禽类嵌合体的制作、行为学特征及其转基因研究
摘要
鸡胚盘细胞在第X期还未分化，所以在引入同期发育的受体胚胎时能够参与受体各种组织的发育。本试验以狼山鸡为供体，从肉鸡为受体，通过赤道开窗法制作了狼山鸡-AA肉鸡的嵌合体。试验共操作了108枚鸡蛋，孵化成活19只，孵化成活率17.6％。获得表型嵌合体鸡胚22枚，嵌合率为20.4％，其中15只孵化成活，成活个体中嵌合体比例为78.9％。对2只嵌合体公鸡解剖发现，有一只的睾丸发育异常，通过PCR扩增禽类W染色体特异性的重复序列发现，两只嵌合体公鸡的性腺都嵌合了供体异性的细胞。这一结果表明我们所采用的嵌合体制作方法对于下一步转基因鸡的制备是可行的。
通过对狼山鸡、AA肉鸡和嵌合体鸡的12种行为观察发现，在站立、蹲卧、站立修饰羽毛、站立啄环境、走动或跑动等行为上，狼山鸡的发生频率比AA肉鸡高，其差异极显著(P＜0.01)。比较发现，嵌合体1的这些行为的发生频率都位于狼山鸡相应行为平均值的95％的置信区间之內，其行为模式倾向于供体狼山鸡，嵌合体2和6分别在洗浴和站立行为的发生频率上与供体狼山鸡相似，而其他3只嵌合体鸡的行为模式却与受体AA肉鸡接近。这一制作嵌合体的方法还可用于禽类神经行为学的研究。
利用脂质体介导法在体外将外源pEGFP质粒转入到鸡X期胚胎细胞中。培养8小时开始有外源基因的表达，体外培养12小时后获得了21.43％的转染效率。将转染后的鸡胚盘细胞移入到受体AA肉鸡的胚下腔，孵化6天后在8个鸡胚中检测到有外源基因的存在，阳性率为40％(8/20)。
The X stage avian blastodermal cells have not yet differentiated, and when they were introduced into the same stage embryos of recipients. They could take part in the development of different tissues of the recipients. In the experiment, the langshan is used as the donor and the AA broiler is the recipient, the langshan-AA chimeras were produced via windowing the recipient eggs. 108 eggs were manipulated, and 19 chickens were hatched, the hatching rate was 17.6%. 22 chimeric embryos were obtained, and the chimera rate was 20.4%. 15 chimeras survived, and the survived chimera rate was 78.9%. Abnormal testises were founded in one of the two male chimeras by dissecting. Hetero-sexual cells were found in the gonads of both chimeras by PCR amplifying the W-specific repeating sequences. The result showed that it was possible to produce transgenic avians by using our method of producing chimeras.
By observing the 10 behaviors of the langshans, the AA broilers and the chimeras, Significant differences (p<0.01) were found on standing, sitting, stand preening, stand environment pecking, walking or running between the two control breeds, and the frenquency on these behaviors of chimeral were all located in 95% confidence intervals for means of the langshan, so its behavioral patterm prefered for the donor langshan. Chimera2 and 4 were similar to the langshan chickens separately on the performance of dustbathing and standing, while the behavioral pattern of the other three chimeras were close to the recipient. The method of producing chimeras can also be used in avian neuroethology.
Plasmid pEGFP was introduced into the X stage avian balstodermal cells via lipofectamine in vitro, the exogenous gene began to express. After cultured for 12h in vitro the transfecting rate reached 21.43%. The transfected blastodermal cells were injected into the subgerminal cavity of the recipient, after bing hatched for 6 days the exogenous gene
were detected in 40% (8/20) of the embryos examined.
通过对狼山鸡、AA肉鸡和嵌合体鸡的12种行为观察发现，在站立、蹲卧、站立修饰羽毛、站立啄环境、走动或跑动等行为上，狼山鸡的发生频率比AA肉鸡高，其差异极显著(P＜0.01)。比较发现，嵌合体1的这些行为的发生频率都位于狼山鸡相应行为平均值的95％的置信区间之內，其行为模式倾向于供体狼山鸡，嵌合体2和6分别在洗浴和站立行为的发生频率上与供体狼山鸡相似，而其他3只嵌合体鸡的行为模式却与受体AA肉鸡接近。这一制作嵌合体的方法还可用于禽类神经行为学的研究。
利用脂质体介导法在体外将外源pEGFP质粒转入到鸡X期胚胎细胞中。培养8小时开始有外源基因的表达，体外培养12小时后获得了21.43％的转染效率。将转染后的鸡胚盘细胞移入到受体AA肉鸡的胚下腔，孵化6天后在8个鸡胚中检测到有外源基因的存在，阳性率为40％(8/20)。
The X stage avian blastodermal cells have not yet differentiated, and when they were introduced into the same stage embryos of recipients. They could take part in the development of different tissues of the recipients. In the experiment, the langshan is used as the donor and the AA broiler is the recipient, the langshan-AA chimeras were produced via windowing the recipient eggs. 108 eggs were manipulated, and 19 chickens were hatched, the hatching rate was 17.6%. 22 chimeric embryos were obtained, and the chimera rate was 20.4%. 15 chimeras survived, and the survived chimera rate was 78.9%. Abnormal testises were founded in one of the two male chimeras by dissecting. Hetero-sexual cells were found in the gonads of both chimeras by PCR amplifying the W-specific repeating sequences. The result showed that it was possible to produce transgenic avians by using our method of producing chimeras.
By observing the 10 behaviors of the langshans, the AA broilers and the chimeras, Significant differences (p<0.01) were found on standing, sitting, stand preening, stand environment pecking, walking or running between the two control breeds, and the frenquency on these behaviors of chimeral were all located in 95% confidence intervals for means of the langshan, so its behavioral patterm prefered for the donor langshan. Chimera2 and 4 were similar to the langshan chickens separately on the performance of dustbathing and standing, while the behavioral pattern of the other three chimeras were close to the recipient. The method of producing chimeras can also be used in avian neuroethology.
Plasmid pEGFP was introduced into the X stage avian balstodermal cells via lipofectamine in vitro, the exogenous gene began to express. After cultured for 12h in vitro the transfecting rate reached 21.43%. The transfected blastodermal cells were injected into the subgerminal cavity of the recipient, after bing hatched for 6 days the exogenous gene
were detected in 40% (8/20) of the embryos examined.
引文
[1] Watt JM, Petitte JN, Etches RJ. Early development of the chick embry[J]. J. Morphol, 1993,215:165-182.
[2] 马芸，陈系古．嵌合体动物技术研究进展[J]．中国实验动物学报，2002，10(4)：249-254．
[3] 燕海峰，Trelil P，肖后南等．转基因鸡研究进展[J]．中国家禽，2000，22(9)：30-32．
[4] 马玉忠，李赞东，沙金等．供体细胞在鸡-麻鸭嵌合体胚胎中的发育．遗传学报，2001，28(11)：1002～1005．
[5] Brazolot, C.L., Petitte, J.N., Etches, R.J., Verrinder Gibbins, A.M. Efficient transfection of chicken cells by lipofection and introduction of tansfected blastodermal cells into the embryo[J]. Mol. Reprod. Dev, 1991.30, 304-312.
[6] Fekete, D.M., Cepko, C.L. Retroviral infection coupled with tissue transplantation limits gene transfer in the chicken embryo. Proc. Natl. Acad. Sci. U S A, 1993,90:2350-2354.
[7] Petitte JN, Clark ME, Luv G. Production of somatic and germ line chimeras in the chicken by transfer of early blastodermal cells[J]. Development, 1990, 108:185-195.
[8] Thoraval P, Lasserre F, Coudert F, et al. Somatic and germline chicken chimeras obtained from Brown and White Leghorns by transfer of early blastodermal cells[J]. Poult Sci, 1994,73(12):1897-1905.
[9] Ono T, Muto S, Mizutani M. Production of quail chimera by transfer of early blastodermal cells and its use for transgenesis[J]. Jap Poult Sci, 1994, 31(2):119-129.
[10] Naito M, Watanabe M, Kinutani M. Production of quail-chick chimeras by blastodermal cell transfer[J]. Br Poult Sci, 1991,31(1):79-86.
[11] Li ZD, Deng H, Liu CH, et al. Production of duck-chicken chimeras by transferring early blastodermal cells[J]. Poult Sci, 2002,81:1360-1364.
[12] Karagenc L, Cinnamon Y, Ginsgurg M, et al. Origin of primordial germ cells in the prestreak chicken embryo[J]. Dev Genet, 1996,19:290-301.
[13] Ginsburg M. Primordial germ cell development in avians. Poult Sci, 1997, 76 : 91～95.
[14] Didier E, Fargeix N. Germinal population of gonads in some chemical embryos obtained by connecting pieces of Japanese quailand domestic chick blastoderms. Experientia, 1976,32:1333～1334.
[15] Roguska T. Behavior of mouse primordial germ cells in chick embryo. J. Embryo. Exp,
Morph, 1971,25:155～164.
[16]Chang HK, Tajima A, Yasuda Y, et al. Simple method for isolation of primordial germ cells from chick embryos[J], cell Bio Int Rep, 1992,15(9):853-857.
[17]Naito M, Tajima A, Tagami T, et al. Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cell[J]. Mol Repro Dev, 1994,39:153-161.
[18]Yasuda Y, Tajima A, Fujimoto T, et al. A method to obtain avain germ line chimera using isolated primordial germ cells[J]. J Reprod Fert, 1992(96):521-528.
[19]Tajima A, Naito M, Yasuda Y, et al. Production of germline chimera by transfer of primordial germ cells in the domestic chicken (Gallus domesticus)[J]. Theriogenology, 1993,40(3):509-519.
[20]Chang Ik, DK Jeong, YK Hong, et al. Production of germline chimeric chickens by transfer of cultured primordial germ cells[J]. Cell Biol Int, 1997,21:495-499.
[21]Carsience RS, Clark ME, Verrinder Ginbbs AM, et al. Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos [J]. Development, 1993,117:669-675.
[22]Maeda T, Clark ME, Etches RJ. Mortality size of the gonads, and ultrastructure of primordial germ cell in chick embryos treated with gamma-irradiation or injected with donor cells[J]. Poult Sci, 1998, 77(6):905-907.
[23]Furuta H, Fujihara N. Production of exogenously injected primordial germ cells (PGCs) into busulfan-treated chicken embryos[J]. Asian J Andro, 1999,1(4):187-190.
[24]Ginsburg M. Primordial gern cell formation in birds. In Germline Development: Ciba Foundation Symposium 182, 1994, 52-67 Eds J Marsh and J Goode. John Wiley and Sons, Chichester.
[25]Kagami H, Tagami T, Matsubara Y, et al. The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken[J]. Mol Repro Dev, 1997, 48(4):501-510.
[26]Kagami H, Iwata J, Nakata A, et al. Substantial evidence to localize the developmental origin of primordial germ cells in the chicken[J]. Ani Sci J, 2000,71(1):38-41.
[27]Naito M, Sano A, Matsubara Y, et al. Localization of primordial germ cells or their precursors in stage X blastoderm of chickens and their ability to differentiate into functional gametes in opposite-sex recipient gonads[J]. Reproduction, 2001, 121:547-552.
[28]Naito M, Matsubara Y, Harumi T, et al. Differentiation of donor primordial germ cells
into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells isolated from embryonic blood[J]. J Reprod Fert, 1999,117:291-298.
[29]Trefil P, Bruno MM, Mikus T, et al. Sexing of chicken feather follicle, blastodermal and blood cells[J]. Folia Bio Phaha, 1999, 45(6):253-256.
[30]Bermudez-Hunaran LG, Garcia-Garcia A, Lea-Garza CH, et al. Melecμlar sexing of monomorphic endangered Ara birds[J]. J Exp Zoo, 2002,292: 677-680.
[31]Saitoh Y, Mizuno S. Distributions of Xho I and EcoR I family repetitive DNA sequences into separate domains in thechicken W chromosome. Chromosoma, 1992,101:474～477.
[32]Kagami H, Clark ME, Yerrinder Gibbins AM, et al. Sexual differention of chimeric chickens containing ZZ and ZW cells in the germline[J]. Mol Reprod Dev, 1995,42:379-387.
[33]Shaw DL, Carscience RS, Etches RJ, et al. The fate of female donor blastodermal cells in male chimeric chicken[J]. Bioch Cell Biol, 1992,70:1218-1229.
[34]Yamaguchi H, Xi YM, Fujihara N. Inter embryonic(homo- or hetero-sexual)transfer of primordial germ cells between chicken embryos[J]. Cytotechnology, 2000,33:101-108.
[35]Tagami T, Matsubara Y, Hanada H, et al. Differentiation of female chicken primordial germ cells into spermatozoa in male gonads[J]. Dev Growth differ, 1997,39:267-271.
[36]Kino K, Pain B, Leibo SP, et al. Production of chimeras from injection of frozon-thawed blastodermal cells[J]. Poult Sci, 1997, 76(5):753-760.
[37]Tajima A, Naito M, Yasuda Y, et al. Production of germ-line chimeras by transfer of cryopreserved gonadal primordial germ cells (gPGCs) in chicken[J]. J Exp Zoo, 1998, 280(3):265-267.
[38]Bednarczyk M, Lakota P, Slomski R, et al. Reconstitution of a chicken breed by inter se mating of germline chimeric birds[J]. Poult Sci, 2002, 81:1347-1353.
[39]Perry MM. A complete culture system for the chick embryo[J]. Nature, 1988, 331:70-72.
[40]Naito M, Nirasawa K, Oishi T. Development in culture of the chick embryo from fertilized ovum to hatching[J]. J Exp Zool, 1990, 254(3):322-326.
[41]李赞东，周国清，周顺伍等．在代用蛋壳中孵化鸡胚[J]．畜牧兽医学报，1994，25(6)：484-484．
[42]Love J, Gribbin C, Mather C, et al. Transgenic birds by DNA microinjection. Biotechnology, 1994, 12(1):60-63.
[43]Furuta H, Kim KB, Fujihara N. Gene transfer to chicken blastodermal by lipofection
or electroporation[J]. J Appl Anim Res, 2000, 17(2):209-216.
[44]Naito M, Sand A, Tagami T, et al. Efficient transfection of chicken blastoderms in vivo by lipofection and electroporation using green fluorescent protein gene as a marker[J]. Anim Sci J, 2000, 71(4):377-385.
[45]Sand A, Tagami T, Harumi T, et al. Introduction of exogenous DNA into gonads of chick embryos bylipofection and electroporation of stage Ⅹ blastoderms in vivo[J]. Bri poult Sci, 2003, 44:36-39.
[46]Ebara F, Fujihara N. Successful transfer of exogenously introduced gene (lacZ/MiwZ and LacZ&GFP/pkkv-LacZ) for generation in chicken[J]. J Reprod Dev, 2000, 46(3):177-182.
[47]Salter DW, Smith EJ, Hμghes SH, et al. Transgenic chickens: insertion of retroviral genes into the chicken germ line[J]. Virology, 1987, 157(1):236-240.
[48]Bosselman RA, Hsu RY, Boggs T, et al. Germline transmission of exogenous genes in the chicken[J]. Science, 1989, 243(4890):533-535.
[49]Briskin MJ, Hsu RY, Boggs T, et al. Heritable retroviral transgenes are highly expressed in chickens. Proc Nat1Acad Sci U S A, 1991, 88(5):1736-1740.
[50]Chen HY, Garber EA, Mills E, et al. Vectors, promoters, and expression of genes in chick embryos[J]. J Reprod Fertil Suppl, 1990, 41:173-182.
[51]Muramatsu T, Mizutani Y, Ohmori Y, et al. Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo[J]. Biochem Biophys Res Commun, 1997, 230(2):376-380.
[52]Ribeiro LA, Mariani PD, Azevedo JL, et al. A biolistic process for in vitro gene transfer into chicken embryos[J]. Braz J Med Biol Res, 2001, 34(9):1115-1124.
[53]Pandey KK, Patchell MR. Genetic transformation in chicken by the use of irradiated male gametes[J]. Mol Gen Genet, 1982,186(3):305-308.
[54]Bumstead N, Messer LI, Freeman BM, et al. Genetic transformation of chickens using irradiated male gametes[J]. Heredity, 1987, 58:25-30.
[55]Lavitrano M, Camaioni A, Fazio VM, et al. Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice[J]. Cell, 1989, 57(5):717-723.
[56]Gruenbaum Y, Revel E, Yarus S, et al Sperm cells as vectors for the generation of transgenic chickens[J]. J Cell Biochem suppl, 1991, 15E: 194.
[57]Squires EJ, Drake O. Liposome mediated and transfer to chicken sperm cells[J]. Anim Biotech, 1993, 4(11):71～88
[58]杜立新，蒋满喜．脂质体介导精子载体制备转基因鸡的研究[J]．山东畜牧兽医，2001，：1-2．
[59]Etches RJ, Carsience RS,Clark ME, et al. Chimeric chickens and their use in manipulation of the chicken genome[J]. Poult Sci, 1993,72: 882-889.
[60]Etches RJ, Clark ME, Zajchowski L, et al. Manipulation of blastodermal cells[J]. Poult Sci, 1997, 76(8):1075-1083.
[61]Chang HK, Yoshiki A, Kusakabe M, et al. Germ line chimera produced by transfer of cultured chicken primordial germ cells[J]. Cell Bio Int, 1995, 19(7):569-576.
[62]Yang GQ, Fjujihara N. Survival and proliferation of refined chicken circulating primordial gern cells cultured in vitro[J]. J Reprod Dev, 1999, 45(2):177-181.
[63]Park TS, Han JY. Derivation and characterization of pluripotent embryonic germ cells in chicken[J]. Mol Eeprod Dev, 2000,56(4):475-482.
[64]Wei Q, Croy BA, Etches RJ. Selection of genetically modified chicken blastodermal cells by magnetic-activated cell sorting[J]. Poult Sci, 2001, 80(12):1671-1678.
[65]Fraser RA, Carsience RS, Clark ME, et al. Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos [J]. Int J Dev Bio, 1993, 37:381-385.
[66]Han JY, Seo DS, Hong YH, et al. Expression of transfected primordial germ cells in the chicken gonad and germline chimeric chicken[J]. European J Immun, 1994, 24(7):108-111.
[67]Naito M, Sakurai M, Kuwana T. Expression of exogenous DNA in the gonads of chimaeric chicken embryos produced by transfer of primordial germ cells transfeced in vitro and subsequent fate of the introduced DNA[J]. J Reprod Fert, 1998, 113:137-143.
[68]Jeong DK, Han JL. Migration activity of chicken gonadal primordial germ cells (gPGC) and post-transfer localization of lacz-transfected gPGC in the embryonic gonads[J]. Asian-Aust J Anim Sci, 2002,15(9):1227-1231.
[69]王晓通，娄义洲，王晓娜．转基因鸡的制作方法及应用[J]．世界农业，2004，2：48-49．
[70]Balaban E, Teillet MA, Douarin NL. Application of the quail-chick chimera system to the study of brain development and behavior[J]. Science, 1988, 241(4871):1339-1342.
[71]Balaban E. Changes in multiple brain regions underlie species differences in a complex, congenital behavior. Proc Natl Acad Sci U S A, 1997, 94(5):2001-2006.
[72]Long KD, Kennedy G, Balaban E. Transferring an inborn auditory perceptual predisposition with interspecies brain transplants. : Proc Natl Acad Sci U S A, 2001, 98(10):5862-5867.
[73]Inoue I, Yanai K, Kitamura D. et al. Impaired locomotor activity and exploratory behavior in mice lacking histamine H1 receptors. Proc Natl Acad Sci U S A, 1996,
93 (23): 13316-20.
[74]Lubin DA, Elliott JC, Black MC. et al. An oxytocin antagonist infused into the central nucleus of the amygdala increases maternal aggressive behavior[J]. Behav Neurosci, 2003, 117(2) : 195-201.
[75]Powell WS, Dominguez JM, Hull EM. An NMDA antagonist impairs copulation and the experience-induced enchancement of male sexual behavior in the rat[J]. Behav Neurosci, 2003, 117(1) :67-95.
[76]Paredes RG. Medial preoptic area/anterior hypothalamus and sexual motivation. [J] Scand J Psychol, 2003, 44(3):203-212.
[77]Bednarczyk M, Lakota P, Siwek M. Improvement of hatchability of chicken eggs injected by blastoderm cells[J]. Poult Sci, 2000, 79(12):1823-8.
[78]贾彦征，熊一力，刘光泽等．鸡胚培养与嵌合体鸡的制备[J]．中国实验动物学杂志，2001，11(2)：93-94．
[79]刘士寻，燕海峰，肖兵南等．鸡蛋开窗法导入供体胚盘细胞对家鸡胚胎发育的影响研究[J]．生命科学研究，2001，5(3)：270-274
[80]Speksnijder G, Ivarie R. A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs[J]. Poult Sci, 2000, 79(10):1430-1433.
[81]Ono T, Muraknmi T, Mochii K, et al. A complete culture system for avian transgenesis, supporting quail embryo s from the single-cell stage to hatching [J]. Dev Biol, 1994, 16 (1)126-130.
[82]杜立新，李善刚，蒋满喜等．转移早期囊胚期细胞制备嵌合体鸡[J]．中国家禽，2001，23(1)：10-12．
[83]Altmann, J. Observational study of behavior: sampling methods[J]. Behavior, 1974,49:227-267.
[84]Vestergaard K, Hogan JA, Kruijt JP. The development of a behavior system: dustbathing in the Burmese red junglefowle Ⅰ. The influence of the rearing environment on the organization of dustbathing[J]. Behaviour, 1990, 112(1):99-116.
[85]Weeks CA., Danbury TD., Davies HC, et al. The Behavior of broiler chickens and its modification by lameness[J]. Appl. Anim. Behav. Sci, 2000, 67:111-125.
[86]张灵芝，汤建，唐朝枢．脂质体作为基因载体的研究进展[J]．国外医学．分子生物学分册，1993，15(1)：10-12．
[87]Kawaura C, Noguchi A, Furuno T, et al. Atomic force microscopy for studying gene transfection mediated by cationic liposomes with a cationic cholesterol derivative[J].
FEBS Lett, 1998, 421(1):69-72.
[88]Spector DL，Goldman RD，Leinewnd LA[M]．著．黄培堂等译．细胞实验指南，科学出版社，2001年，第一版：786．
[89]Pain B, Chenevier P, Samarut J. Chicken Embryonic Stem cells and transgenic startegies[J]. Cells Tissues Organs, 1999,165(3-4):212-219.
[90]易学瑞，祖萍，刘慧萍．增强型绿色荧光蛋白基因在鸡胚盘细胞中的表达[J]．上海实验动物科学，2001，24(3)：137-140．
[91]刘默芳，王恩多，绿色荧光蛋白[J]．生物化学与生物物理进展，2000．27(3)：238-243．
[2] 马芸，陈系古．嵌合体动物技术研究进展[J]．中国实验动物学报，2002，10(4)：249-254．
[3] 燕海峰，Trelil P，肖后南等．转基因鸡研究进展[J]．中国家禽，2000，22(9)：30-32．
[4] 马玉忠，李赞东，沙金等．供体细胞在鸡-麻鸭嵌合体胚胎中的发育．遗传学报，2001，28(11)：1002～1005．
[5] Brazolot, C.L., Petitte, J.N., Etches, R.J., Verrinder Gibbins, A.M. Efficient transfection of chicken cells by lipofection and introduction of tansfected blastodermal cells into the embryo[J]. Mol. Reprod. Dev, 1991.30, 304-312.
[6] Fekete, D.M., Cepko, C.L. Retroviral infection coupled with tissue transplantation limits gene transfer in the chicken embryo. Proc. Natl. Acad. Sci. U S A, 1993,90:2350-2354.
[7] Petitte JN, Clark ME, Luv G. Production of somatic and germ line chimeras in the chicken by transfer of early blastodermal cells[J]. Development, 1990, 108:185-195.
[8] Thoraval P, Lasserre F, Coudert F, et al. Somatic and germline chicken chimeras obtained from Brown and White Leghorns by transfer of early blastodermal cells[J]. Poult Sci, 1994,73(12):1897-1905.
[9] Ono T, Muto S, Mizutani M. Production of quail chimera by transfer of early blastodermal cells and its use for transgenesis[J]. Jap Poult Sci, 1994, 31(2):119-129.
[10] Naito M, Watanabe M, Kinutani M. Production of quail-chick chimeras by blastodermal cell transfer[J]. Br Poult Sci, 1991,31(1):79-86.
[11] Li ZD, Deng H, Liu CH, et al. Production of duck-chicken chimeras by transferring early blastodermal cells[J]. Poult Sci, 2002,81:1360-1364.
[12] Karagenc L, Cinnamon Y, Ginsgurg M, et al. Origin of primordial germ cells in the prestreak chicken embryo[J]. Dev Genet, 1996,19:290-301.
[13] Ginsburg M. Primordial germ cell development in avians. Poult Sci, 1997, 76 : 91～95.
[14] Didier E, Fargeix N. Germinal population of gonads in some chemical embryos obtained by connecting pieces of Japanese quailand domestic chick blastoderms. Experientia, 1976,32:1333～1334.
[15] Roguska T. Behavior of mouse primordial germ cells in chick embryo. J. Embryo. Exp,
Morph, 1971,25:155～164.
[16]Chang HK, Tajima A, Yasuda Y, et al. Simple method for isolation of primordial germ cells from chick embryos[J], cell Bio Int Rep, 1992,15(9):853-857.
[17]Naito M, Tajima A, Tagami T, et al. Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cell[J]. Mol Repro Dev, 1994,39:153-161.
[18]Yasuda Y, Tajima A, Fujimoto T, et al. A method to obtain avain germ line chimera using isolated primordial germ cells[J]. J Reprod Fert, 1992(96):521-528.
[19]Tajima A, Naito M, Yasuda Y, et al. Production of germline chimera by transfer of primordial germ cells in the domestic chicken (Gallus domesticus)[J]. Theriogenology, 1993,40(3):509-519.
[20]Chang Ik, DK Jeong, YK Hong, et al. Production of germline chimeric chickens by transfer of cultured primordial germ cells[J]. Cell Biol Int, 1997,21:495-499.
[21]Carsience RS, Clark ME, Verrinder Ginbbs AM, et al. Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos [J]. Development, 1993,117:669-675.
[22]Maeda T, Clark ME, Etches RJ. Mortality size of the gonads, and ultrastructure of primordial germ cell in chick embryos treated with gamma-irradiation or injected with donor cells[J]. Poult Sci, 1998, 77(6):905-907.
[23]Furuta H, Fujihara N. Production of exogenously injected primordial germ cells (PGCs) into busulfan-treated chicken embryos[J]. Asian J Andro, 1999,1(4):187-190.
[24]Ginsburg M. Primordial gern cell formation in birds. In Germline Development: Ciba Foundation Symposium 182, 1994, 52-67 Eds J Marsh and J Goode. John Wiley and Sons, Chichester.
[25]Kagami H, Tagami T, Matsubara Y, et al. The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken[J]. Mol Repro Dev, 1997, 48(4):501-510.
[26]Kagami H, Iwata J, Nakata A, et al. Substantial evidence to localize the developmental origin of primordial germ cells in the chicken[J]. Ani Sci J, 2000,71(1):38-41.
[27]Naito M, Sano A, Matsubara Y, et al. Localization of primordial germ cells or their precursors in stage X blastoderm of chickens and their ability to differentiate into functional gametes in opposite-sex recipient gonads[J]. Reproduction, 2001, 121:547-552.
[28]Naito M, Matsubara Y, Harumi T, et al. Differentiation of donor primordial germ cells
into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells isolated from embryonic blood[J]. J Reprod Fert, 1999,117:291-298.
[29]Trefil P, Bruno MM, Mikus T, et al. Sexing of chicken feather follicle, blastodermal and blood cells[J]. Folia Bio Phaha, 1999, 45(6):253-256.
[30]Bermudez-Hunaran LG, Garcia-Garcia A, Lea-Garza CH, et al. Melecμlar sexing of monomorphic endangered Ara birds[J]. J Exp Zoo, 2002,292: 677-680.
[31]Saitoh Y, Mizuno S. Distributions of Xho I and EcoR I family repetitive DNA sequences into separate domains in thechicken W chromosome. Chromosoma, 1992,101:474～477.
[32]Kagami H, Clark ME, Yerrinder Gibbins AM, et al. Sexual differention of chimeric chickens containing ZZ and ZW cells in the germline[J]. Mol Reprod Dev, 1995,42:379-387.
[33]Shaw DL, Carscience RS, Etches RJ, et al. The fate of female donor blastodermal cells in male chimeric chicken[J]. Bioch Cell Biol, 1992,70:1218-1229.
[34]Yamaguchi H, Xi YM, Fujihara N. Inter embryonic(homo- or hetero-sexual)transfer of primordial germ cells between chicken embryos[J]. Cytotechnology, 2000,33:101-108.
[35]Tagami T, Matsubara Y, Hanada H, et al. Differentiation of female chicken primordial germ cells into spermatozoa in male gonads[J]. Dev Growth differ, 1997,39:267-271.
[36]Kino K, Pain B, Leibo SP, et al. Production of chimeras from injection of frozon-thawed blastodermal cells[J]. Poult Sci, 1997, 76(5):753-760.
[37]Tajima A, Naito M, Yasuda Y, et al. Production of germ-line chimeras by transfer of cryopreserved gonadal primordial germ cells (gPGCs) in chicken[J]. J Exp Zoo, 1998, 280(3):265-267.
[38]Bednarczyk M, Lakota P, Slomski R, et al. Reconstitution of a chicken breed by inter se mating of germline chimeric birds[J]. Poult Sci, 2002, 81:1347-1353.
[39]Perry MM. A complete culture system for the chick embryo[J]. Nature, 1988, 331:70-72.
[40]Naito M, Nirasawa K, Oishi T. Development in culture of the chick embryo from fertilized ovum to hatching[J]. J Exp Zool, 1990, 254(3):322-326.
[41]李赞东，周国清，周顺伍等．在代用蛋壳中孵化鸡胚[J]．畜牧兽医学报，1994，25(6)：484-484．
[42]Love J, Gribbin C, Mather C, et al. Transgenic birds by DNA microinjection. Biotechnology, 1994, 12(1):60-63.
[43]Furuta H, Kim KB, Fujihara N. Gene transfer to chicken blastodermal by lipofection
or electroporation[J]. J Appl Anim Res, 2000, 17(2):209-216.
[44]Naito M, Sand A, Tagami T, et al. Efficient transfection of chicken blastoderms in vivo by lipofection and electroporation using green fluorescent protein gene as a marker[J]. Anim Sci J, 2000, 71(4):377-385.
[45]Sand A, Tagami T, Harumi T, et al. Introduction of exogenous DNA into gonads of chick embryos bylipofection and electroporation of stage Ⅹ blastoderms in vivo[J]. Bri poult Sci, 2003, 44:36-39.
[46]Ebara F, Fujihara N. Successful transfer of exogenously introduced gene (lacZ/MiwZ and LacZ&GFP/pkkv-LacZ) for generation in chicken[J]. J Reprod Dev, 2000, 46(3):177-182.
[47]Salter DW, Smith EJ, Hμghes SH, et al. Transgenic chickens: insertion of retroviral genes into the chicken germ line[J]. Virology, 1987, 157(1):236-240.
[48]Bosselman RA, Hsu RY, Boggs T, et al. Germline transmission of exogenous genes in the chicken[J]. Science, 1989, 243(4890):533-535.
[49]Briskin MJ, Hsu RY, Boggs T, et al. Heritable retroviral transgenes are highly expressed in chickens. Proc Nat1Acad Sci U S A, 1991, 88(5):1736-1740.
[50]Chen HY, Garber EA, Mills E, et al. Vectors, promoters, and expression of genes in chick embryos[J]. J Reprod Fertil Suppl, 1990, 41:173-182.
[51]Muramatsu T, Mizutani Y, Ohmori Y, et al. Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo[J]. Biochem Biophys Res Commun, 1997, 230(2):376-380.
[52]Ribeiro LA, Mariani PD, Azevedo JL, et al. A biolistic process for in vitro gene transfer into chicken embryos[J]. Braz J Med Biol Res, 2001, 34(9):1115-1124.
[53]Pandey KK, Patchell MR. Genetic transformation in chicken by the use of irradiated male gametes[J]. Mol Gen Genet, 1982,186(3):305-308.
[54]Bumstead N, Messer LI, Freeman BM, et al. Genetic transformation of chickens using irradiated male gametes[J]. Heredity, 1987, 58:25-30.
[55]Lavitrano M, Camaioni A, Fazio VM, et al. Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice[J]. Cell, 1989, 57(5):717-723.
[56]Gruenbaum Y, Revel E, Yarus S, et al Sperm cells as vectors for the generation of transgenic chickens[J]. J Cell Biochem suppl, 1991, 15E: 194.
[57]Squires EJ, Drake O. Liposome mediated and transfer to chicken sperm cells[J]. Anim Biotech, 1993, 4(11):71～88
[58]杜立新，蒋满喜．脂质体介导精子载体制备转基因鸡的研究[J]．山东畜牧兽医，2001，：1-2．
[59]Etches RJ, Carsience RS,Clark ME, et al. Chimeric chickens and their use in manipulation of the chicken genome[J]. Poult Sci, 1993,72: 882-889.
[60]Etches RJ, Clark ME, Zajchowski L, et al. Manipulation of blastodermal cells[J]. Poult Sci, 1997, 76(8):1075-1083.
[61]Chang HK, Yoshiki A, Kusakabe M, et al. Germ line chimera produced by transfer of cultured chicken primordial germ cells[J]. Cell Bio Int, 1995, 19(7):569-576.
[62]Yang GQ, Fjujihara N. Survival and proliferation of refined chicken circulating primordial gern cells cultured in vitro[J]. J Reprod Dev, 1999, 45(2):177-181.
[63]Park TS, Han JY. Derivation and characterization of pluripotent embryonic germ cells in chicken[J]. Mol Eeprod Dev, 2000,56(4):475-482.
[64]Wei Q, Croy BA, Etches RJ. Selection of genetically modified chicken blastodermal cells by magnetic-activated cell sorting[J]. Poult Sci, 2001, 80(12):1671-1678.
[65]Fraser RA, Carsience RS, Clark ME, et al. Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos [J]. Int J Dev Bio, 1993, 37:381-385.
[66]Han JY, Seo DS, Hong YH, et al. Expression of transfected primordial germ cells in the chicken gonad and germline chimeric chicken[J]. European J Immun, 1994, 24(7):108-111.
[67]Naito M, Sakurai M, Kuwana T. Expression of exogenous DNA in the gonads of chimaeric chicken embryos produced by transfer of primordial germ cells transfeced in vitro and subsequent fate of the introduced DNA[J]. J Reprod Fert, 1998, 113:137-143.
[68]Jeong DK, Han JL. Migration activity of chicken gonadal primordial germ cells (gPGC) and post-transfer localization of lacz-transfected gPGC in the embryonic gonads[J]. Asian-Aust J Anim Sci, 2002,15(9):1227-1231.
[69]王晓通，娄义洲，王晓娜．转基因鸡的制作方法及应用[J]．世界农业，2004，2：48-49．
[70]Balaban E, Teillet MA, Douarin NL. Application of the quail-chick chimera system to the study of brain development and behavior[J]. Science, 1988, 241(4871):1339-1342.
[71]Balaban E. Changes in multiple brain regions underlie species differences in a complex, congenital behavior. Proc Natl Acad Sci U S A, 1997, 94(5):2001-2006.
[72]Long KD, Kennedy G, Balaban E. Transferring an inborn auditory perceptual predisposition with interspecies brain transplants. : Proc Natl Acad Sci U S A, 2001, 98(10):5862-5867.
[73]Inoue I, Yanai K, Kitamura D. et al. Impaired locomotor activity and exploratory behavior in mice lacking histamine H1 receptors. Proc Natl Acad Sci U S A, 1996,
93 (23): 13316-20.
[74]Lubin DA, Elliott JC, Black MC. et al. An oxytocin antagonist infused into the central nucleus of the amygdala increases maternal aggressive behavior[J]. Behav Neurosci, 2003, 117(2) : 195-201.
[75]Powell WS, Dominguez JM, Hull EM. An NMDA antagonist impairs copulation and the experience-induced enchancement of male sexual behavior in the rat[J]. Behav Neurosci, 2003, 117(1) :67-95.
[76]Paredes RG. Medial preoptic area/anterior hypothalamus and sexual motivation. [J] Scand J Psychol, 2003, 44(3):203-212.
[77]Bednarczyk M, Lakota P, Siwek M. Improvement of hatchability of chicken eggs injected by blastoderm cells[J]. Poult Sci, 2000, 79(12):1823-8.
[78]贾彦征，熊一力，刘光泽等．鸡胚培养与嵌合体鸡的制备[J]．中国实验动物学杂志，2001，11(2)：93-94．
[79]刘士寻，燕海峰，肖兵南等．鸡蛋开窗法导入供体胚盘细胞对家鸡胚胎发育的影响研究[J]．生命科学研究，2001，5(3)：270-274
[80]Speksnijder G, Ivarie R. A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs[J]. Poult Sci, 2000, 79(10):1430-1433.
[81]Ono T, Muraknmi T, Mochii K, et al. A complete culture system for avian transgenesis, supporting quail embryo s from the single-cell stage to hatching [J]. Dev Biol, 1994, 16 (1)126-130.
[82]杜立新，李善刚，蒋满喜等．转移早期囊胚期细胞制备嵌合体鸡[J]．中国家禽，2001，23(1)：10-12．
[83]Altmann, J. Observational study of behavior: sampling methods[J]. Behavior, 1974,49:227-267.
[84]Vestergaard K, Hogan JA, Kruijt JP. The development of a behavior system: dustbathing in the Burmese red junglefowle Ⅰ. The influence of the rearing environment on the organization of dustbathing[J]. Behaviour, 1990, 112(1):99-116.
[85]Weeks CA., Danbury TD., Davies HC, et al. The Behavior of broiler chickens and its modification by lameness[J]. Appl. Anim. Behav. Sci, 2000, 67:111-125.
[86]张灵芝，汤建，唐朝枢．脂质体作为基因载体的研究进展[J]．国外医学．分子生物学分册，1993，15(1)：10-12．
[87]Kawaura C, Noguchi A, Furuno T, et al. Atomic force microscopy for studying gene transfection mediated by cationic liposomes with a cationic cholesterol derivative[J].
FEBS Lett, 1998, 421(1):69-72.
[88]Spector DL，Goldman RD，Leinewnd LA[M]．著．黄培堂等译．细胞实验指南，科学出版社，2001年，第一版：786．
[89]Pain B, Chenevier P, Samarut J. Chicken Embryonic Stem cells and transgenic startegies[J]. Cells Tissues Organs, 1999,165(3-4):212-219.
[90]易学瑞，祖萍，刘慧萍．增强型绿色荧光蛋白基因在鸡胚盘细胞中的表达[J]．上海实验动物科学，2001，24(3)：137-140．
[91]刘默芳，王恩多，绿色荧光蛋白[J]．生物化学与生物物理进展，2000．27(3)：238-243．