鸡蛋开窗法对嵌合体鸡制备效率的影响

将受体种蛋分为3组,分别经换壳法、钝端开窗法和赤道面开窗后,比较3种开窗方法对孵化率的影响;分离培养鸡胚胎干细胞(ESCs),体外培养传代后用线性化的质粒p EGFP-N1转染鸡ESCs,比较显微注射时3种不同的处理方法对孵化率的影响;对获得的嵌合体鸡分别提取血液和组织DNA后进行PCR检测。结果表明:赤道面开窗法的孵化率显著高于换壳法和钝端开窗法(P0.01);开窗后,显微注射经转染的ESCs组孵化率显著低于其他2组(P0.01);PCR检测结果显示,有四只嵌合体鸡的部分器官表达了EGFP基因,其中两只鸡的性腺发生EGFP基因的嵌合。表明赤道面开窗后,对受体鸡胚下腔显微注射经转染的ESCs可以生产嵌合体鸡,产生嵌合体鸡的效率为4.17%。     

制作石蜡切片验证鸡胚盘显微注射外源DNA的效果

胚盘下腔是禽类胚胎发育到囊胚时出现的特有腔室,通过向禽类胚盘下腔注射外源细胞或基因是常用的制备嵌合体和转基因禽类的方法。本试验通过制备石蜡切片,验证胚盘下腔显微注射效果的方法。以白来航鸡种蛋为模型,向胚盘下腔中微注射少量的标记物,经过琼脂糖包埋脱水后制作石蜡切片,获得完整的鸡胚Ⅹ期胚盘切片。经过HE染色,可以观察到鸡胚Ⅹ期胚盘的上下胚层及胚盘下腔,鉴定标记物是否准确注入胚盘下腔中。结果表明利用本方法可以获取完整胚盘各个切面,准确检验胚盘显微注射效果,为改进显微注射方法和提高转基因禽类制备效率提供新的有利途径。     

鸡胚大头开孔原壳体外培养方法的建立

通过原壳体外培养和抽蛋清的方法,就胚龄、蛋清抽取量对鸡胚发育的影响进行了研究。结果表明:①0日龄鸡胚抽取1mL、3mL和6mL蛋清后,其孵化率分别为38.6%,30.35%和8.6%。抽取蛋清1mL组(P<0.01)和3mL组种蛋(P<0.05)的孵化率明显优于抽取6mL组。②对3日龄鸡胚,抽取9mL蛋清后,鸡胚的发育受到的影响最大,其孵化率仅为33.3%,明显低于3mL和6mL组(P<0.01);而抽取3mL和6mL蛋清对3日龄鸡胚的影响较小,其孵化率仍高达74.3%和76.5%。③抽取6mL蛋清并在大头顶端开直径为1.0cm的圆孔进行鸡胚原壳体外培养获得成功,3日龄鸡胚的孵化率为18.5%。     

利用CRISPR/Cas9腺病毒载体在鸡胚中进行基因敲入研究

为研究CRISPR/Cas9腺病毒载体在鸡胚中进行基因敲入的可行性,将包装不同滴度增强型绿色荧光报道基因(Enhance green fluorescent protein,EGFP)的腺病毒载体和慢病毒载体显微注射到HH14时期鸡胚的外周血管中,对胚胎发育至3.5 d和9 d鸡胚存活、各器官中EGFP荧光强度等指标进行检测;将包装CRISPR/Cas9系统的腺病毒载体和Donor片段的腺病毒载体共同显微注射到鸡胚后,对成年公鸡各器官中EGFP荧光长期表达、EGFP基因敲入等指标进行检测。结果显示:两种病毒载体对鸡胚心脏和性腺的转染效率呈现显著的滴度依赖性,但1×1011TU/mL的EGFP腺病毒载体将9 d鸡胚存活率从93.18%显著降低至64.11%(P<0.05);选择滴度为1×1010TU/mL的CRISPR/Cas9腺病毒和Donor腺病毒载体进行EGFP的基因敲入,注射后成年公鸡多个组织均能稳定表达绿色荧光,PCR检测证明该荧光来自EGFP的基因敲入。研究提示:腺病毒载体比慢病毒载体具有更高的鸡胚转染效率,使用CRISPR/Cas9腺病毒载体可以在鸡胚中实现高效的EGFP基因敲入。     

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。     

鸡胚胎干细胞的分离及其嵌合体制备条件探索

分离培养鸡胚胎干细胞(ESCs),体外培养传代后,进行碱性磷酸酶活性检测和SSEA-1染色鉴定;并用线性化的质粒pEGFP-N1转染鸡ESCs。受体种蛋经赤道面开窗后,将经转染的ESCs注射到受体鸡胚胚下腔,以便制作嵌合体鸡;对获得的嵌合体鸡分别提取血液和组织DNA后进行PCR检测。结果表明:5只存活的的嵌合体鸡血液中没有发生EGFP基因嵌合,但有4只嵌合体鸡的部分器官表达了EGFP基因,其中有2只鸡的性腺发生嵌合,表明利用赤道面开窗后对受体鸡进行胚下腔显微注射可以生产嵌合体鸡。     

胚蛋注射L-精氨酸对蛋雏鸡孵化性能、1~14日龄肠道发育及血清生化指标的影响

本试验旨在研究胚蛋注射L-精氨酸(L-Arg)对蛋雏鸡孵化性能、1~14日龄肠道发育和血清生化指标的影响。选取47周龄京红1号蛋鸡种蛋1080枚,随机分为3组,分别为未注射对照(NC)组、生理盐水注射对照(SC)组和L-Arg注射(Arg)组,每组8个重复,每个重复45枚种蛋。在孵化第17.5天时,Arg组每枚蛋注射0.1 mL 10%的L-Arg溶液(0.85%生理盐水作溶剂,10 mg L-Arg/枚蛋),SC组注射相同剂量的0.85%生理盐水。出壳后,每组选取体重相近的健康母雏120只,随机分为8个重复。试验期14 d。结果表明:1)与NC和SC组相比,胚蛋注射L-Arg对1日龄蛋雏鸡的孵化率和出孵重无显著影响(P>0.05),但显著降低卵黄囊重(P<0.05)。2)与NC和SC组相比,胚蛋注射L-Arg显著提高1日龄蛋雏鸡的空肠长度、3日龄时的十二指肠长度和14日龄时的空肠和回肠长度(P<0.05),对3日龄蛋雏鸡空肠内容物脂肪酶活性有增加趋势(P=0.084)。3)与NC组相比,胚蛋注射L-Arg显著提高14日龄蛋雏鸡的空肠上皮细胞增殖指数(P<0.05)。4)与NC组相比,胚蛋注射L-Arg显著降低3和14日龄蛋雏鸡的血清甘油三酯(TG)含量(P<0.05),显著增加3和14日龄时的血清总胆固醇(TC)和低密度脂蛋白(LDL)含量(P<0.05)。与SC组相比,胚蛋注射L-Arg显著增加14日龄蛋雏鸡的血清高密度脂蛋白(HDL)和葡萄糖(GLU)含量(P<0.05)。由此可见,胚蛋注射10 mg L-Arg对蛋雏鸡的孵化率和出孵重等孵化性能无不良影响;胚蛋注射10 mg L-Arg促进蛋雏鸡的脂质代谢和糖代谢,为早期肠道发育提供更多能量,提高空肠上皮细胞增殖指数,增加肠道长度,促进早期肠道发育。     

种蛋开窗封口方法对转基因鸡胚孵化和发育的影响

试验旨在优化一种方便快捷高效的开窗封口方法,检测其对鸡胚孵化和生长发育的影响。将种蛋孵化至HH14阶段,在钝端开一小孔进行胚胎外源DNA显微注射,然后分别用医用胶布、医用胶布+石蜡膜两种方法进行封口,继续孵化至出雏。检测孵化第7、14和21天的鸡胚成活率、孵化率及初生体重。结果表明,胶布封口孵化率高于胶布+石蜡膜,两组孵化率虽然显著低于正常种蛋孵化率,但优于传统封口方法。两种封口方法对雏鸡初生重影响不明显,与对照组差异不显著。研究为转基因鸡制备提供了一种方便有效的开窗封口方法,为提高转基因鸡效率奠定基础。     

