显微注射法制备转基因鼠和转基因猪的技术特点比较

本文采用显微注射技术制作转基因鼠和转基因猪并对其进行比较,阐明了用显微注射方法制作两种转基因动物的技术原理和实验方法。对两者相似点进行了归纳和总结,同时也对两种转基因动物制作过程中的不同之处进行了比较和分析。根据两种不同的实验动物,描述了它们各自不同的实验特点,技术方法和实验要求,本文同时也在理论上和实践中对转基因动物的制作进行了探索。     

水平显微注射法制备转基因小鼠的研究

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。     

转基因技术研究进展

随着胚胎生物学和发育生物学研究的不断深入，在实验室内生产转基因胚胎已成为可能。本文综合有关文献资料，主要介绍生产转基因动物的程序与方法及其鉴定方法和应用前景以及存在的问题。     

动物转基因与核移植技术的新进展

本文就动物转基因与核移植技术进行文献回顾，原核注射是一种可靠、使用广泛的动物转基因方法，但该方法存在花费时间长、整合效率低及不能定点整合的问题。所以近年来出现了一些新的转基因方法，包括精子介导、反转录病毒介导、携带外源基因体细胞的核移植、ES细胞基因打靶技术等。这些方法中动物核移植技术贡献较大，很有前途。核移植技术解决了同源重组在大动物上的限制，可以应用于生物制药动物保种、预防遗传病、辅助生殖及治疗性克隆。     

显微注射制备转基因小鼠的优化

显微注射法是目前应用最广泛的转基因小鼠制备技术,但其效率较低,提高显微注射法制备转基因小鼠的效率有重要意义。外源DNA浓度、小鼠品系均是影响转基因小鼠制备效率的因素,但目前较多文献采用KM小鼠进行转基因小鼠制备效率的优化,对C57/BL6小鼠研究较少。本实验以C57/BL6小鼠为对象,分别进行2.5 ng/μl、5.0 ng/μl的外源DNA浓度原核注射。结果显示,2.5 ng/μl组的怀孕率、出生率、小鼠阳性率、转基因效率均显著高于5.0 ng/μl组的结果(P0.05)。结论:通过原核注射方式获得C57/BL6转基因小鼠,注射的外源DNA浓度2.5 ng/μl效果较好。     

转基因技术研究进展及在动物生产中的应用

随着新技术的发展和对基因功能的了解,转基因技术获得了更加广泛的应用.本文综合有关文献资料,主要介绍生产转基因动物的程序与方法、鉴定方法及其在动物生产中的应用前景.     

单精注射法生产转基因小鼠的研究

精子胞质内显微受精技术（ICSI）作为辅助受精的一种手段,将体外受精研究和胚胎的显微操作技术结合起来,比以往对精子质量的要求大大降低,使其无论在畜牧业生产实践还是在哺乳动物的生殖生理基础研究中都有着十分重要的意义。本研究用小鼠精子及小鼠精子与GFP基因孵育后对小鼠卵母细胞进行ICSI,获得子代鼠。提取鼠尾基因组DNA,应用PCR、Southern blot进行整合检测。在发育至成年的11只小鼠中经PCR和Southernblot检测到3只阳性（27.3%）。结果表明,利用ICSI技术可以高效地生产转基因小鼠。     

转基因鸡制备方法的研究进展

在获得转基因哺乳动物的基础上,由于禽类(鸡)作为生物反应器本身所具有的优势,许多研究者投向制备转基因鸡的研究上。本文简要叙述了常用的制备转基因鸡的方法。其中,结合本实验室的情况,重点介绍了胚盘直接注射法。     

显微注射转基因用猪胚胎获得研究

本文从猪胚胎的体外成熟和体内成熟两方面对显微注射用猪胚胎的获取进行了初步研究。实验结果表明 ;超数排卵母猪的手术冲胚是获取显微注射用猪胚胎的最佳方法 ,该方法获得的胚胎数量多 ,质量好 ,转基因效率高。体外成熟法获得可供显微注射用猪胚胎的效率低 ,基因转移的总效率也很低。     

