黏蛋白1基因敲除小鼠模型的构建

黏蛋白1(MUC1)属黏蛋白家族成员,分布于上皮细胞膜表面,由于在免疫炎症反应以及肿瘤发生中的重要作用而日益受到重视.为了进一步深入研究MUC1的生物学功能,构建了Muc1基因敲除小鼠模型.首先,根据小鼠Muc1基因组序列设计基因剔除策略,将2个loxP位点分别插在外显子2和3两侧,构建基因剔除载体Muc1-ABRLFn-pBR322.以电穿孔方法将载体导入胚胎干细胞(ES细胞),用G418和更昔洛韦进行正负筛选获得4个同源重组的ES细胞克隆.挑选其中一个阳性ES克隆行囊胚显微注射,获得16只嵌合率大于50%的雄鼠;其次,利用嵌合雄鼠与C57BL/6J野生型雌鼠交配后获得11只floxP杂合子小鼠(10雄1雌),通过杂合子小鼠回交,并进一步与EⅡa-Cre小鼠交配,最终成功得到Muc1全身敲除小鼠,其中纯合子小鼠未出现胚胎致死现象.初步表型观察未发现Muc1基因敲除相关器官组织结构的异常改变.本研究为MUC1的生物学功能的挖掘,尤其是MUC1在肿瘤发生转移中的作用机制的揭示提供了实验平台.     

Grx-1基因敲除鼠的繁殖及子代基因型鉴定

目的探讨glutaredoxin-1(Grx-1)基因敲除小鼠的优化繁殖及子代鼠的鉴定方法,为进一步研究Grx-1在支气管肺发育不良(BPD)中的作用奠定基础。方法将从美国哈佛医学院引进的纯合子Grx-1基因敲除小鼠与野生型小鼠进行交配后得到的子一代小鼠同代间相互交配,繁殖出的子二代中将出现纯合子、杂合子以及野生型3种基因型。从出生起观察其生长发育情况,2周龄时剪尾提取基因组DNA,用PCR方法扩增目的基因片段,琼脂糖凝胶电泳结果判定基因型。结果 Grx-1纯合子小鼠的饲养繁殖取得成功,获得了一批Grx-1基因敲除纯合子小鼠。结论正确的饲养繁殖以及鉴定方法是获得Grx-1基因敲除纯合子小鼠的有效途径,为相关研究提供动物实验模型奠定了基础。     

Presenilin-1/Presenilin-2双基因敲除小鼠的繁育及基因型鉴定

目的:繁殖及鉴定Presenilins双基因敲除小鼠,为进一步研究阿尔茨海默症(AD)奠定基础。方法:将引进的野生型及PS1/PS2双基因敲除小鼠进行饲养并繁殖,繁殖成功的子代小鼠基因型有野生型、杂合子和纯合子3种。提取子代小鼠鼠尾基因组DNA,用PCR法和琼脂糖凝胶电泳鉴定基因类型。结果:PS1/PS2双基因敲除小鼠的饲养和繁殖均获得成功,繁殖结果符合孟德尔遗传规律,同时获得更多基因型小鼠和Presenilins双基因敲除小鼠。结论:正确的饲养繁殖以及鉴定方法是获得PS1/PS2双基因敲除小鼠的有效途径。     

肝特异性LCMT1基因敲除小鼠模型的制备及鉴定

目的 建立肝特异性LCMT1基因敲除小鼠模型。方法 运用CRISPR/Cas9技术构建肝特异性LCMT1-KO小鼠。通过RT-PCR、实时定量PCR、Western Blot和HE染色等方法鉴定及比较野生和敲除小鼠的差异;观察和分析两组小鼠的一般情况、繁殖能力和子代存活率。结果 成功鉴定子代的基因型;LCMT1-KO小鼠肝LCMT1 mRNA和蛋白质水平显著低于对照组小鼠;两组小鼠的饮食、饮水、体重、繁殖能力、子代存活率、肝外观和HE染色无明显差异;LCMT1-KO小鼠心脏、大脑和肾组织中LCMT1表达与对照组相比没有显著变化;LCMT1-KO小鼠sgRNA未发生脱靶。结论 成功构建肝特异性LCMT1基因敲除小鼠,为研究LCMT1基因在疾病中的调控作用提供实验手段。     

