胚胎移植技术对特种突变小鼠的净化研究

目的通过净化特种突变小鼠来建立SPF级种子库。方法利用胚胎移植技术对四种突变无毛小鼠和一种白内障小鼠的桑葚胚进行子宫内移植。结果四种突变无毛小鼠和白内障小鼠均产出仔鼠,获得移植成功,其中白内障小鼠的产仔率最高为25.5%。结论建立了SPF级特种突变小鼠种群,为进一步研究与开发利用提供了可靠保证。     

昆明小鼠精子冷冻的研究(简报)

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受体,生长发育并产出幼仔。胚胎体外培养获得足够的晚桑葚胚和早期囊胚是胚胎干细胞建立与制备嵌合体小鼠的成功关键,暂时不用的胚胎应仍能耐受冷冻保存,待复苏移植,建立新的繁殖群。但小鼠精子冷冻研究在我国开展的十分有限,缺乏相关资料数据。本实验对不同周龄SPF级昆明(KM)小鼠进行精子冷冻及冻融精子IVF,并将IVF获得的2-细胞胚胎分别进行胚胎移植、体外培养、胚胎冷冻及复苏后移植,应用冻融小鼠精子进行胚胎工程实践。     

使用超净工作台实施子宫摘除术净化KM小鼠的研究

目的建立SPF级湖北封闭群KM小鼠生产群.方法使用超净工作台实施子宫摘除术净化小鼠的方法,建立了SPF级湖北封闭群KM小鼠生产群,并对子一代进行了微生物及寄生虫的检测.结果使用上述方法实施手术67例,成功57例,得净化仔鼠509只,育成的460只小鼠符合SPF级标准.结论使用生物净化的方法可以保存优良实验动物品种.     

小鼠不同阶段胚胎玻璃化冷冻保存的比较研究

采用玻璃化冷冻法对ICR、C57BL/6、DBA~*C57BL/6杂交F1代三种品系小鼠的不同阶段胚胎进行冷冻保存,比较胚胎解冻后形态良好率、体外发育率和移植后的出生率,结果表明解冻后各品系小鼠胚胎从2细胞到桑椹胚形态良好率在75%以上,其中8细胞胚胎形态良好率在83%以上,而囊胚的形态良好率仅在40%左右。解冻后胚胎体外培养的发育率随胚胎发育阶段的提高而提高,桑椹胚的发育达93%以上。体外受精2细胞冷冻胚与体内受精2细胞冷冻胚比较,二者形态良好率差异无显著意义(74%∶75%),但体内受精冷冻胚的发育率明显高于体外受精冷冻胚(76%:40%,p<0.01);胚胎经过三次反复冻融后形态良好率无显著差别;冷冻2细胞胚移植后的受孕率与仔鼠出生率分别达64%和40%,但均低于新鲜2细胞胚。     

IVF-20培养液用于大鼠体外受精的实验研究

目的探讨人用体外受精液用于实验大鼠体外受精的可行性,为实验大鼠的胚胎保种提供参考。方法选用人体外受精IVF-20培养液作为大鼠精子获能和受精培养液,对SD、Wistar、GK、F344等四种不同品系的实验大鼠进行体外受精;用mR1ECM培养液对体外受精所得胚胎进行体外培养实验。同时,将所得2-细胞胚胎移植给经假孕处理的受体鼠。结果四个品系大鼠体外受精后的卵裂率分别可达83.2%、72.6%、87.8%和71.6%。体外受精卵裂胚能够在体外进一步发育,SD大鼠囊胚发育率为16.7%,与体内受精胚胎在体外培养的囊胚发育率(24.5%)差异不显著。80枚2-细胞胚胎移植给2只受体鼠后均妊娠成功,产下正常仔鼠10只,产仔率12.5%。结论用于人体外受精的IVF-20培养液同样适合于实验大鼠精子的体外获能和受精,可以在多种品系获得较高的卵裂率,所得卵裂胚能够在体外发育到囊胚,并且所得卵裂胚在移植给受体鼠后能够产下正常仔鼠。     

无菌APPswe/PS1ΔE9双转基因小鼠模型建立及脑内斑块变化初步观察

目的 使用剖宫产净化方法建立无菌APPswe/PS1ΔE9(PAP)双转基因小鼠模型并对动物脑内斑块沉积情况进行初步观察,为研究肠道菌群与阿尔茨海默症关系提供新的动物模型。方法 选择阳性PAP雄性杂合子鼠与经产的C57野生型雌鼠1∶2进行交配。怀孕母鼠在超净工作台内行剖宫产手术,用无菌ICR小鼠代乳。术后每个月进行无菌状态检测;PCR方法检测剖宫产所得PAP仔鼠的基因型;免疫组化方法定量检测9月龄PAP小鼠脑内斑块变化情况。结果 实施剖宫产手术12例,获仔鼠66只,剖宫产存活率及离乳存活率分别为95.45%(63/66)和95.24%(60/63),净化后按国标检测无菌状态均为合格。免疫组化结果显示9月龄无菌PAP小鼠海马内斑块较同月龄SPF级动物减少。结论 通过剖宫产净化技术去除了PAP小鼠携带的菌群,9月龄无菌PAP小鼠脑内斑块减少。     