转基因技术研究进展

随着胚胎生物学和发育生物学研究的不断深入,在实验室内生产转基因胚胎已成为可能。本文综合有关文献资料,主要介绍生产转基因动物的程序与方法及其鉴定方法和应用前景以及存在的问题。     

自体移植情况下移入胚数对猪基因导入效率的影响

为了探讨在自体移植的情况下,每头受体移入的微注射基因胚胎的数量对猪基因导入总效率的影响,在导入ＰＥＦｈＤＡＦ（Ｈｕｍａｎｄｅｃａｙａｃｃｅｌｅｒａｔｉｎｇｆａｃｔｏｒ,人类补体衰减加速因子）基因的实验过程中,将受体母猪按称入胚数分成三组：第一组１２～１５枚,第二组１６～２０枚,第三组２１～３２枚,其受孕率分别为８０％,９０．９％,和７０％,三组之间的差异不显著（Ｐ〉０．０５）;共产仔率分别为１９．     

Compensatory Growth and its Effect on Muscularity and Technological Meat Quality in Growing Pigs

This study investigated the effect of various feeding levels from weaning (day 28) to day 170 of age on growth, muscularity and technological meat quality in female pigs. From day 28 to day 90 of age (growing period) and from day 90 to day 170 of age (finishing period), the pigs were fed either ad libitum (A) or restrictively (R) in a 2 2 2 factorial design with treatments named AA, AR, RA and RR. In the growing period, the growth rate of A pigs was 35% higher than that of R pigs. In the finishing period, the growth rate was dependent on the feed intake in the growing period, i.e. pigs fed restrictively in the growing period had 6-8% higher growth rate in the finishing period (RA and RR) than pigs fed ad libitum in the growing period (AA and AR). Furthermore, despite RA pigs being 11 kg lighter at day 90 of age they produced as much muscle tissue at slaughter as did AA pigs, but less subcutaneous fat, which resulted in a 5% higher meat content of the carcass. The increased muscle growth of RA pigs in the finishing period (compensatory growth) was probably accomplished by increased satellite cell proliferation (muscle DNA accumulation) and increased capacity for protein synthesis, as indicated by a higher RNA concentration. Feeding level did not affect the lightness of meat, the ultimate pH or the drip loss. However, a change in feeding level at day 90 of age (RA and AR) led to a reduction in meat redness. The present data suggest that feed restriction in the growing period results in compensatory growth of muscle tissue in the finishing period if ad libitum feeding was applied during this period, accomplished by increased satellite cell proliferation and increased capacity for protein synthesis, without significantly affecting the technological meat quality.     

Transgenic Pigs with Pancreas-specific Expression of Green Fluorescent Protein

The development and regeneration of the pancreas is of considerable interest because of the role of these processes in pancreatic diseases, such as diabetes. Here, we sought to develop a large animal model in which the pancreatic cell lineage could be tracked. The pancreatic and duodenal homeobox-1 (Pdx1) gene promoter was conjugated to Venus, a green fluorescent protein, and introduced into 370 in vitro-matured porcine oocytes by intracytoplasmic sperm injection-mediated gene transfer. These oocytes were transferred into four recipient gilts, all of which became pregnant. Three gilts were sacrificed at 47–65 days of gestation, and the fourth was allowed to farrow. Seven of 16 fetuses obtained were transgenic (Tg) and exhibited pancreas-specific green fluorescence. The fourth recipient gilt produced a litter of six piglets, two of which were Tg. The founder Tg offspring matured normally and produced healthy first-generation (G1) progeny. A postweaning autopsy of four 27-day-old G1 Tg piglets confirmed the pancreas-specific Venus expression. Immunostaining of the pancreatic tissue indicated the transgene was expressed in β-cells. Pancreatic islets from Tg pigs were transplanted under the renal capsules of NOD/SCID mice and expressed fluorescence up to one month after transplantation. Tg G1 pigs developed normally and had blood glucose levels within the normal range. Insulin levels before and after sexual maturity were within normal ranges, as were other blood biochemistry parameters, indicating that pancreatic function was normal. We conclude that Pdx1-Venus Tg pigs represent a large animal model suitable for research on pancreatic development/regeneration and diabetes.     

