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1.
由于传统显微注射法具有操作繁琐、技术要求高和外源基因导入率低等局限性,本试验对常规显微注射法进行改进,旨在建立一套比较完善的水平显微注射方法。采用水平微注射系统,将外源DNA片段导入胚胎的雄原核中,获得子代鼠,分娩后3周提取鼠尾基因组DNA,PCR法筛选转基因阳性鼠。本试验采用Puc/BLG IN S和Chym os in基因,共使用710枚鼠原核胚进行研究,胚胎经注射后移植了25只受体,产下仔鼠77只;妊娠率分别为33.3%(5/15)和50%(5/10),胚胎成活率分别为9.47%(41/433)和l3.00%(36/277),阳性率分别为4.88%(2/41)和8.33%(3/36)。将阳性雌鼠配种妊娠后,对外源基因整合情况进行检测,结果表明初步获得了转基因阳性小鼠。 相似文献
2.
本试验探讨了不同辅助激活方法(Calciumionophore A23187激活、Calciumionophore A23187+6-DMAP联合激活和电激活)、不同精子预处理方法(液氮冻融处理和0.1%Triton X-100处理)和在添加半胱氨酸的胚胎培养液中培养不同时间(0 h、4 h、12 h和168 h)对猪卵母细胞内单精子注射(ICSI)胚胎体外发育的影响。结果显示:与无辅助激活相比,A23187+6-DMAP联合激活和电激活均能显著提高ICSI卵母细胞的激活率、卵裂率和囊胚率(P0.05),A23187+6-DMAP联合激活能显著提高ICSI卵母细胞的受精率(P0.05)。液氮冻融精子组ICSI卵母细胞的雄原核形成率显著高于活精子组(P0.05)。在添加半胱氨酸的胚胎培养液中培养4 h的ICSI卵母细胞受精率、雄原核形成率和囊胚率显著高于0 h组(P0.05)。以上结果表明,猪卵母细胞在ICSI后需要辅助激活来启动胚胎顺利发育,A23187+6-DMAP激活效果较好。液氮冻融精子可以促进ICSI后雄原核的形成。半胱氨酸处理4 h对猪ICSI卵母细胞受精和发育均有促进作用。 相似文献
3.
试验旨在探讨单精子胞浆内注射（intracytoplasmic sperm injection,ICSI）法生产猪体外受精卵和应用猪ICSI受精卵进行胞质注射生产转基因胚胎的可行性。首先对比了猪体外受精（in vitro fertilization,IVF）受精卵与ICSI受精卵的胚胎发育效率;然后观察了猪ICSI受精卵的双原核形成时间及效率,对精子注射到胞质后6~18 h分6个时间段进行地衣红染色,对比精子进入卵胞质后的状态及原核形成;最后对猪IVF受精卵受精后8~10 h及ICSI受精卵受精后12~14 h进行EGFP-N1质粒（20 ng/μL）胞质注射,观察胚胎发育效率及转基因效率。结果表明,ICSI受精卵的胚胎发育率（卵裂率89.4%和67.9%、囊胚率36.5%和16.1%）显著优于IVF组（P<0.05）,适合用于猪的体外受精卵试验;猪ICSI受精卵双原核在精子注射到卵胞质后12~14 h形成,双原核形成率为54.90%,显著高于其余5个试验组（P<0.05）;ICSI受精卵胞质注射组胚胎卵裂率（86.2%和66.3%）、囊胚率（30.0%和13.6%）及转基因效率（18.5%和0）均显著高于IVF受精卵胞质注射试验组（P<0.05）。本试验结果为采用ICSI受精卵进行胞质注射生产转基因猪的研究奠定了基础。 相似文献
4.
