論文名稱:來亨雞卵管細胞組織特異性啟動子之選殖及活性分析
Cloning and characterization of tubular gland cell tissue-specific promoters of White-Leghorn Chicken
研究生:陳長欣
Chang-Hsin Chen
指導教授:戴謙
Chein Tai
張敏政
Min-Cheng Chang
口試委員:黃木秋
M-Chrang Huang
學位類別:碩士
學院:生物科學與科技學院
College of Bioscience & Biotechnology
系所名稱:生物科技研究所
Institute of Biotechnology
畢業學年度:92
學期:2
論文出版年:93
語文別:中文
論文頁數:100
中文關鍵詞:卵白蛋白、伴白蛋白、啟動子
英文關鍵詞:ovalbumin、conalbumin、promoter
  乳鐵蛋白(lactoferrin, LF) 為一78 kDa的醣蛋白,廣泛存在於動物外分泌腺體之分泌液中,其中又以哺乳動物乳腺之分泌量較高;但種別間亦有差異存在。由前人研究得知乳鐵蛋白具有多種生理特性,包括抗菌能力、增強免疫力、抗氧化、改善腸內菌相、調整鐵的吸收能力和調節發炎反應等功能,為有益人體的蛋白質之一。本試驗室從台灣特有種台灣獼猴(Macaca Cyclopis)之乳腺細胞中選殖出台灣獼猴乳鐵蛋白基因,期望利用家禽當作生物工廠(bioreactor),量產此一高實用性的蛋白質。在家禽基因轉殖技術開發過程中,多數使用病毒啟動子驅動家禽表現外源蛋白。雖然病毒啟動子具有高效率且高穩定性的特點,但基於生物安全性的考量,恐對人體造成危害。於是本文之目的即為開發家禽具有高專一性且能大量在雞蛋內表現外源蛋白之啟動子,即卵白蛋白(ovalbumin)和伴白蛋白(conalbumin)之啟動子。此二啟動子在雞卵管膨大部細胞(magnum tubular gland cell)具有高度活性,且在基因轉錄時會受類固醇荷爾蒙(steroid hormone)調控。在本試驗中,我們從基因庫中找出此二啟動子之序列,並將之構築結合於螢光蛋白及冷光酶報導基因,再利用電穿孔法(electroporation)轉染至來亨雞之不同原代細胞(primary cell)中進行表現,包括雞的卵管膨大部細胞、肝細胞、肌肉細胞、纖維母細胞,結果證實此二啟動子會專一的在卵管膨大部細胞驅動報導基因的表現,且經荷爾蒙處理之後,兩啟動子的活性至少約提高2.5倍以上。我們進一步將此二啟動子構築台灣彌猴乳鐵蛋白基因,轉染至來亨雞的卵管膨大部細胞與肝原代細胞中,48小時後收取細胞萃取液,以西方點漬法分析,證實了此二啟動子可成功的在來亨雞的卵管膨大部細胞與肝原代細胞中趨動台灣彌猴乳鐵蛋白表現。從本論文的試驗結果得知:在細胞層次上,我們所選殖到的此二啟動子具有組織特異性;可受荷爾蒙調控;更可驅動台灣彌猴乳鐵蛋白表現。因此,未來我們將應用此二啟動子於基因轉殖家禽之平臺,取代目前的病毒啟動子,期待可在雞蛋中得到高濃度之台灣彌猴乳鐵蛋白,以達到利用禽蛋生產高價值人類藥用蛋白的目的。


  Lactoferrin (LF) belongs to transferrin family, whose major function is regulating the iron concentration in blood stream and gastrointestinal tract. Several studies indicated that LF has a remarkable antibacterial effect and plays an important role in the regulation of immune response. In a previous study, a full-length cDNA of 2,133 bp from Macaca Cyclopis lactoferrin gene (mLF) had been cloned and sequenced. The nucleotide sequence of mLF showed high identity (93%) to that of human lactoferrin gene (hLF). In order to establish a transgenic chicken line that could overexpress mLF in its eggs, an effective promoter that can specifically drive the gene of egg is necessary, besides the viral promoters under developing. Conalbumin and ovalbumin are the major proteins in egg white. The promoter regions of these two genes were cloned from chromosome of White-Leghorn Chicken in this study. We tested their activities and tissue-specificity in primary cell cultures of White-Leghorn Chicken by fluorescence and luciferase assays. The results showed that the two promoters did not function in chicken muscle cells and fibroblast cells. However, conalbumin promoter was active in chicken hepatocytes and oviduct tubular gland cells; and ovalbumin was only active in chicken primary oviduct tubular gland cells. Both of the two promoters were regulated by the hormones mixture, including β-estradiol, progesterone and corticosterone. Furthermore, we applied the two promoters to drive mLF expressing in chicken primary hepatocytes and oviduct tubular gland cells. The mLF recombinant protein was detected by Western blotting. It proved that ovalbumin and conalbumin promoters cloned in this study had function in the chicken primary cell culture level. In future, promoters will be further studied or re-constructed for the application of gene transfer in chicken.