还原型谷胱甘肽在AG129小鼠生物净化及快速繁殖中的应用

目的 AG129小鼠自然产仔困难,采用常规体外受精试剂无法获得足够数量用于移植的胚胎。使用还原型谷胱甘肽(GSH)调节小鼠体外受精液成分,完成对AG129小鼠体外受精,为体外受精困难品系的基因工程小鼠生物净化及快速扩繁提供思路。方法设计3个含不同浓度GSH的精子获能及受精液的实验组,浓度分别0. 5、1、2 mmol/L,1个不含GSH的精子获能液及受精液的对照组,每组超排5只小鼠,重复3次。结果不同GSH实验组及对照组间平均受精率差异具有显著性(P 0. 05),在精子获能及受精液中GSH浓度为1 mmol/L时,AG129小鼠体外受精效果最佳,平均受精率为(43. 35±1. 76)%,胚胎状态良好。将获得胚胎移植受体小鼠,受体动物均妊娠并顺利产仔。结论体外受精液中的GSH浓度为1 mmol/L时,能够显著提高AG129小鼠体外受精效率,为类似体外受精困难品系的小鼠生物净化及快速繁殖提供解决方案。     

SPF级小鼠超数排卵效果

目的探求小鼠周龄、品系和季节对SPF级小鼠超数排卵效果的影响。方法在春、夏、冬季对4～10周龄的SPF级BALB／c小鼠和KM小鼠进行超数排卵处理,通过见栓率、产胚率、3．5d胚胎获得量及胚胎类型等方面进行统计学分析。结果小鼠周龄、品系和季节对SPF级BALB／c小鼠和KM小鼠见栓率没有影响;对产胚率、3．5d胚胎获得量和胚胎类型等方面有一定影响。结论SPF级BALB／c小鼠宜用9、10周龄的进行超数排卵处理以供应3．5d胚胎为需要所用,而SPF级KM小鼠使用4～10周龄的都可以;另外,SPF级小鼠超数排卵处理仍宜在春、夏季进行,可以保证获得最佳的效果。     

我国商业化SPF级小鼠病原体污染分析

目的按现行国标《实验动物微生物学检测方法》和《实验动物寄生虫学检测方法》对我国商品化的无特殊病原体（specific pathogen free,SPF）小鼠进行微生物状况检测,为生产高质量的实验动物提供依据。方法对五家主要实验动物生产单位生产的ICR、KM、C57BL/6、BALB/c及BALB/c-nu品系的SPF级小鼠进行随机抽检。检测抽检小鼠所携带的微生物和寄生虫情况。结果在抽检的SPF级小鼠中,病毒污染主要包括呼肠孤病毒Ⅲ型（Reo-3）、小鼠肺炎病毒（PVM）和多瘤病毒（POLY）;主要细菌污染为肺炎克雷伯杆菌、金黄色葡萄球菌和绿脓杆菌。这些微生物病原体中,除条件性致病菌绿脓杆菌外,其他病原体对小鼠本身和实验研究均具有一定的影响。     

不同品系小鼠的体外受精、胚胎冷冻及移植的比较研究

目的　探讨不同品系小鼠的体外受精、胚胎和精子的低温保存效果。方法　本实验分别在中国科学院上海实验动物中心 (SLAC)和日本熊本大学动物资源开发中心 (CARD)对 13个品系小鼠 (C57BL 6J、BALB c、C3H HeJ、ICR、KM、FVB、MRL、NOD、CBA、DBA 2、CD 1、BDF1、B6C3F1)的体外受精 (IVF)率、胚胎培养及移植成绩进行了比较研究。结果　各品系小鼠新鲜精子的IVF率 15 1%～ 87 9% ,冻融精子的IVF率 8%～ 80 % ;冷冻胚胎的复苏率4 2 6 %～ 83 9% ;冻融胚胎移植后的产仔率在 17 8%～ 5 1 8%。结论　遗传背景不同的小鼠体外受精率、冷冻胚胎复苏率和胚胎移植的产仔率差异有显著性。但同一品系两个实验室间的新鲜精子的IVF率、冷冻胚胎的复苏率及移植产仔率差异无显著性 (P >0 0 5 ) ;冻融精子的体外受精率CARD明显高于SLAC(P <0 0 1)。     

从兽用生物制品企业角度看我国SPF鸡质量控制现状

SPF鸡和鸡胚是兽用生物制品生产和检验环节的主要原材料,直接影响到产品质量和检验结果,2010年版《中国兽药典》（三部）附录《生产、检验用动物标准》明确要求,用于禽类制品毒种制备与鉴定、病毒活疫苗生产与检验、灭活疫苗检验的鸡和鸡胚应符合国家无特定病原体（SPF级）动物标准.SPF鸡的质量控制包括遗传、微生物、环境和营养四个方面,本文主要从兽用生物制品生产企业的角度,分析国内SPF鸡质控指标,结合禽用活疫苗生产和检验实践,提出对SPF鸡质量控制的思考和建议.     