Fmr1基因敲除对小鼠生理发育和繁殖性能的影响

目的对清洁级FVB.KO和FVB的生理发育指标和繁殖性能进行分析比较,探讨Fmr1基因敲除对小鼠生理发育和繁殖性能的影响。方法挑选10周龄清洁级FVB.KO和FVB各20只（雌雄各半）,采取1∶1同居,全部同胞兄妹近交繁殖,测定新生仔鼠生理发育指标和品系繁殖性能。结果 FVB.KO在耳廓分离、体毛长出、门齿萌发、眼睑开裂等生理发育指标上和FVB比较接近,在平均每窝产仔数和离乳率等方面偏低,在雌雄比例上有显著差异。结论 Fmr1基因敲除对小鼠生理发育和繁殖性能影响较小,对后代雌雄比例可能有一定的影响。     

敲除NDRG2 基因的AD模型小鼠的构建与鉴定

目的:构建与鉴定NDRG2基因全身敲除的阿尔茨海默症(AD)小鼠模型。方法:将NDRG2-/-、APP/PS1进行饲养杂交繁殖,将通过PCR技术鉴定基因型为NDRG2+/-APP/PS1的子一代小鼠再与NDRG2-/-小鼠回交获得子二代小鼠,提取子二代小鼠的基因组DNA再利用PCR方法:扩增NDRG2和APP/PS1基因片段并进行琼脂糖凝胶电泳检测获得4种基因型的小鼠。结果:选取基因型为NDRG2-/-APP/PS1的小鼠即为全身NDRG2敲除且淀粉样蛋白基因过表达的阿尔茨海默症模型小鼠。应用PCR方法:鉴定NDRG2基因全身敲除的AD小鼠模型。成功获得NDRG2基因全身敲除的AD小鼠,该基因型小鼠有繁殖能力,其繁殖符合孟德尔遗传规律。结论:成功构建NDRG2基因敲除的阿尔茨海默症模型小鼠,为进一步研究NDRG2基因在阿尔茨海默症病理发展过程中的作用机制及新的治疗方法的研究提供模型基础。     

胸腺上皮细胞特异性敲除GSK-3β基因小鼠模型的鉴定及免疫学特征观察

目的 构建并鉴定胸腺上皮细胞(thymic epithelial cells, TECs)特异性敲除GSK-3β基因小鼠模型,评价其免疫学特征。方法 采用小鼠胚胎干细胞(ES细胞,embryonic stem cells)打靶基因敲除技术,得到F0代的GSK-3β^flox/flox(GSK-3β^f/f)小鼠;再采用Cre-LoxP系统进行小鼠的繁殖;PCR鉴定,筛选出基因型为GSK-3β^f/fFoxN1-Cre^+/-(GSK-3β^-/-)小鼠,即为在TECs特异性敲除GSK-3β基因的小鼠。观察基因敲除鼠的一般生物学特征、繁殖能力和子代存活率。应用HE染色、流式细胞术及免疫荧光技术,比较基因敲除鼠和野生型小鼠免疫器官结构、胸腺、脾及外周血中免疫细胞比例及增殖能力差异。结果 成功构建TECs特异性敲除GSK-3β基因小鼠模型。与野生型(wild type, WT)小鼠相比,GSK-3β^-/-小鼠一般生物学特征无明显差异,子代存活率> 90%;衰老进程中...     

北京地区食蟹猴的室内试验性繁殖初报

目的食蟹猴在北京地区的适应性驯养与繁殖。方法从2004年底开始,建立了一个20只雄猴,140只雌猴的食蟹猴室内生产繁殖种群。每个室内饲养单元内包括两只雄猴,14只雌猴,采取自由交配的方式进行繁殖。同时对室内饲养食蟹猴繁殖的季节性、妊娠期、月经周期、仔猴出生体重等进行了观察、记录和统计。结果从2005年4月到2008年3月的3年时间,母猴怀孕305例,流产59例,生产仔猴246只。平均妊娠率、产仔率分别为74.0%和59.7%。室内饲养食蟹猴的繁殖没有明显的季节性,平均月经周期为（28.5±3.3）d（n=30）,平均妊娠期为（167±12）d（n=30）,仔猴的平均出生体重为（350±120）g（n=30）。结论食蟹猴能够在温带地区的室内进行成功的驯养和繁殖。     

乳腺上皮细胞特异性敲除Scrib基因杂合子小鼠的制作与鉴定

目的利用Cre．LoxP重组酶系统构建乳腺上皮细胞特异性敲除Serib基因杂合子小鼠,并进行鉴定,为进一步在动物整体水平研究Scrib基因在乳腺癌中的作用提供研究平台。方法将Scrib条件敲除杂合子小鼠（Scrib＋/ft小鼠）进行繁殖并鉴定,然后将鉴定结果为阳性的子代Scrib＋/ft小鼠与乳腺上皮细胞特异性表达Cre重组酶的MMTV．Cre纯合子小鼠进行杂交,鉴定其子代小鼠的基因型。结果成功繁育Scrib条件敲除小鼠和MMTV．Cre小鼠,并通过鉴定得到Scrib＋/ft小鼠,与MMTV-Cre小鼠杂交并繁殖,获得基因型为Scrib＋/ft;MMTVCre＋/-小鼠5只。结论本研究利用Cre．LoxP重组酶系统成功构建了乳腺上皮细胞特异性敲除Scrib基因杂合子小鼠,为进一步研究极性蛋白Scrib表达下调在乳腺癌发生中的作用提供了良好的动物模型。     

不同繁育饲养设施对Beagle犬生产性能的影响

目的探索不同繁育饲养设施对Beagle犬主要生产性能的影响。方法使用两种不同Beagle犬繁育饲养设施（设施Ⅰ、设施Ⅱ）对母犬产仔状况、仔犬初生重、60 d离乳犬只存活率、60 d离乳犬只体重、1～6月龄犬只体重月增长等生产性能进行了比较研究。结果两种设施比较母犬产仔状况,仔犬初生重无差异（P〉0.05）;设施Ⅱ比设施Ⅰ的60 d离乳犬只存活率高18.4%,差异极显著（P〈0.01）,60日龄离乳犬只体重平均多（0.62±0.13）kg,差异极显著（P〈0.01）;1～6月龄犬只体重月增长平均多0.29±0.14 kg,差异近显著（P=0.08〉0.05）。结论设施Ⅱ优于设施Ⅰ。     

凝血因子IX基因剔除小鼠的培育历史与建系方法

目的　培育凝血因子IX基因剔除小鼠近交系。方法　采用全同胞兄妹对交配 ,每代选用第一胎小鼠留种 ,记录繁殖性能 ;第 8代开始筛选纯合子。结果　凝血因子IX基因剔除小鼠已近交培育到第 12代 ,交配年龄 70 - 90d ,平均每窝产仔数 5只以上 ,离乳数 3只以上。结论　纯合子FIX剔除基因小鼠在子代鼠中能稳定遗传     

Effect of birth litter size, birth parity number, growth rate, backfat thickness and age at first mating of gilts on their reproductive performance as sows

The present study was performed to evaluate retrospectively the influence of birth litter size, birth parity number, performance test parameters (growth rate from birth to 100kg body weight and backfat thickness at 100kg body weight) and age at first mating (AFM) of gilts on their reproductive performance as sows. Traits analysed included remating rate in gilts (RRG), litter size, weaning-to-first-service interval (WSI), remating rate in sows and farrowing rate (FR). Data were collected from 11 Swedish Landrace (L) and 8 Swedish Yorkshire (Y) nucleus herds and included 20712 farrowing records from sow parities 1-5. Sows that farrowed for the first time during 1993-1997, having complete records of performance test and AFM, were followed up to investigate their subsequent reproductive performance until their last farrowing in 1999. Analysis of variance and multiple regression were applied to continuous data. Logistic regression was applied to categorical data. The analyses were based on the same animals and the records were split into six groups of females, i.e. gilts, primiparous sows, and sows in parities 2-5, respectively. Each additional piglet in the litter in which the gilt was born was associated with an increase of her own litter size of between 0.07 and 0.1 piglets per litter (P<0.001). Gilts born from sow parity 1 had a longer WSI as primiparous sows compared with gilts born from sow parity 4 (0.3 days; P<0.05) or parity 5 (0.4 days; P<0.01). Gilts with a higher growth rate of up to 100kg body weight had a larger litter size (all parities 1-5; P<0.05), shorter WSI (all parities 1-5; P<0.05) and higher FR (parities 2 and 5; P<0.05) than gilts with a lower growth rate. Gilts with a high backfat thickness at 100kg body weight had a shorter WSI as primiparous sows (P<0.001) compared with low backfat gilts, and 0.1 piglets per litter more as second parity sows (P<0.01). A 10 day increase in AFM resulted in an increase in litter size of about 0.1 piglet for primiparous sows (P<0.001) and a decrease (P<0.05) for sow parities 4 and 5.     

