共查询到20条相似文献,搜索用时 15 毫秒
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Shangang Li Yi Guo Jianjun Shi Chunguang Yin Fengying Xing Lingyang Xu Chuanshan Zhang Tao Liu Yao Li Hongbin Li Lixin Du Xuejin Chen 《Transgenic research》2009,18(2):227-235
Live rabbits have previously been generated through nuclear transfer using adult cells as nuclear donors. We demonstrated
in this study that transfected adult rabbit fibroblasts are also capable of supporting full-term development. The fibroblasts
were transfected with a pEGFP-C1 plasmid using lipofectamine™ 2000, and the transgenic cells were derived from conditioned medium. The transgenic fibroblasts were cultured until confluent
and then serum-starved prior to be used as nuclear donors. After nuclear transfer and activation, 22% (12/55) of the transgenic
cloned embryos developed to the blastocyst stage. A total of 114 embryos at the 4- to 8-cell stage were transferred to the
oviducts of 8 pseudo-pregnant mothers; 5 of these animals became pregnant, and 3 of the 5 mother rabbits carried the pregnancy
to term. Caesarean section was performed on the 3 pregnant mothers, yielding 4 kits, one of which has survived for more than
9 months. Green fluorescence could be detected in the toenails of the living cloned rabbit and the offspring from the living
cloned rabbit under ultraviolet light. DNA analyses confirmed that all 4 cloned rabbits were genetically identical to the
transgenic donor cells, and that they all carried the EGFP gene. The present study demonstrated that transgenic rabbits can
be generated through nuclear transfer. These results may facilitate future developments in the genetic engineering of rabbits. 相似文献
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利用转基因克隆技术实现外源基因的导入宿主染色体基因组内稳定整合,并能遗传给后代,已在基因表达与调控的理论研究、人类遗传病动物模型的建立、药用蛋白的生产、抗病育种、人类移植用的器官的研究等方面得到广泛应用。转基因动物的研究与应用也已经成为21世纪生命科学领域最活跃、最具有实际应用价值的方向之一,尤其是作为生物反应器和医学上为人类提供所用器官方面,其经济价值和社会效益将是不可估量。在查阅大量近年来国内外相关资料的基础上,本文以转基因动物克隆为中心,对转基因动物克隆所采用显微注射技术、核移植技术、基因打靶与真核BAC表达载体制备等主要研究技术,以及转基因动物克隆在异种器官移植、构建生物反应器等方面的应用进行了综合性论述与分析,同时阐述了各种转基因技术的优点与缺点,以其为转基因动物克隆研究提供理论基础与技术支撑。 相似文献
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Chrenek P Ryban L Vetr H Makarevich AV Uhrin P Paleyanda RK Binder BR 《Transgenic research》2007,16(3):353-361
Transgenic founder rabbits carrying a gene construct consisting of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb
of the human clotting factor VIII (hFVIII) cDNA and 4.6 kb of 3′ flanking sequences of mWAP gene were crossed for three generations.
All transgenic animals showed stable transgene transmission. Transgenic females showed high level of recombinant hFVIII (rhFVIII)
mRNA expression in biopsed mammary gland tissues, while marginal expression of rhFVIII mRNA was observed in the spleen, lung
and brain. No adverse effects of ectopic expression on the physiology of the rabbits were observed. Expression was not detected
in the liver, kidney, heart and skeletal muscle. In transgenic females derived from three generations, rhFVIII protein was
secreted from the mammary gland of lactating females, as shown by Western blotting. Biological activity of rhFVIII protein,
as revealed in clotting assays was ranged from 0.012 to 0.599 IU/ml corresponding to 1.2% and 59.9% of the hFVIII level in
normal human plasma. No apparent effect of secreted rhFVIII on the milk performance of rabbits was observed. Our results confirm
the possibility of producing a significant amount of a biologically active rhFVIII in the mammary gland of established transgenic
rabbit lines. 相似文献
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The production of recombinant proteins in the milk of transgenic animals is an alternative to traditional cell culture methodology. Transgenic rabbits can serve in the small-scale production of recombinant proteins, underscoring the need to maintain valuable transgenic lines. In this study, the authors used cryopreserved transgenic rabbit semen to artificially inseminate does, demonstrating the utility of this method for the reestablishment of a transgenic rabbit herd. 相似文献
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L. Hiripi D. Negre F.-L. Cosset K. Kvell T. Czömpöly M. Baranyi E. Gócza O. Hoffmann B. Bender Zs. Bősze 《Transgenic research》2010,19(5):799-808
Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit. 相似文献
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外源基因在转基因动物中遗传和表达的稳定性 总被引:2,自引:0,他引:2
转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。 相似文献
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R Fluhr 《Developmental genetics》1990,11(3):197-204
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Baranyi M Hiripi L Szabó L Catunda AP Harsányi I Komáromy P Bosze Z 《Journal of biotechnology》2007,128(2):383-392