鸡胚盘内转染外源基因的研究

采用显微注射法，分别将人生长激素(hGH)基因和人组织激肽释放酶基因(KLK1)直接注射到孵化24h的鸡胚中，继续孵化。在注射的320枚鸡胚中，孵化前7d死亡194枚，8～18d死亡108枚，孵出雏鸡18只，孵出率为5.6％(18/320)。8只雏鸡分别于出壳后的第1、2、3、5天死亡。7只于3月龄死亡。3只鸡(1公2母)存活至今，并开始产蛋。死亡雏鸡具有脚瘫、体弱、斜颈、站立不稳等异常表现，存活鸡无外观异常，但有产蛋紊乱现象。从孵化4d后死亡的150放鸡胚和18只出壳雏鸡的组织提取基因组DNA，并用DNA杂交试验进行基因整合检测，结果发现其中的3枚鸡胚和3只雏鸡为人生长激素基因整合阳性，基因整合率为12.5％(6/48)；18枚鸡胚和6枚雏鸡为KLK1整合阳性，整合率为22.2％(24/108)。结果表明：鸡胚盘内细胞通过显微注射法可以转染外源基因。     

胚盘下腔显微注射慢病毒载体制备转基因鹌鹑

利用显微注射法给每枚新鲜鹌鹑种蛋胚盘下腔注射滴度为1×109 TU/mL人类免疫缺陷病毒I型(human immunodeficiency virus-1,HIV-1)慢病毒载体1μL,直接封口后孵化出雏,应用倒置荧光显微镜及分子生物学方法检测。结果显示:在注射慢病毒后第4天,倒置荧光显微镜下观察到发育胚胎卵黄膜上绿色荧光蛋白较强表达;试验操作240枚鹌鹑种蛋,孵化出雏34只,孵化率为14.1%;对新出雏鹌鹑解剖后,可检测到喙部、眼部、羽毛、肝脏、肾脏、盲肠、肺脏、小肠、大肠、输卵管、心脏、胸肌及大脑等内脏器官中绿色荧光蛋白广泛性表达,但在性腺中绿色荧光蛋白表达信号较弱;对发育到性成熟期的G0代19只雄性鹌鹑精液基因组聚合酶链式反应(polymerase chain reaction,PCR)检测,其中3只为阳性,阳性率为15.7%(3/19);在3只阳性雄性鹌鹑的236只后代中,有4只经PCR及印迹杂交(Southern-blot)检测为阳性,阳性率为1.69%(4/236)。结果表明:利用慢病毒载体胚盘下腔注射成功生产出转基因鹌鹑,为转基因禽类制备提供了一种简便易行的好方法。     

Fluorescence expression by bovine embryos after pronuclear microinjection with the EGFP gene.

Fluorescence expression by bovine embryos was examined after pronuclear microinjection with an enhanced green fluorescent protein (EGFP) cDNA under control of the chicken beta-actin promoter and cytomegalovirus enhancer, as a first step in evaluating the applicability of EGFP for non-invasive selection of transgenic bovine embryos. After injection, developmental competence of the embryos was reduced, and light was emitted in 11.9% of them (37/310) under a fluorescence microscope. Although 2.9% of the injected embryos developed to the fluorescent blastocysts (9/310), a majority of the fluorescent embryos showed mosaic expression including the negative blastomeres (26/37, 70.3%). These results suggest the feasibility of EGFP for in vitro selection of transgenic bovine embryos by fluorescence microscopy. However, the impaired development and high frequency of mosaicism were observed in these injected embryos.     

Transgenic chimera quail production by microinjecting lentiviral vector into the blood vessel of the early embryo

In the past, several strategies have been used to generate transgenic birds. The most successful method has proven to be injection of lentiviral vector into the subgerminal cavity of the newly laid egg. In this study, we directly injected lentiviral vector into the blood vessel of HH13–15 quail embryos to produce transgenic chimeras. In the manipulated, hatched birds, the green fluorescent protein (GFP) gene driven by a cytomegalovirus (CMV) promoter was extensively expressed. All tissues analyzed were GFP‐positive, and gonad cells from some of the manipulated embryos expressed GFP. The semen genome of 21.4% of mature male birds was determined to be GFP‐positive by PCR, indicating these male birds were transgenic chimeras.     

影响猪ICSI转基因技术效率的主要因素研究

以猪体外成熟卵子和冷冻解冻的死精子为材料,以pEGFP-N1为模式基因,探讨注射台温度、激活后6-DMAP的处理和精子与PEGFP-N1孵育液添加BSA(牛血清白蛋白)对精子胞质内注射(ICSI)转基因效率的影响。结果表明:注射台温度为30℃时的阳性率为40.07%,而38.5℃时为20.97%,差异极显著(P<0.01)。添加BSA的囊胚转基因率为55.56%,对照组为33.33%,差异极显著(P<0.01)。6-DMAP处理组与对照组的转基因率分别为52.53%和26.25%,差异极显著(P<0.01);而且6-DMAP处理组的囊胚率(9.96%)显著高于(P<0.05)对照组(2.30%)。研究表明注射台温度对转基因效率有明显影响,温度高转基因率低;精子与PEGFP-N1孵育液添加BSA对转基因胚胎发育有一定促进和保护作用,有利于提高囊胚转基因率;激活后用6-DMAP处理能提高转基因率和囊胚率。     

ZB transposon and chicken vasa homologue (Cvh) promoter interact to increase transfection efficiency of primordial germ cells in vivo

ABSTRACT

1. In order to increase the efficiency of generating transgenic chicken, this trial focused on two points: primordial germ cells (PGCs）transfection in vivo and a germline-specific promoter.

2. In order to transfect PGCs in vivo, two plasmids (pZB-CAG-GFP, pCMV-ZB）were co-injected into chicken embryos via the subgerminal cavity at Hamburger and Hamilton (HH) stage 2–3 or via blood vessel at HH stage 13–14. Results showed that the percentage of GFP+ embryos, viability and hatching rate of embryos injected at HH stage 13–14 were significantly higher than that at HH stage 2–3.

3. Two plasmid transposon systems were used for chicken embryo micro-injections. The donor plasmid, with a green fluorescent protein (GFP) reporter gene, was mediated by the ZB transposon. The helper plasmid was a transposase expression vector driven by the promoter of the chicken vasa homologue (Cvh) gene or Human cytomegalovirus (CMV) promoter. Results showed that 60.98% of gonads in Cvh group expressed GFP, which was 52.50% higher than seen in the CMV group. Only gonad tissue from the Cvh group showed any GFP signal, whereas both gonads and other tissues in the CMV group showed green fluorescence.

4. The data suggested that ZB transposon-mediated gene transfer was efficient for transfecting PGCs in vivo; the Cvh promoter drove the transposase gene specifically in the germline and increased the efficiency of germline transmission. Blood vessels injection at HH stage 13–14 may be a more efficient route for PGCs transfection in vivo.     

钙调素基因启动子序列多态性对京海黄鸡产蛋性状和蛋壳性状的遗传效应

本试验旨在分析钙调素(Calmodulin,CAM)基因启动子区域碱基多态性与京海黄鸡产蛋性状和鸡蛋蛋壳质量性状间的关系。采用PCR-SSCP法检测京海黄鸡CAM基因启动子区域碱基多态性。结果表明:京海黄鸡CAM基因启动子区域有3个SNPs(G326A、A327G、C366T),PCR扩增产物SSCP出现6种基因型:AA、AB、AC、BB、BC和CC。AA基因型个体的300日龄产蛋数和66周龄产蛋数显著高于BB型个体(P<0.05)。AA型个体的蛋壳强度和蛋壳重显著小于AB、BB、AC和BC型个体(P<0.05)。CAM基因座对京海黄鸡300日龄蛋重、蛋型指数、蛋壳强度和壳重百分率的主效应指数(MEI)均大于3%。初步推断CAM基因座可能是影响京海黄鸡产蛋数和蛋壳质量性状重要候选基因。     

鸡鸭异种间转基因嵌合体的制备

为研究异种间嵌合体的制作方法及供体和受体的嵌合情况,同时探讨绿色荧光蛋白(pEGFP-N3)基因作为报告基因在转基因动物制作中的应用价值。本研究利用脂质体介导法将外源pEGFP-N3质粒转入到北京鸭原始生殖细胞(PGCs)中,将转染后的PGCs以微注射法转移至受体北京油鸡的胚下腔,探索转基因鸡鸭嵌合体的制作方法。结果显示:PGCs在转染后6 h开始有外源基因的表达,体外培养24 h后获得了33.6%的转染效率。120枚蛋在整个孵化期中共有33枚鸡胚发育,但最终无孵化成活鸡。在13个鸡胚中检测到有外源基因的存在,阳性率为10.8%。PCR扩增禽类W染色体特异性的重复序列发现,8只嵌合体公鸡的性腺都嵌合了供体异性的细胞。研究结果表明所采用的嵌合体制作方法制备转基因禽类是可行的。鸭PGCs能够迁移并定居到鸡胚性腺中,并有可能在鸡性腺中增殖分化成有功能的配子。     

Lethality and bone alterations in chicken embryos and newly hatched chickens given bone-active agents

Studies were undertaken to assess the chicken embryo and newly hatched chicken as models for studying the effects of bone-active agents. Initially, 1,25-dihydroxycholecaliferol (1,25[OH]2D3), sodium fluoride (NaF), parathyroid extract, epidermal growth factor, and prostaglandin E2, were tested for lethality over a broad dose range. One or 3 injections of 1,25(OH)2D3 into the yolk sac of chicken embryos resulted in death of embryos given greater than or equal to 0.1 ng/injection, whereas 0.01 ng was tolerated by the embryos. Administering 1,25(OH)2D3 intraperitoneally to newly hatched chickens as a single injection or weekly for 3 weeks resulted in no deaths at doses up to 50 ng. One or 3 IV injections of 800 micrograms of NaF were lethal to embryos, whereas injections of less than or equal to 400 micrograms were tolerated by the embryo. Giving chickens feed and water containing 2.4 g of NaF/kg was lethal, but no deaths occurred when chickens were given feed containing less than or equal to 1.2 g of NaF/kg. Mortality associated with the administration of epidermal growth factor to embryos was inconsistent, in that death occurred in embryos given a single injection of greater than or equal to 250 ng, but no deaths occurred in embryos given 3 injections at similar doses. Parathyroid extract and prostaglandin E2 were not lethal when administered to embryos and chickens in a single-injection or multiple-injection regimen. Overall, lethality in chicken embryos given a particular agent reflected the dose of bone-active agent injected, rather than the number of injections. Three of the bone-active agents were selected to characterize their microscopic bone effects in chicken embryos and chickens.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-irradiation Removes Endogenous Primordial Germ Cells (PGCs) and Increases Germline Transmission of Donor PGCs in Chimeric Chickens

Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P<0.05). No embryos irradiated with 9 Gy of X-rays survived to hatching. X-irradiation significantly reduced the number of endogenous PGCs in the embryonic gonads at stage 27 in a dose-dependent manner compared with nonirradiated controls. The numbers of endogenous PGCs in the 3-, 6- and 9-Gy-irradiated groups were 21.0, 9.6 and 4.6% of the nonirradiated control numbers, respectively. Sets of 100 donor PGCs were subsequently transferred intravascularly into embryos irradiated with 3 Gy X-rays and nonirradiated control embryos. Genetic cross-test analysis revealed that the germline transmission rate in the 3-Gy-irradiated group was significantly higher than in the control group (27.5 vs. 5.6%; P<0.05). In conclusion, X-irradiation reduced the number of endogenous PGCs and increased the germline transmission of transferred PGCs in chimeric chickens.     

中草药复合添加剂对高龄蛋鸡生产性能、蛋品质及蛋壳超微结构的影响

研究旨在探讨中草药复合添加剂对高龄蛋鸡生产性能、蛋品质及蛋壳超微结构的影响.选取900只440日龄海兰褐蛋鸡,分为对照组、试验组,每组各5个重复,每个重复90只鸡.对照组饲喂基础饲粮,试验组在基础饲粮中添加0.2％中草药复合添加剂,预饲期7d,试验期56 d.结果 显示:与对照组相比,试验组死淘率与破(软)蛋率分别降低...