Somfai T Kashiwazaki N Ozawa M Nakai M Maedomari N Noguchi J Kaneko H Nagai T Kikuchi K 《The Journal of reproduction and development》2008,54(3):149-155
The aim of the present study was to investigate the effects of centrifugation pretreatment on the viability and nuclear status of porcine in vitro matured (IVM) oocytes and on the developmental competence of in vitro fertilized (IVF) oocytes (zygotes) after cryopreservation by vitrification (Solid Surface Vitrification; SSV). Mature oocytes having the first polar body after IVM and zygotes having the second polar body at 10 h after IVF were centrifuged at 10,000 x g at 37 C for 20 min and then subjected to SSV. Their viability was evaluated by morphological appearance and fluorescein diacetate staining. The nuclear status of oocytes was evaluated 6 h after vitrification. The developmental ability to the blastocyst stage of vitrified zygotes was evaluated after 6 days of in vitro culture. Although centrifugation did not damage the oocytes directly, it drastically reduced the rate of live oocytes after SSV. The rates of vitrification-induced parthenogenetic activation were similar in both centrifuged and non-centrifuged oocytes (42.4 and 47.4%, respectively). Centrifugation had no significant effects on the viability of pronuclear oocytes. The development of vitrified zygotes to the blastocyst stage was significantly lower than that of the control irrespective of centrifugation pretreatment. There was no difference in the cleavage and blastocyst rates between the control and centrifuged zygotes after vitrification. There was also no difference in the total cell numbers of blastocysts between the control and centrifuged zygotes irrespective of vitrification. These results reveal that, in IVM porcine oocytes, centrifugation pretreatment is highly detrimental to cryotolerance; however, in zygotes, it has only a slight effect on viability and does not alter the developmental competence of surviving zygotes. 相似文献
5.
M Murakami NWK Karja P Wongsrikeao B Agung M Taniguchi H Naoi T Otoi 《Reproduction in domestic animals》2005,40(6):511-515
This study was conducted to examine whether cat spermatozoa stored in ethanol for 1 month was capable of developing into pronuclei and of supporting normal embryonic development. In vitro matured oocytes were injected with frozen-thawed spermatozoa and ethanol-stored spermatozoa. The status of oocytes and sperm nuclei was examined at 4 and 18 h after injection of spermatozoa, and the presumptive zygotes were cultured for 7 days to assess the development of oocytes injected with each storage sperm. The percentage of enlarged sperm head at 4 h after injection was higher in ethanol-stored spermatozoa than in frozen-thawed spermatozoa, but there was no significant difference in the development of oocytes and sperm nuclei at 18 h after injection between the two groups. The development of oocytes to the blastocyst stage after injection of spermatozoa was observed only in oocytes with frozen-thawed spermatozoa. Of oocytes injected with ethanol-stored spermatozoa, two (2.8%) oocytes developed to the 16-cell stage. These results indicate that cat spermatozoa stored in ethanol can decondense and form male pronuclei after intracytoplasmic injection. However, the oocytes injected with ethanol-stored spermatozoa did not have the ability to develop to the blastocyst stage. 相似文献
6.
Transgenic pigs for xenotransplantation for humans] 总被引：1，自引：0，他引：1
H Niemann 《DTW. Deutsche tier?rztliche Wochenschrift》1999,106(4):141-146
Transgenic livestock have been generated via microinjection of DNA-constructs into pronuclei of zygotes. However, efficiency is low and only 1-3% transgenic offspring are to be obtained. Integration of the transgene occurs at random and expression is independent from the number of integrated copies but can be affected by the integration site. To overcome the shortage of human organs, transgenic pigs have been generated that express human complement regulatory genes. This approach enables to overcome the hyperacute rejection response as shown by an average survival rate (40-90 days) of the immunosuppressed primate recipients receiving a heart from a transgenic pig. It is expected that transgenic pigs would be available as organ donors in the next 5-10 years. A major prerequisite, however, is the prevention of the potential transfer of pathogenic microorganisms, in particular porcine endogenous retroviruses (PERV). Improvements of the efficiency in the generation of transgenic pigs will be achieved by the use of genetically modified donor cells in nuclear transfer technology (cloning). 相似文献
7.