Patients suffering certain metabolic diseases (e.g. phenylketonuria) need a low-phenylalanine diet throughout their lives. Transgenic rabbits were created to express low-phenylalanine kappa-casein in their milk. The aim was to demonstrate for the first time the feasibility of producing a modified milk protein in addition to normal milk proteins. A gene construct containing the coding region of the rabbit kappa-casein gene was modified by site-specific oligonucleotide directed mutagenesis. Four of the five phenylalanine amino acids present in the mature protein were mutated and the gene construct was used to create two transgenic rabbit lines. The transgenic rabbits produced the recombinant kappa-casein at a high level in their milk causing a reduction in the average size of the casein micelles. The low-phenylalanine kappa-casein was digestible with chymosin and it was separated from its native counterpart and from the other milk proteins by a one-step HPLC method on a reversed-phase column. In the future, low-phenylalanine casein produced in transgenic animals could be used as dietary replacements to meet the special requirements of certain consumer groups. 相似文献
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次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(hypoxanthine guanine phosphoribosyltransferase,HGPRT)的功能缺失与痛风、肾结石和雷纳综合症(Lesch-Nyhan Syndrome)等疾病相关.制作HGPRT基因表达降低的模式动物,将有利于人们对这种疾病的发病机理和治疗做进一步的研究.构建了针对HGPRT基因表达的shRNA干扰载体,并将质粒转染兔成纤维细胞,获得携带该干扰片段的转基因细胞系,经PCR鉴定转基因成纤维细胞克隆阳性率为83.3%.RT-PCR及Western blot检测结果表明转基因干扰成纤维细胞系HGPRTmRNA和蛋白质表达量明显降低.最后,以转基因成纤维细胞进行核移植,囊胚率为27.8%,与正常来源的成纤维细胞囊胚率相比较差异不显著.说明,通过RNAi可稳定干扰兔成纤维细胞HGPRT基因的表达,为进一步通过核移植技术建立HGPRT RNAi转基因兔模型创造条件. 相似文献
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Transgenic Research - This study aims to produce transgenic cattle expressing lysine-rich polypeptide in milk by somatic cell nuclear transfer. Lysine is the first limiting amino acid in cereal... 相似文献
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M Massoud R Bischoff W Dalemans H Pointu J Attal H Schultz D Clesse M G Stinnakre A Pavirani L M Houdebine 《Journal of biotechnology》1991,18(3):193-203
A DNA construct containing the human alpha 1-antitrypsin gene including 1.5 and 4 kb of 5' and 3' flanking sequences, was microinjected into the pronucleus of rabbit embryos. The recombinant human protein was (a) expressed in the blood circulation of F0 and F1 transgenic rabbits at an average concentration of 1 mg ml-1, (b) shown to be fully active and (c) shown to be separable from its rabbit counterpart. Transgenic rabbits might represent a novel source of human proteins of therapeutic interest. 相似文献
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Transgenic animals, especially mice, have been used quite extensively as models for various human diseases. At first, the
level of scientific inquiry was driven by the need to establish the model. In many cases, these models may be considered quite
crude because of their limitations. More recently, transgenic models of disease have become more refined and are currently
being used to study the pathological mechanisms behind the disease rather than to just provide a model of the disease. Using
some examples from the recent literature, we will document the current level and complexity of inquiry using transgenic animals.
New techniques and techniques that may prove promising will be discussed.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
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应用RNA干扰技术体外抑制兔成纤维细胞HGPRT的表达及其核移植研究 总被引:2,自引:0,他引:2
次黄嘌呤-鸟嘌呤磷酸核糖基转移酶( hypoxanthine guanine phosphoribosyltransferase,HGPRT )的功能缺失与痛风、肾结石和雷纳综合症(Lesch-Nyhan Syndrome)等疾病相关.制作HGPRT基因表达降低的模式动物,将有利于人们对这种疾病的发病机理和治疗做进一步的研究.构建了针对HGPRT基因表达的shRNA干扰载体,并将质粒转染兔成纤维细胞,获得携带该干扰片段的转基因细胞系,经PCR鉴定转基因成纤维细胞克隆阳性率为83.3%.RT-PCR及Western blot检测结果表明转基因干扰成纤维细胞系HGPRT mRNA和蛋白质表达量明显降低.最后,以转基因成纤维细胞进行核移植,囊胚率为27.8%,与正常来源的成纤维细胞囊胚率相比较差异不显著.说明,通过RNAi可稳定干扰兔成纤维细胞HGPRT基因的表达,为进一步通过核移植技术建立HGPRT RNAi转基因兔模型创造条件. 相似文献
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Coulibaly S Besenfelder U Fleischmann M Zinovieva N Grossmann A Wozny M Bartke I Tögel M Müller M Brem G 《FEBS letters》1999,444(1):111-116
Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF-beta) cDNA were generated. Expression of hNGF-beta mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF-beta was purified from the milk by a two-step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF-beta depicted a molecular weight of 13,261 Da per subunit. The biological activity of the hNGF-beta was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF-beta demonstrated full biological activity when compared to commercial recombinant hNGF-beta. 相似文献
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In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene. Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays. In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants. A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system. 相似文献
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Transgenic mice and rats have become popular tools to study the regulation of gene expression and the consequences of protein over-production. Over the past decade, numerous transgenic models have been developed to study the mechanisms of human renin gene expression and the participation of the renin-angiotensin system in the development of hypertension. Herein we will provide an overview of what has been learned from the use of transgenic models for studying the human renin gene. 相似文献