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.     

Risk of transmission of Mycobacterium avium ssp. paratuberculosis by embryo transfer of in vivo and in vitro fertilized bovine embryos

Over a 5-year interval, experiments were conducted to determine if Mycobacterium avium ssp. paratuberculosis (Map) is associated with in vivo and in vitro fertilized (IVF) embryos and whether it can be transmitted by embryo transfer. The present studies included: collection of embryos from five asymptomatic, naturally infected donors and transfer to uninfected recipients; collection of oocytes from two naturally infected donors with overt clinical signs; exposure of in vivo and IVF embryos to Map and transfer to uninfected recipients; and the inoculation (transfer) of "clean" IVF embryos to the uterine lumen of infected cows. The presence of Map was confirmed in the uterine horns of all asymptomatic, infected donors. None of the tested embryos, which were not used for embryo transfer, or unfertilized ova (two per batch), were positive for Map, as determined by culture (n = 19) or by PCR (n = 13). However, all in vivo fertilized embryos exposed to Map in vitro (and subsequently sequentially washed) tested positive for Map, by both culture (12 batches) and PCR (15 batches), whereas IVF embryos treated in the same manner tested positive on culture (51%, 18/35 batches) and by PCR (28%, 20/71 batches). Transferring both in vivo embryos and IVF embryos potentially contaminated with Map into 28 recipients resulted in 13 pregnancies and eight calves born without evidence of disease transmission to either the recipients or the offspring over the following 5-year period. In samples collected from one of the clinically infected animals, two of seven (28%) cumulus oocyte complexes (COC) and follicular fluid tested positive by PCR and 10/10 cumulus oocyte complexes on culture for Map. From the second clinically infected cow, three of five batches of IVF embryos (n = 20) were positive on PCR and two of four batches containing unfertilized oocytes and embryos were positive on culture. Only 10% of embryos reached the morula and blastocyst stage 10 days after fertilization. In conclusion, Map is unlikely to be transmitted by embryo transfer when the embryos have been washed as recommended by the International Embryo Transfer Society.     

Normal reproductive capacity of heifers that originated from in vitro fertilized embryos cultured with an antiviral compound

Bovine viral diarrhea virus (BVDV) can associate with in vitro fertilized (IVF) bovine embryos despite washing and trypsin treatment. An antiviral compound, DB606 (2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan), inhibits the replication of BVDV in bovine uterine tubal epithelial cells, Madin Darby bovine kidney cells, and fetal fibroblast cells. As well, DB606 in in vitro culture medium does not affect embryonic development. Antiviral-treated-IVF embryos placed into recipients developed into clinically normal calves. The objective of this project was to determine if these resultant heifer calves were capable of reproducing. Seven heifers from each of the treatment groups (natural breeding, IVF embryo, and IVF embryo cultured in DB606) of the previous study were used. At 20-27 months of age, the heifers were exposed to a fertile bull in a single pasture during a 63 d breeding season. Five of the seven heifers originating from natural breeding were pregnant 35 d after removal of the bull and calved. All of the heifers resulting from transfer of untreated IVF embryos were pregnant at 35 d; however, one aborted the fetus at 5-7 months of gestation. All of the heifers derived from transfer of IVF embryos cultured in DB606 were pregnant and calved. Offspring from dams of all treatment groups were clinically normal at birth. Adjusted 205 d weaning weights were not significantly different among the offspring of the treated and untreated dams. These results indicate that culture of bovine-IVF embryos in DB606 does not impair future reproductive capacity of resulting heifers.     

精子介导法建立sFat-1转基因小鼠模型

利用精子介导的基因转移(Sperm-mediated gene transfer,SMGT)方法,将sFat-1 DNA片段直接和小鼠精子孵育,再通过体外受精、胚胎移植等技术建立sFat-1转基因小鼠模型。经PCR检测,16只后代中发现2只阳性个体,Southern blot进一步鉴定的结果显示有2只后代整合了sFat-1基因,成功建立sFat-1转基因小鼠模型,转基因率为12.5%。本研究运用精子介导的转基因方法将sFat-1基因整合到小鼠基因组中,为进一步研究sFat-1的生物学功能提供了实验动物模型。     

Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT-PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1-cell, 2-cell, 6-8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK-L, PFK-M, and PFK-P. PFK-L and PFK-P were not detected in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrated that PFK was not detected in 8-cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH-A and LDH-B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH-C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH-C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer.