禽类嵌合体技术及其在转基因中应用的研究进展

本文综述了禽类以囊胚层细胞（ＢＣ）和原生殖细胞（ＰＧＣ）为供体细胞的嵌合体制作技术及其在禽类转基因中应用的研究进展。嵌合体技术作为禽类转基因的手段之一具有诸多优点。迄今,禽类ＢＣ和ＰＧＣ为供体细胞的嵌合体制作,体细胞和种系嵌合程度分析的方法已建立,且具有较高的操作成功率和准确性。嵌合体性别分化规律和繁殖机能已作了初步研究。嵌合体技术应用于禽类转基因的方法路线已建立,导入外源基因的供体细胞在形成嵌合     

转移早期囊胚期细胞制备的嵌合体鸡

用显微注射方法将尼拉商品鸡鸡胚X期细胞液注入同期海兰鸡的囊胚腔中，再经体外培养至出雏，获得尼拉--海兰嵌合体鸡，嵌合体率为5%（3/60），出雏率为15%（9/60）。另一项实验是将受体鸡胚经过600rad^60Co辐射处理后再注入供体细胞并体外培养至出雏，结果嵌合体率为14.0%(8/57），孵化率为15.8%(9/57），其中两只毛色嵌合体公鸡存活至今已经8个月。嵌合体鸡的黑色羽毛可出现在多个部位，有两只鸡的腿部和喙的角质也出现黑色斑。嵌合体鸡血液DNA具有供体鸡的特征带。本研究为进一步制备转基因鸡奠定了基础，也为鸡的发育生物学研究和遗传学研究提供了实验材料。     

猪原始生殖嵴细胞(PGCs)建系因素的研究

从五指山猪(WSZP)近交系第8～13代培育群中，先后选用21头5～10月龄青年母猪，分别于授精后25～30d采集胎儿106个，进行原始生殖嵴(PGCs)细胞分离、培养等建系技术研究。以DMEM F10(1:1)为基础培养液，按添加或不添加生长因子，将培养液分为A、B、C3种，并以STO细胞作饲养层，在38℃、5.0％CO2和湿润的气相中进行培养建系。结果获得胚胎生殖嵴细胞(EG)细胞系6个细胞株，其中1个EG细胞株传至11代、2个传至5代、1个传至4代、2个传至3代冻存。并进行了AKP染色、体外分化、冷冻-解冻复苏和嵌合体制作等鉴定研究。研究发现：不同胚龄对EG细胞建系具有一定影响，不同培养液对EG细胞建系效果不同，STO细胞饲养层的质量是建株、传代、冷冻-解冻复苏的关键因素之一。EG细胞系的初步建立，为今后筛选进入种系的EG细胞系、实施体外基因操作提供了可能。     

囊胚注射转基因ES细胞制作嵌合体的研究

在小鼠胚胎成纤维细胞制作的饲养层上培养并成功的维持了携带LacZ基因的胚胎干细胞系（S8），在此基础上，以S8为供体细胞，以远交系昆明白小鼠3．5d胚胎为受体，通过显微注射法将供体细胞转移到受体的囊胚腔内，经过恢复培养，移植到代孕鼠昆明白雌鼠的子宫中；后代在嵌合体出生一周后进行判定。本试验用8～13代的S8细胞共注射胚胎597枚，经1～3h恢复培养，有585枚胚胎重新具有膨大的囊胚腔，细胞轮廓分明，滋养层细胞间连接也清晰可见，胚胎成活率为97％；胚胎移植后，代孕母鼠共移植胚胎228枚，经17～19d的妊娠期后，产仔37只（2只死胎），产仔率为16％；有35只仔鼠（雄鼠18只，雌鼠17只）存活到可以判断毛色，共获得8只S8细胞毛色嵌合体小鼠，嵌合体的产生率为21．6％。结果表明用S8细胞经囊胚注射后能够获得嵌合体，并且嵌合体明显发生了性偏离现象。本试验为国内利用囊胚注射携带LacZ基因的胚胎干细胞获得嵌合体小鼠的首例报道。     

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。     

胚胎干细胞及种系嵌合体的研究进展

胚胎干细胞是着床前的囊胚内细胞团或早期胎儿的原始生殖细胞经体外分化抑制培养建立的多能性细胞系 ,具有与胚胎细胞相似的形态特征和分化潜能 ,体外培养时保持未分化状态 ,可以传代增殖。改变维持胚胎干细胞不分化的培养条件 ,胚胎干细胞可自发分化成多细胞结构。在一定诱导下 ,胚胎干细胞可向多个方向分化 ,并生成多种功能细胞。胚胎干细胞注入到胚泡期胚胎或与桑椹期胚胎聚合 ,可以参与包括性腺在内的各种组织的嵌合体的形成。胚胎干细胞在细胞分化与调控 ,胚胎发育 ,遗传病 ,肿瘤 ,免疫和组织或器官移植等研究中显示着广泛的应用前景。而种系嵌合体的获得是实现 ES细胞途径的决定步骤 ,低的种系嵌合率则是制约 ES细胞应用的关键。提高供体 PGCs在受体生殖腺中的比例 ,缩短 ES细胞的体外培养时间 ,以及注入早期发育阶段的受体胚胎等都能提高种系嵌合率。文章从多个方面综述了胚胎干细胞的最新研究成果 ,并着重以禽类 ES细胞为例论述了种系嵌合体的检测方法 ,种系嵌合率的影响因素以及提高种系嵌合率的方法     

利用ES细胞制作嵌合体的研究进展

嵌合体一词起源于古希腊神话,是指狮头、羊体、蛇尾的怪物.早在1901年Spemann就对两栖动物(蛙)进行了嵌合体研究,最早培育出嵌合体个体.大约经过半个世纪,人们才开始进行鸟类及哺乳动物嵌合体的研究.     

转基因鸡的研究现状及其应用展望

与哺乳动物的转基因相比，禽类的转基因研究遇到了更大阻力，尽管如此，一些很有希望的生产转基因鸡的方法还是在人们的不断努力下产生了．不少人用基因做注射的方法得到了转基因鸡．转基因鸡的潜在利用价值极大，本综述对目前鸡的转基因研究进行总结并探讨了转基因技术在家禽抗菌育种中的应用及转基因鸡的某些特殊利用价值．     

Secondary sexual traits and semen characteristic of chicken germline chimeras

Birds obtained by embryo engineering are used to study embryo development and to produce transgenic birds. As this method of producing birds still generate strong emotions of the public opinion head ornaments, testes and semen characteristics of sex chimera roosters were examined to check whether they differ from chickens obtained by non‐manipulated methods. Measurements of head ornaments, testes and semen were correlated with each other. Semen quality factor (SQF) was calculated, as well as the level of fluctuating asymmetry (FA) of bilateral traits (wattles and testes). Positive correlation was found for comb width and wattle length and comb thickness and sperm concentration. Semen characteristics and FA did not exceed the level encounter in other chicken lines. Results obtained indicate that germline chimeras are similar in appearance of secondary sexual traits, and semen and testes characteristics to chickens produced in non‐manipulated way.     

Migration and differentiation of gonadal germ cells under cross-sex germline chimeras condition in domestic chickens

A series of experiments was conducted to investigate migration, proliferation and differentiation of gonadal germ cells (GGCs) collected from the gonads of 7-day-old chick embryos under cross-sex germline chimera conditions. The migratory and proliferative abilities of exogenous GGCs were examined by transferring 50 fluorescently labeled GGCs collected from White Leghorn (WL) embryos into the blood of 2-day-old Rhode Island Red (RIR) embryos. No significant difference was observed in the number of fluorescently labeled GGCs in the gonads of recipient embryos among any of the four possible donor and recipient sex combinations. Cross-sex germline chimeras were produced to examine the differentiation of GGCs by transferring 100 GGCs from WL embryos into 2-day-old RIR embryos. Exogenous-GGC-derived progeny were obtained from both male and female recipients, except when female GGCs were transferred into male recipients. The migratory ability of GGCs recovered from the 7-day-old embryonic gonad was not influenced by cross-sex germ cell transfer conditions, whereas the differentiation of the GGCs was affected by the sex combinations of GGCs donors and recipients.     

Development of avian embryo manipulation techniques and their application to germ cell manipulation

The development of chicken embryo culture techniques, from single‐cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single‐cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non‐viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary.     

Production of ovine chimeras by inner cell mass transplantation

Ovine chimeras were produced by micro-injection of isolated inner cell masses (ICM) into recipient blastocysts. Inner cell masses were isolated by immunosurgery. A total of 57 chimeric embryos was produced, 52 of which were transferred to recipient ewes. Thirty-seven live lambs were born, of which 15 were determined to be chimeric on the basis of blood type analysis. One lamb, although not a blood chimera, exhibited overt signs of chimerism. An additional six lambs were determined to have developed solely from the injected ICM. The rate of chimerism in live lambs was 43% (16/37) while the survival rate of injected ICM was 59% (22/37). The method presented allows the production of relatively large proportion of viable, chimeric embryos without the use of an intermediate recipient.     