Lack of the intestinal Muc1 mucin impairs cholesterol uptake and absorption but not fatty acid uptake in Muc1-/- mice

Before cholesterol and fatty acid molecules in the small intestinal lumen can interact with their possible transporters for uptake and absorption, they must pass through a diffusion barrier, which may modify the kinetics of nutrient assimilation. This barrier includes an unstirred water layer and a surface mucous coat, which is located at the intestinal lumen-membrane interface. In the present study, we investigated whether disruption of the mucin gene (Muc)1 may influence intestinal uptake and absorption of cholesterol and fatty acid in male Muc1(-/-) mice. The wild-type mice displayed relatively high levels of Muc1, Muc2, Muc3, and Muc4 mRNAs and relatively low levels of Muc5ac and Muc5b mRNAs in the small intestine. The absence of Muc1 mRNA and protein in the small intestines of Muc1(-/-) mice confirmed complete knockout of the Muc1 gene, but the mRNA expression for other mucin genes remained unchanged. Intestinal uptake and absorption of cholesterol but not palmitic acid were significantly reduced in Muc1(-/-) mice compared with the wild-type mice. However, knockout of the Muc1 gene did not impair either expression levels of the genes that encode intestinal sterol efflux transporters Abcg5 and Abcg8 and fatty acid transporter Fatp4 or small intestinal transit rates. We conclude that physiological levels of the epithelial mucin produced by the Muc1 gene are necessary for normal intestinal uptake and absorption of cholesterol in mice. Our study implies that because cholesterol absorption efficiency is reduced by approximately 50% in Muc1-deficient mice, there may be one or more additional pathways for cholesterol absorption.     

Targeted disruption of the murine mucin gene 1 decreases susceptibility to cholesterol gallstone formation

Gallbladder mucins play a critical role in the pathogenesis of cholesterol gallstones because of their ability to bind biliary lipids and accelerate cholesterol crystallization. Mucin secretion and accumulation in the gallbladder is determined by multiple mucin genes. To study whether mucin gene 1 (Muc1) influences susceptibility to cholesterol cholelithiasis, we investigated male Muc1-deficient (Muc1(-/-)) and wild-type mice fed a lithogenic diet containing 1% cholesterol and 0.5% cholic acid for 56 days. Gene expression of the gallbladder Muc1 and Muc5ac was significantly reduced in Muc1(-/-) mice in response to the lithogenic diet. Muc3 and Muc4 levels were upregulated and were similar between Muc1(-/-) and wild-type mice. Little or no Muc2 and Muc5b mRNAs were detected. Muc1(-/-) mice displayed significant decreases in total mucin secretion and accumulation in the gallbladder as well as retardation of crystallization, growth, and agglomeration of cholesterol monohydrate crystals. At 56 days of feeding, gallstone prevalence was decreased by 40% in Muc1(-/-) mice. However, cholesterol saturation indices of gallbladder bile, hepatic secretion of biliary lipids, and gallbladder size were comparable in Muc1(-/-) and wild-type mice. We conclude that decreased gallstone formation in mice with disrupted Muc1 gene results from reduced mucin secretion and accumulation in the gallbladder.     