鼻咽癌来源潜伏膜蛋白1的表达诱发转基因小鼠鼻咽不典型增生 总被引：2，自引：0，他引：2
为建立鼻咽癌来源潜伏膜蛋白1（N－LMP1）转基因小鼠模型，从整体的角度研究N－LMP1的功能，进一步阐明EB病毒在鼻咽癌变过程中的作用，应用DNA重组技术将角质细胞特异性启动子EDL2与N－LMP1连接，构建转基因EDL2－N－LMP1；并用显微注射技术，将转基因EDL2－N－LMP1注射入小鼠受精卵前核，再将注射后的受精卵植入假孕母鼠，获得转基因鼠，然后观察目的的基因在鼻咽部的表达和病理变化。本实验共获得53只转基因鼠，PCR法证明其中6只小鼠有N－LMP1基因扩增带，Suothern杂交显示PCR阳性小鼠有特异的杂交信号，整合率为11．3％，1只4月龄的转基因鼠出现了鼻咽部不典型增生，说明N－LMP1单独就能诱发小鼠鼻咽部发生癌前病变，为EB病毒可能是鼻咽癌的主要病因提供了有力的证据。 相似文献
8.
菌糠在兔饲料中的应用效果研究 总被引：2，自引：0，他引：2
本研究分冬季（全喂饲合饲料）和夏季（增喂一定量的青草）两次试验，每次试验选用比利时兔和日本大耳白兔各３６只，将菌糠以１０％和２０％的比例添加于饲料中，探讨了菌糠对兔的增重、产肉性能等的影响。结果表明，饲料中添加１０％的菌糠，在增重、料肉比、屠宰率和经济效益等方面与对照组无显著差异，添加２０％的菌糠有不良影响。同时还表明，比利时兔比大耳白兔能更好地利用菌糠，在冬季全喂配合饲料时能获得较好的饲养效果。 相似文献
9.
The present investigation was undertaken to study the effect of addition of dbc AMP on bovine oocyte maturation and fertilization in vitro. The bovine oocytes isolated from 2–8 mm follicles were cultured for 26 h in TCM-199. The maturation rate (71.4 %) did not significantly increase after supplementation of the culture medium with dbc AMP (86.3 %.) or FSH + hCG (86.3 %). The in vitro fertilization rate of oocytes based on sperm penetration and presence of sperm tail in the ooplasm increased significantly in the dbc AMP (34.7 %) and the dbc AMP + FSH + hCG (33.9 %) treated groups when compared with untreated controls (17.9 %). However, dbc AMP treated oocytes were not able to secure the formation of male pronucleus 20 h after in vitro fertilization, while in oocytes matured in dbc AMP free medium both pronuclei were present in approximately 15 % of the penetrated oocytes. Also, the sperm head decondensation was blocked or slowed down by the dbc AMP treatment. It is concluded (1) that dbc AMP may improve the condition for the interaction of oocytes with spermatozoa, and (2) that the ooplasm of such dbc AMP treated oocytes apparently is not able to decandense the sperm head and transform it to the male pronucleus. 相似文献
10.
利用5-甲基胞嘧啶（5-methylcytosine，5mC）和5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5-hrnC）特异性抗体对小鼠原核时期胚胎进行免疫荧光染色。同时使用荧光定量PCR方法检测Tet（Ten eleven translocation）基因在小鼠早期胚胎中的表达。免疫荧光染色结果显示早期原核阶段（PN1到PN3），雄原核中5mC的含量逐渐减少，而5hmC的含量逐渐增加。但是雌原核中5mC和5hmC的含量基本不变。在原核后期阶段（PN4到PN5）5hmC主要存在于雄原核中。荧光定量结果显示在小鼠MⅡ卵母细胞和植入前胚胎中Tet 1和Tet 2基因表达量较低，但是Tet 3在卵母细胞和原核时期表达量较高，随着胚胎的发育其表达量逐渐降低。结果表明，在小鼠原核时期阶段5mC到5hmC的转变过程主要是由TET3蛋白催化的，并且此过程参与小鼠雄原核的DNA主动去甲基化。 相似文献
11.