Production of sheep-goat chimeras by inner cell mass transplantation

Embryos were surgically flushed from goats and sheep on d 6 and 7, respectively, following the first day of estrus (d 0). After enzymatic removal of the zonae pellucidae, inner cell masses were isolated from caprine blastocysts by immunosurgery. The intact inner cell masses were injected into ovine blastocysts with the aid of a micromanipulator. Twenty-two manipulated blastocysts were surgically transferred into 12 ovine recipients. Nine ewes gave birth to a total of 13 young (59% embryo survival). Ten were classified by serum electrophoretic assays or karyotypes as lambs, one as a kid, and two as interspecific chimeras.     

Virulence of five live vaccines against avian infectious laryngotracheitis and their immunogenicity and spread after eyedrop or spray application

Five live virus vaccines against avian infectious laryngotracheitis were studied with regard to safety, immunogenicity and route of administration. Significant differences in virulence between the vaccine strains were found. Reduced virulence was accompanied by a reduction of immunogenicity and capacity to spread. After eyedrop application, a low virulent vaccine induced 90-100% flock immunity for the first 10 weeks after vaccination (PV), followed by a slow decline to 50% at 31 weeks PV, whereas flock immunity induced with the more virulent types remained at about 90% till the end of the experiments (24 and 48 weeks PV). Aerosol vaccination induced 70-100% flock immunity but vaccine reactions were severe. Application of vaccine in a coarse spray did not result in adverse vaccine reactions but induced a maximal protection rate of only 50%. Microneutralisation titres provided a useful indicator of immunity from the onset of immunity until immunity started to decline. A vaccine virus carrier state was demonstrated by means of sentinel birds.     

H5亚型禽流感单克隆抗体的研制及应用

以禽流感H5亚型病毒分别免疫BABL／C小鼠，取其脾细胞与SP2／0骨髓瘤细胞融合，用血凝抑制试验(HI)和间接ELISA检测细胞上清液，结果获得了3株抗禽流感H5亚型病毒特异性单克隆抗体，分别命名为186，1D4，7D1；其中1D4株为抗禽流感H5亚型病毒血凝素特异性单克隆抗体细胞株．186株和7D1株为针对禽流感病毒NP蛋白的单克隆抗体细胞株。经3次亚克隆后，100％杂交瘤细胞保持了分泌抗禽流感病毒抗体的能力。这些单克隆抗体小鼠腹水ELISA效价为10^5，HI效价为2^11-12。研究结果表明，所有这些单克隆抗体仅与相应的禽流感病毒株发生特异性反应，而不与鸡新城疫病毒、产蛋下降综合征病毒、鹅副粘病毒等反应。所有这些单抗在禽流感诊断中将发挥重要作用。     

禽脑脊髓炎病毒单克隆抗体的制备与初步应用

利用提纯的禽脑脊髓炎病毒（AEV）Van Roekel株作为免疫原，免疫6周龄BALB／c小鼠，采取其脾细胞与SP2／0骨髓瘤细胞融合，用间接ELISA法筛选，间接免疫荧光法（IFA）和免疫组织化学法鉴定，经3次亚克隆得到了稳定分泌抗AEV单克隆抗体的杂交瘤细胞株F11和G2，制备了腹水，并利用该单克隆抗体初步建立了Dot—ELISA、间接ELISA和IFA等特异性检测AEV抗原的方法。     

Production and characterization of monoclonal antibodies against an avian group A rotavirus.

Fifteen monoclonal antibodies (MAbs) against an avian group A rotavirus were cloned and characterized. Eight of the 15 MAbs had neutralizing activity (N-MAbs). Five of the N-MAbs (1G1, 5B8, 4E2, 3G1, 2E3) were VP4-specific by radioimmunoprecipitation assay (RIPA), and two N-MAbs (2D11, 6E8) were possibly VP7-specific (faint bands by RIPA). One N-MAb (4H12) of undefined protein specificity cross-reacted with serotype 3 simian rotaviruses. The other seven N-MAbs did not cross-react with any of the eight distinct serotypes of human and mammalian rotaviruses tested. Of the seven non-neutralizing MAbs, three were VP6-specific (3H10, 4B12, 5F6), two were VP8-specific (6C9, 1D1), one was VP4-specific (4E9), and one was of undefined protein specificity (1B11). Four non-neutralizing MAbs recognized only avian group A rotavirus in cell-culture immunofluorescence tests (6C9, 1D1, 4E9 and 5F6), whereas two MAbs (3H10 and 4B12) cross-reacted with all human and animal rotaviruses tested. The MAb 1B11 did not recognize any human rotavirus serotypes but cross-reacted with all nonhuman animal rotavirus serotypes. The MAbs produced in this study should be useful for the detection and further characterization of avian group A rotaviruses.