Genetic parameters for litter size at birth and kidding interval in the West African Dwarf goats

A total of 587 kidding records of West African Dwarf (WAD) goats over 10 years (1982–1991) were used. Mean kidding interval and litter size at birth were 275.68 ± 6.08 days and 1.79 ± 0.05 kids, respectively. Effects of parity, season and year of birth were significant (P < 0.05) for kidding interval. Parity also had significant effect (P < 0.05) on litter size at birth. Heritability estimates of 0.35 ± 0.05 and 0.32 ± 0.07 were obtained from sire and sire-dam groups, respectively, for litter size at birth. Heritability estimate for kidding interval was 0.03 ± 0.01 from sire-dam group. Repeatability estimates of 0.38 ± 0.05 and 0.33 ± 0.03 were obtained for litter size at birth while 0.06 ± 0.04 and 0.04 ± 0.02 were obtained for kidding interval from dam and sire-dam group, respectively. Selection for multiple birth is likely to result in larger litter size while culling of does for long kidding interval should be based on more than two records.     

Body weight loss during lactation and its influence on weaning-to-service interval and ovulation rate in Landrace and Yorkshire sows in the tropical environment of Thailand

The aim of this study was to investigate the ovulation rate and the weaning-to-service interval (WSI) of sows in relation to their body weight loss during lactation in tropical climatic conditions. Effect of lactation length (LL), number of total born piglets, number of live born piglets, litter birth weight, average piglet birth weight, number of pigs weaned, litter weaning weight and average pig weaned weight on sow weight loss during lactation were also studied. This study was conducted in two commercial purebred sow herds (A, B) in the central part of Thailand from August to December 1997. The herds had both Landrace (L) and Yorkshire (Y) sows. The 123 sows (55 L and 68 Y) in herd A and 153 sows (95 L and 58 Y) in herd B, parity 1-4, were weighed within 4 days after farrowing and at weaning. Lactation length, litter size at birth and at weaning, litter weight at birth and at weaning, and WSI were recorded for each of these sows. In herd A, 52 sows (20 L and 32 Y) were examined once by laparoscopy between days 8 and 14 after AI-service. These sows had farrowed at least seven piglets in the previous parturition. The numbers of corpora lutea (CL) in both ovaries were counted, and were assumed to equal the ovulation rate. L-sows had significantly (P < 0.05) higher relative weight loss during lactation (RWL) than Y-sows. The RWL increased by 0.7% for each extra pig weaned. When LL increased by 1 day, within the interval of 17-34 days, RWL decreased by 0.6%. Sows with a high weight loss had significantly (P < 0.05) longer WSI than sows with medium or low weight loss. Weight loss had a significant (P < 0.05) effect on WSI in parity 1 and 2 sows. Y-sows had more CL than L-sows (15.7 versus 14.0) (P < 0.05). RWL, parity and regression on lactation length had no significant effect on number of CL. In conclusion, sows with higher number of pigs weaned lose more weight. Under the restricted feeding regime applied, high weight loss during lactation prolongs WSI in parity 1 and 2 sows, but has no influence on the ovulation rate at first oestrus after weaning. The ovulation rate is higher in Yorkshire than in Landrace sows. The ovulation rate is independent of parity.     

The importance of farrowing to service interval in sows served during lactation or after shorter lactation than 28 days

In a retrospective study, based on data from the national litter recording system, farrowing rate and litter size of sows served (inseminated or mated) during the lactation period (n = 574) or after a lactation period shorter than 28 days (n = 14,219) were analysed. The results were compared with the corresponding figures for sows with lactation length between 28 and 35 days and weaning to first service interval of 4 or 5 days (reference group; n = 41,741). The farrowing rate of the reference group was 80.9% and subsequent litter size was 13.7 total piglets born. Among sows served prior to weaning, the farrowing rates and litter sizes were significantly lower for those served earlier than 22 days post-farrowing compared to those served later (P < 0.05). Shorter lactations than 28 days and service within 10 days post-weaning led to lower farrowing rates than in the reference group (P < 0.01). Significant differences were seen after different lactation lengths. After correction for weaning to service interval, preceding litter size weaned, parity, breed and the interaction between parity and breed, litter size was significantly and positively associated with the preceding lactation length. The study shows that service within the first 3 weeks post-farrowing results in reduced reproductive performance.     