12.
Kato M Ishikawa A Hochi S Hirabayashi M 《The Journal of reproduction and development》2004,50(2):191-195
The present study was conducted to examine the developmental potential to offspring of rat embryos cultured from 1-cell to morula/blastocyst stage. Pronuclear zygotes from Wistar x Wistar or (SD x DA) x Wistar strains were cultured in modified rat 1-cell embryo culture medium (mR1ECM) for 96 h in 5% CO(2) in air at 37 C. The proportion of the 3-way cross hybrid zygotes developing into morula/blastocyst stage (74%) was higher than that of the Wistar zygotes (66%). Day-5 morulae/blastocysts developed in vitro were transferred into Day-3 or -4 pseudopregnant recipients of Wistar or SD x DA strain. The transfer of cultured embryos resulted in the birth of offspring at 13-59%, while that of non-cultured control blastocysts showed birth rates of 35-65%. The best offspring rate of cultured embryos (59%) was obtained when the hybrid 1-cell zygotes were cultured in mR1ECM medium and transferred into the 2-days earlier uteri of SD x DA recipients. These results suggest that genetic background of recipients as well as donors is a possible factor affecting full-term development of rat morulae/blastocysts derived from 1-cell stage zygotes cultured in vitro. 相似文献
13.
转基因动物研究进展 总被引：7，自引：0，他引：7
转基因动物技术始于 2 0世纪 80年代 ,近 3 0年来 ,随着研究的深入 ,转基因动物的制作技术得到了突破性的发展。最初的原核注射法是应用最普遍、最可靠、效果最稳定的一种方法。但该方法存在价格昂贵 ,整合率低及不能定点整合的问题 ,所以近几年来转基因动物技术已出现了胚胎干细胞法 ,精子载体法 ,体细胞核移植法和人工酵母染色体法等一系列新方法。随着这些技术的不断发展 ,转基因动物技术应用正以其突出的优越性指导着多个领域的工作。目前 ,它的应用已渗透到基础理论、疾病动物模型、人异种器官移植、制药、畜牧兽医等领域。转基因动物应用正走向产业化的道路 ,具有十分广阔的前景。但作为一个新兴的技术 ,转基因动物研究还面临着一些急需解决的问题 相似文献
14.
Genetic engineering of mammalian embryos 总被引：2，自引：0，他引：2
R E Hammer V G Pursel C E Rexroad R J Wall D J Bolt R D Palmiter R L Brinster 《Journal of animal science》1986,63(1):269-278
A gene consisting of the mouse metallothionein I promoter/regulator (MT) fused to the human growth hormone (hGH) structural gene (MThGH) was microinjected into rabbit, sheep and pig eggs. Visualization of nuclear structures was accomplished by interference-contrast (I-C) microscopy for rabbit and sheep eggs and by centrifugation and I-C microscopy for pig eggs. The gene integrated into the chromosomes of each species with an efficiency of 13% in rabbits, 1% in sheep and 10% in pigs. Human GH mRNA was detected in the liver of transgenic rabbits as well as tail and ear samples of pigs. Immunoreactive hGH was present in the serum of a transgenic rabbit and plasma of most transgenic pigs. In several pigs hGH levels increased between birth and 90 d of age. The presence of substantial quantities of hGH in plasma of pigs did not increase postnatal somatic growth rates. Founder animals will be bred and their transgenic and control progeny used to assess the effects of hGH on feed efficiency and carcass composition. These experiments demonstrate the feasibility of introducing foreign genes into the genome of several animal species by microinjection of eggs. 相似文献
15.
Wheeler MB 《Journal of animal science》2003,81(Z3):32-37
The introduction of specific genes into the genome of farm animals and its stable incorporation into the germ line has been a major technological advance in agriculture. Transgenic technology provides a method to rapidly introduce "new" genes into cattle, swine, sheep, and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Methods to produce transgenic animals have been available for more than 20 yr, yet recently lines of transgenic livestock have been developed that have the potential to improve animal agriculture and benefit producers and/or consumers. There are a number of methods that can be used to produce transgenic animals. However, the primary method to date has been the microinjection of genes into the pronuclei of zygotes. This method is one of an array of rapidly developing transgenic methodologies. Another method that has enjoyed recent success is that of nuclear transfer or "cloning." The use of this technique to produce transgenic livestock will profoundly affect the use of transgenic technology in livestock production. Cell-based, nuclear transfer or cloning strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition, and increased disease resistance. One practical application of transgenics in swine production is to improve milk production and/or composition. To address the problem of low milk production, transgenic swine over-expressing the milk protein bovine alpha-lactalbumin were developed and characterized. The outcomes assessed were milk composition, milk yield, and piglet growth. Our results indicate that transgenic overexpression of milk proteins may provide a means to improve swine lactation performance. 相似文献
16.
Transgenic animals constitute an important tool with many biotechnological applications. Although there have been advances in this field, we propose a novel method that may greatly increase the efficiency of transgenic animal production and thereby its application. This new technique consists of intracytoplasmic injection of liposomes, in bovine oocytes and zygotes, to introduce exogenous DNA. In the first experiment, we evaluated embryo development and EGFP expression in In Vitro Fertilization (IVF) embryos injected with different concentrations of exogenous DNA–liposome complexes (0.5, 5, 50, 500 ng pCX‐EGFP/μl). The highest EGFP‐embryos rates were obtained using 500 ng pCX‐EGFP/μl. In the second experiment, we evaluated embryo development and EGFP expression following the injection of DNA–liposome complexes into pre‐fertilized oocytes and presumptive zygotes, 16 and 24 h post‐fertilization. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp–liposome was injected 16 h post‐fertilization. The percentages of positive embryos for the 24‐h post‐fertilization and pre‐fertilization groups were 30.1 and 6.3, respectively. Blastocysts that developed from injected zygotes were analysed by PCR, confirming the presence of transgene in all embryos. Finally, we examined the embryo development and EGFP expression of parthenogenetic embryos that resulted from the injection of egfp–liposome complexes into pre‐activated oocytes, and 3 and 11 h post‐activated oocytes. The group with the highest expression rate (48.4%) was the one injected 3 h post‐activation. In summary, this study reports the efficient, reproducible and fast production of IVF and parthenogenetic embryos expressing EGFP, by the intracytoplasmic injection of liposomes to introduce the foreign DNA. 相似文献
17.
不同周龄大耳白兔血液生理生化指标的测定 总被引：5，自引：0，他引：5
为了明确大耳白兔中国兽药监察所封闭群的生物学特性，对其血液生理生化指标进行了测定。选5、10及13周龄的雄兔和雌兔。空腹采血测定红细胞、白细胞等16项血液生理指标和血糖、总蛋白等16项生化指标。10周龄的红细胞数、白细胞数、红细胞压积和平均红细胞血红蛋白含量高于5周龄和13周龄，雌雄间血液生理指标无明显差异，血清丙氨酸转氨酶含量随着周龄的增加而升高，碱性磷酸则随着周龄的增加而下降。与日本大耳白兔相比，主要血液生理指标比较接近，丙氨酸转氨酸、白蛋白、尿素氮、直接胆红素等血液生化指标相近，其他指标有所不同。测定结果表明，该封闭群具有不同于日本大耳白兔（Slc:JW/CSK)的生物学特性。 相似文献
18.
A. Lindner Dr med vet l; P. von Wittke Dr. med vet; M. Schmald J. Kusserow H. Sommer Dr habil Dr med vet 《Journal of Equine Veterinary Science》1992,12(1)
Lactate kinetics in whole blood of horses was investigated after exercise of differing velocities and duration. The following categories of exercise were used: A: <11 m/second and >180 seconds (n=35), B: >11 m/second and <180 seconds (n=17) and C: <11 m/second and <180 s (n=10). The mean peak lactate concentration determined in horses in category A was 4.49 ± 2.21 mmol/1, in B, 16.32 ± 4.81 mmoVl and in C, 4.58 ± 1.59 mmol/l. While the maximum lactate concentrations in categories A and C were always found immediately after the exercise, the peaks in category B were measured between the first and tenth minute after exercise. Mean lactate concentrations measured at 2-minute intervals after bouts of category-B exercise tended to stabilize 3 to 10 minutes after exercise; however, mean lactate concentrations measured during the intervals before and after the peak value differed significantly. The lactate concentration returned to pre-exercise levels within 20 minutes after exercise bouts of category C, but remained above pre-exercise levels up to 60 minutes after bouts of category-A and -B exercise. It was concluded that, for an evaluation of lactate data after intensive anaerobic exercise, sequential blood sampling at 2-minute intervals for a period of up to 12 minutes after exercise is necessary. Less frequent sampling may be a reason for the often described irreproducibility of lactate concentrations in horses. After aerobic or mild anaerobic exercise, one sample is sufficient, but it has to be taken as soon as possible after exercise. 相似文献
19.
The damaging effect at the injection site (muscle) of a series of different preparations of penicillin C with increasing content of citrate buffer has been investigated in rabbits in order to improve the local tolerance of the drug preparation. The injections were given in M.sacrospinal and 3 days after injection the animals were sacrified and the areas of necrotic muscle tissue were isolated and weighed. The damage from the different preparations was determined on the basis of these weighing results. It was concluded that Novocillin 3.15 g and 6.3 g in 20 ml aqueous solution could be improved by adding 3x the original content of citrate buffer to the preparation resulting in a 38% decrease of the mean weight of necrotic areas at the injection site. Moreover, it was shown that the mean weight of necrotic areas at the injection site was diminished by 41% when injecting the improved 6.3 g concentration in a volume of 0.1 ml compared to injecting the improved 3.15 g concentration in a volume of 0.2 ml, which provides the same dose of penicillin G. 相似文献
20.
Ahmad Ali Shahid Ibrahim Bala Salisu Amina Yaqoob Abdul Qayyum Rao Inayat Ullah Tayyab husnain 《Journal of animal physiology and animal nutrition》2020,104(1):343-351
Various feeding studies have been conducted with the different species of animals to evaluate the possible transfer of transgenic DNA (tDNA) from genetically modified (GM) feed into the animal tissues. However, the conclusions drawn from most of such studies are sometimes controversial. Thus, in the present study, an attempt has been made to evaluate the fate of tDNA in rabbits raised on GM cotton-based diet through PCR analysis of the DNA extracted specifically from blood, liver, kidney, heart and intestine (jejunum). A total of 48 rabbits were fed a mixed diet consisting variable proportions of transgenic cottonseeds meal (i.e. 0% w/w, 20% w/w, 30% w/w and 40% w/w) for 180 days. The presence of transgenic DNA fragments (Cry1Ac, Cry2A and CP4 EPSPS) or plant endogenous gene (Sad1) was traced in those specific tissues and organs. The presence of β-actin (ACTB) was also monitored as an internal control. Neither the transgenic fragments (459 bp of Cry1Ac gene, 167 bp of Cry2A gene and111 bp of CP4 EPSPS gene) nor cotton endogenous reference gene (155 bp of Sad1) could be detected in any of the DNA samples extracted from the rabbit's tissues in both control and transgenic groups. However, 155 bp fragment of the rabbit's reference gene (ACTB) was recovered in all the DNA samples extracted from rabbit tissues. The results obtained from this study revealed that both plant endogenous and transgenic DNA fragments have same fate in rabbit's tissues and were efficiently degraded in the gastrointestinal tract (GIT). 相似文献