滇南小耳猪与DLY商品猪背最长肌营养成分比较分析

本试验为比较研究滇南小耳猪与DLY商品猪肉的营养价值,分别检测了53头滇南小耳猪和58头DLY猪背最长肌肉的水分、粗蛋白质、粗脂肪、灰分、氨基酸及脂肪酸等营养成分含量,比较分析其差异性。结果显示,滇南小耳猪背最长肌的水分含量极显著低于DLY商品猪背最长肌（P < 0.01）,粗蛋白质、粗脂肪含量极显著高于DLY商品猪（P < 0.01）,灰分含量差异不显著（P > 0.05）;其中滇南小耳猪背最长肌的7种必需氨基酸（苏氨酸、缬氨酸、蛋氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸）、肉豆蔻酸、棕榈酸、油酸、顺式异油酸、11-顺-二十碳烯酸,饱和脂肪酸、单不饱和脂肪酸、总脂肪酸含量均极显著高于DLY商品猪（P < 0.01）。综上所述,滇南小耳猪与DLY猪相比具有更高的营养价值和开发价值。     

Nutrient Composition Comparison of Longissimus Dorsi between Diannan Small-ear Pigs and DLY Commercial Pigs

This study was conducted to compare the nutrient composition of longissimus dorsi between Diannan Small-ear pigs and DLY commercial pigs.53 Diannan Small-ear pigs and 58 DLY commercial pigs were chosen and longissimus dorsi samples were collected to determine the content of moisture,crude protein,crude fat,ash,amino acids and fatty acids.The results showed that the moisture content of Diannan Small-ear pigs was extremely significantly lower than DLY commercial pigs (P < 0.01).However,the content of crude protein and crude fat were extremely significantly higher than DLY commercial pigs (P < 0.01).And there was no significant difference in ash content (P > 0.05).The levels of threonine,valine,methionine,isoleucine,leucine,lysine and phenylalanine in longissimus dorsi of Diannan Small-ear pigs were extremely significantly higher than that of DLY commercial pigs (P < 0.01),and the content of myristic acid,palmitic acid,oleic acid,cis different oleic acid,11-cis-20 carbon olefine acid,saturated fatty acids,monounsaturated fatty acids and the total fatty acids in longissimus dorsi of Diannan Small-ear pigs were also extremely significantly higher than that of DLY commercial pigs (P < 0.01).In conclusion,Diannan Small-ear pigs had a better value in nutrient and development.     

显微注射生产转人胰岛素原基因奶山羊

将构建的绵羊β－乳球蛋白基因（BLG）调控人胰岛素原基因的重组基因通过显微注射生产转基因奶山羊。同期化超排处理供体奶山羊15只，排卵262枚，回收卵221枚，移植206枚注射卵，移植受体68只，怀孕21只（妊娠率33％，其中成年母羊妊娠率为58．62％，周岁母羊为9．4％），产羔33只，其中母羊羔8只，公羔25只。经PCR检测及酶切分析，证明有5只为阳性，阳性整合率15．15％，但5只全为雄性。     

贵州白香猪与3种杂交猪脏器系数及血液生理指标比较

试验对贵州白香猪及贵州白香猪与大约克猪、杜洛克猪、长白猪3种杂交猪的体重、脏器系数和血液生理指标进行了比较。结果显示,贵州白香猪体重极显著低于3种杂交猪（P＜0.01）;贵州白香猪的脏器指标除心脏外与3种杂交猪均存在差异;贵州白香猪红细胞总数(RBC)和红细胞压积(HCT)极显著高于大约克猪×香猪（YX）（P＜0.01）;贵州白香猪的平均红细胞血红蛋白浓度(MCHC)、白细胞中淋巴细胞数量(LYM)、白细胞中淋巴细胞比率(LYM,%)及白细胞中中性粒细胞比率(GRA,%)与长白猪×香猪（LX）间存在显著差异（P＜0.05）。