Reproductive performance of Farafra ewes in the subtropics

The present study was carried out to assess the reproductive performance of Farafra ewes—a breed with potential economic importance in the subtropics. A total of 262 ewes were categorized according to age, parity, and postpartum interval. Ewes were introduced for breeding in fall, winter and late spring seasons. Fertility, prolificacy, lamb birth weight, and ultrasonic fetal parameters were recorded. Results obtained showed that litter size was increased with age and parity. Lamb birth weight was affected by season. None of the parameters studied had a clear-cut effect on fertility. However, an interaction between season and parity was detected. Measurement of fetal size in the first half of gestation did not predict birth weight. The obtained data represents the first record of the reproductive performance of Farafra ewes in the subtropics, which could be used to anticipate their performance in different seasons.     

Detailed O-glycomics of the Muc2 mucin from colon of wild-type, core 1- and core 3-transferase-deficient mice highlights differences compared with human MUC2

The heavily O-glycosylated mucin MUC2 constitutes the major protein in the mucosal layer that acts as a physical barrier protecting the epithelial layer in the colon. In this study, Muc2 was purified from mucosal scrapings from the colon of wild-type (WT) mice, core 3 transferase knockout (C3Gnt(-/-)) mice and intestinal epithelial cell-specific core 1 knockout (IEC C1Galt1(-/-)) mice. The Muc2 O-glycans were released by reductive β-elimination and analyzed with liquid chromatography-mass spectrometry in the negative-ion mode. Muc2 from the distal colon of WT and C3Gnt(-/-) knockout mice carried a mixture of core 1- or core 2-type glycans, whereas Muc2 from IEC C1Galt1(-/-) mice carried highly sialylated core 3- and core 4-type glycans. A large portion of NeuAc in all mouse models was positioned on disialylated N-acetyllactosamine units, an epitope not reported on human colonic MUC2. Mass spectra and proton NMR spectroscopy revealed an abundant NeuAc linked to internally positioned N-acetylglucosamine on colonic murine Muc2, which also differs markedly from human MUC2. Our results highlight that murine colonic Muc2 O-glycosylation is substantially different from human MUC2, which could be one explanation for the different commensal microbiota of these two species.     

Some Factors Influencing Pregnancy Rate and Subsequent Litter Size in Primiparous Sows

The pregnancy rate and the subsequent litter size were studied in 332 Swedish Yorkshire primiparous sows, fed according to a commercial Swedish feeding regime during lactation. The sows were weighed and backfat depth was recorded at the first farrowing, at weaning, and at mating. Oestrous detection was performed once daily after weaning, and the interval from weaning to first oestrus (IWO) was recorded. Blood samples for determination of plasma progesterone were drawn regularly after the first weaning. Statistical analyses were only performed on sows with an IWO of 3-8 days. Of these 206 sows were mated on their first (OE1 sows) and 87 sows on their second (87 OE2 sows) oestrus after weaning. The pregnancy rate was 85.4% for OE1 sows and 75.9% for OE2 sows (p=0.048). There was no significant difference in pregnancy rate between OE1 sows with an IWO of 3-5 days and OE1 sows with an interval of 6-8 days. OE2 sows with an IWO of 6-8 days, on the other hand, had a significantly lower pregnancy rate compared with OE2 sows with an interval of 3-5 days. The pregnancy rate in sows that lost more than 30 kg during the first lactation period did not differ from that of sows losing less than 30 kg. In sows with a first litter size of more than 9 piglets alive at birth, the pregnancy rate decreases significantly if mating is delayed until the second oestrus after weaning. OE2 sows had a significantly larger second litter size at birth than OE1 sows (+ 2.0). The litter size at six weeks did not, on the other hand, differ significantly (+ 0.4). There was a positive correlation between the IWO and 2nd litter size, although significant only for OE1 sows between the IWO and litter size alive at birth. In the OE1 group, sows losing 20 kg or less during lactation had significantly larger second litters at birth than the sows losing 21-30 kg, but not significantly larger than the sows losing more than 30 kg. One piglet more, at birth, in the first litter resulted in 0.25 piglet more in the second litter. For sows with a large first litter there was a low probability of also having a large second litter.