种器官移植而激活的人类补体的串联反应是猪异种器官人源化修饰的另一途径。然而,猪内源性逆转录病毒（porcine endogenous retrovirus,PERV）造成的公共卫生问题,给异种器官移植的前景投下了阴影。因此,即要剔除导致人类排异反应的猪细胞表面的α-1,3GT及其相关的分子, 又要确保猪器官异种移植的安全性, 是尚待研究的重大课题。 Abstract:Xenotransplantation (XP) from pig into human has been considered as means to overcome the great lack of donor organ available in transplantation surgery.In order to weaken rejection between human and pig,approaches of gene targeting have been proposed to eliminate “ rejection gene”α-1,3GT from porcine cells directly and accurately.α-1,3GT knockout pigs can be produced by nuclear transfer cloning with the porcine cells(knocking out α-1,3GT).Besides the genetic modification of α-1,3GT in porcine cells,there is another technical way to interdict activity of complement in series for human by XP.However,porcine endogenous retroviruses (PERV) during XP has been thought to not be negligible in being transmitted with the xenograft to the human recipient.Therefore,it is importance task that we should not only knockout α-1,3GT and relative molecules from pigs,but also ensure safety in public health of XP from PERV.     

A preliminary analysis of antineoplastic activity of parvovirus MVMp NS—1 proteins

Human gastric cancer MKN-45 cells were transfected with pULB 3238,a plasmid carrying MVMp MS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR.After the integration and expression of NS-1 gene,some of the transfectants died,while others remained alive,but the growth features of survived cells were changed.For further study on the antineoplastic function of parvoviral NS-1 protein in vivo,transgenic mice carrying NS-1 genes were established by conventional method.Among 4 founders,one of them was found to be able to transmit the transgene to around 50% of their offsprings.RT-PCR was performed to indicate the expression of NS-1 gene in transgenic mice and its mRNA appeared in a variety of tissues.The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.     

Genetically Modified Pig Models for Human Diseases

Genetically modified animal models are important for understanding the pathogenesis of human disease and developing therapeutic strategies.Although genetically modified mice have been widely used to model human diseases,some of these mouse models do not replicate important disease symptoms or pathology.Pigs are more similar to humans than mice in anatomy,physiology,and genome. Thus,pigs are considered to be better animal models to mimic some human diseases.This review describes genetically modified pigs that have been used to model various diseases including neurological,cardiovascular,and diabetic disorders.We also discuss the development in gene modification technology that can facilitate the generation of transgenic pig models for human diseases.     

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITCconjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germcells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flowcytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes ofepithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphologywas normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.     

Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter I （GAT1）

It is well documented that 7-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABAA and GABAB receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter I (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wild-type mice. Our findings provided the first evidence that abnormal expression of GAT1 could result in dysgenesis,and indicated that GAT1 might be therapeutically targeted for contraception or dysgenesis treatment.     

Suppression of type 1 Insulin-like growth factor receptor expression by small interfering RNA inhibits A549 human lung cancer cell invasion in vitro and metastasis in xenograft nude mice

Cancer invasion and metastasis, involving a variety of pathological processes andcytophysiological changes,contribute to the high mortality of lung cancer.The type 1 insulin-like growthfactor receptor (IGF-1R),associated with cancer progression and invasion,is a potential anti-invasion andanti-metastasis target in lung cancer.To inhibit the invasive properties of lung cancer cells,we successfullydown-regulated IGF-1R gene expression in A549 human lung cancer cells by small interfering RNA (siRNA)technology,and evaluated its effects on invasion-related gene expression,tumor cell in vitro invasion,andmetastasis in xenograft nude mice.A549 cells transfected with a plasmid expressing hairpin siRNA forIGF-1R showed a significantly decreased IGF-1R expression at the mRNA level as well as the proteinlevel.In biological assays,transfected A549 cells showed a significant reduction of cell-matrix adhesion,migration and invasion.Consistent with these results,we found that down-regulation of IGR-1Rconcomitantly accompanied by a large reduction in invasion-related gene expressions,including MMP-2,MMP-9,u-PA,and IGF-1R specific downstream p-Akt.Direct tail vein injections of plasmid expressinghairpin siRNA for IGF- 1R significantly inhibited the formation of lung metastases in nude mice.Our resultsshowed the therapeutic potential of siRNA as a method for gene therapy in inhibiting lung cancer invasionand metastasis.     

The Mesenchymal Stem Cells Derived from Transgenic Mice Carrying Human Coagulation Factor VIII Can Correct Phenotype in Hemophilia A Mice

Hemophilia A （HA） is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII （FVIII） deficiency. Previous studies showed that introduction of mesenchymal stem cells （MSCs） modified by FVIll-expressing retrovims may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-de/eted human FVIll （hFVHIBD） vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVlllBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak （77 ng/ mL） at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT （activated partial thromboplastin time） value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice （45.5 s vs. 91.3 s）. Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice.     

Indoleamine 2, 3-dioxygenase （IDO） is essential for dendritic cell activation and chemotactic responsiveness to chemokines

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs),and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC functions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflammatory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regulated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.