中文摘要…………………………………………………………………I
Abstract…………………………………………………………………II
誌謝……………………………………………………………………III
目錄………………………………………………………………………V
圖目錄…………………………………………………………………VIII
附錄目錄………………………………………………………………X
縮寫檢索表……………………………………………………………XI
文獻檢討………………………………………………………………1
一、乳鐵蛋白 (lactoferrin)………………………………………1
二、基因轉殖家禽 (transgenic chicken)………………………6
三、雌禽之生殖 (reproduction)…………………………………8
四、卵白蛋白質之組織特異性 (tissue-specificity)……………13
材料與方法………………………………………………………………17
一、試驗流程圖………………………………………………17
二、試驗材料與方法……………………………………………………………17
1.自白色來亨雞的血液中取得基因組核酸(genomic DNA)…………………17
1.1使用之原代細胞(primary cells)來源…………………………………17
1.2萃取家禽genomic DNA……………………………………………………18
2.構築由conalbumin和ovalbumin啟動子啟動報導基因(GFP、luciferase)或台灣彌猴乳鐵蛋 白之載體…………………………………………………………………………19
2.1使用之菌株、載體及培養基……………………………………………20
2.2白色來亨雞組織特異性啟動子之構築(conalbumin promoter & ovalbumin promoter)…………………………………………………………………………20
2.3以白色來亨雞組織特異性啟動子(conalbumin promoter ovalbumin promoter)接合報導基因(EYFP、luciferase)之構築………………………………………………21
2.4以白色來亨雞組織特異性啟動子(conalbumin promoter、ovalbumin promoter)接合台灣彌猴乳鐵蛋白基因之構築……………………………………………………25
2.5限制酵素切割質體DNA…………………………………………………27
2.6鹼性去磷酸酵素(BAP)處理載體………………………………………28
2.7接合反應 (ligation)…………………………………………………29
2.8大腸桿菌之形質轉換 (transformation)……………………………29
2.9製備少量質體DNA………………………………………………………30
3.自白色來亨雞中取得原代細胞……………………………………………31
3.1細胞之收集……………………………………………………………31
3.2細胞繼代培養 (附著型細胞 adherent cell)………………………33
3.3細胞保存…………………………………………………………………34
3.4細胞解凍…………………………………………………………………34
4.以電穿孔法轉染已構築好之各個載體轉至不同原代細胞中……………35
4.1電穿孔法轉染細胞 (electroporation)………………………………35
5.偵測報導基因(GFP、luciferase)在不同原代細胞中之表現情形………37
5.1冷光酶分析 (luciferase Assay)……………………………………37
5.2 β-Galactosidase Enzyme Assay……………………………………38
6.以西方點漬法分析台灣彌猴乳鐵蛋白基因之表現…………………………38
6.1蛋白質濃度的定量………………………………………………38
6.2 SDS-PAGE(蛋白質分子量分析)………………………………39
6.3西方點漬法 (western blot)………………………………40
結果與討論………………………………………………………………43
一、構築ovalbumin和conalbumin啟動子…………………………43
二、藉由黃綠色螢光蛋白(EYFP)的表現以分析啟動子之活性………44
三、藉由冷光酶(luciferase)的表現以分析啟動子之活性…………45
四、利用西方點漬法偵測mLF-EGFP重組蛋白…………………………47
五、以ELISA之方法偵測mLF-EGFP重組蛋白表現情形………………48
結論………………………………………………………………………50
圖表………………………………………………………………………51
參考文獻……..…………………………………………………………82
1. Anderson BF, Baker HM, Norris GE, Rice DW, and Baker EN. 1989. Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution. J Mol Biol 209: 711-34.
2. Arao Y, Miyatake N, Yamamoto E, Usiku H, Matsuura A, Ninomiya Y, Masushige S, Hasegawa T, and Kato S. 1996. Steroid hormones differentially induce transcription of the chicken ovalbumin gene, but stabilize the mRNA with the same half-life. J Biochem (Tokyo) 120: 710-5.
3. Arnold RR, Russell JE, Champion WJ, Brewer M, and Gauthier JJ. 1982. Bactericidal activity of human lactoferrin: differentiation from the stasis of iron deprivation. Infect Immun 35: 792-9.
4. Bellamy W, Takase M, Wakabayashi H, Kawase K, and Tomita M. 1992a. Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J Appl Bacteriol 73: 472-9.
5. Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K, and Tomita M. 1992b. Identification of the bactericidal domain of lactoferrin. Biochim Biophys Acta 1121: 130-6.
6. Benoist C, O'Hare K, Breathnach R, and Chambon P. 1980. The ovalbumin gene-sequence of putative control regions. Nucleic Acids Res 8: 127-42.
7. Bezault J, Bhimani R, Wiprovnick J, and Furmanski P. 1994. Human lactoferrin inhibits growth of solid tumors and development of experimental metastases in mice. Cancer Res 54: 2310-2.
8. Bi BY, Leveugle B, Liu JL, Collard A, Coppe P, Roche AC, Nillesse N, Capron M, Spik G, and Mazurier J. 1994. Immunolocalization of the lactotransferrin receptor on the human T lymphoblastic cell line Jurkat. Eur J Cell Biol 65: 164-71.
9. Chang DC, Chassy BM, Saunders JA, and Sowers AE. 1992. Guide to Electroporation and Electrofusion. Academic Press, Inc, San Diego.
10. Chang DC. 1997. Experimental strategies in efficient transfection of mammalian cells. Electroporation. Methods Mol Biol 62: 307-18.
11. Cochet M, Perrin F, Gannon F, Krust A, Chambon P, McKnight GS, Lee DC, Mayo KE, and Palmiter R. 1979. Cloning of an almost full-length chicken conalbumin double-stranded cDNA. Nucleic Acids Res 6: 2435-52.
12. Coonrod A, Li FQ, and Horwitz M. 1997. On the mechanism of DNA transfection: efficient gene transfer without viruses. Gene Ther 4: 1313-21.
13. Davidsson L, Kastenmayer P, Yuen M, Lonnerdal B, and Hurrell RF. 1994. Influence of lactoferrin on iron absorption from human milk in infants. Pediatr Res 35: 117-24.
14. Dean DM, Jones PS, and Sanders MM. 1996. Regulation of the chicken ovalbumin gene by estrogen and corticosterone requires a novel DNA element that binds a labile protein, Chirp-1. Mol Cell Biol 16: 2015-24.
15. Dean DM, Berger RR, and Sanders MM. 1998. A winged-helix family member is involved in a steroid hormone-triggered regulatory circuit. Endocrinology 139: 4967-75.
16. Dierich A, Gaub MP, LePennec JP, Astinotti D, and Chambon P. 1987. Cell-specificity of the chicken ovalbumin and conalbumin promoters. Embo J 6: 2305-12.
17. Dillner NB, and Sanders MM. 2002. Upstream stimulatory factor (USF) is recruited into a steroid hormone-triggered regulatory circuit by the estrogen-inducible transcription factor delta EF1. J Biol Chem 277: 33890-4.
18. Dionysius DA, and Milne JM. 1997. Antibacterial peptides of bovine lactoferrin: purification and characterization. J Dairy Sci 80: 667-74.
19. Etches RJ, and Verrinder Gibbins AM. 1997. Strategies for the production of transgenic chicken. Methods Mol Biol 62: 433-50.
20. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, and Danielsen M. 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A 84: 7413-7.
21. Finkelstein RA, Sciortino CV, and McIntosh MA. 1983. Role of iron in microbe-host interactions. Rev Infect Dis 5 Suppl 4: S759-77.
22. Fukuda K, Sakamoto N, Narita T, Saitoh K, Kameda T, Iba H, and Yasugi S. 2000. Application of efficient and specific gene transfer systems and organ culture techniques for the elucidation of mechanisms of epithelial-mesenchymal interaction in the developing gut. Dev Growth Differ 42: 207-11.
23. Gannon F, O'Hare K, Perrin F, LePennec JP, Benoist C, Cochet M, Breathnach R, Royal A, Garapin A, Cami B, and Chambon P. 1979. Organisation and sequences at the 5' end of a cloned complete ovalbumin gene. Nature 278: 428-34.
24. Haecker SA, Muramatsu T, Sensenbaugh KR, and Sanders MM. 1995. Repression of the ovalbumin gene involves multiple negative elements including a ubiquitous transcriptional silencer. Mol Endocrinol 9: 1113-26.
25. Hager LJ, McKnight GS, and Palmiter RD. 1980. Glucocorticoid induction of egg white mRNAs in chick oviduct. J Biol Chem 255: 7796-800.
26. Hynes NE, Groner B, Sippel AE, Jeep S, Wurtz T, Nguyen-Huu MC, Giesecke K, and Schutz G. 1979. Control of cellular content of chicken egg white protein specific RNA during estrogen administration and withdrawal. Biochemistry 18: 616-24.
27. Ikeda M, Sugiyama K, Tanaka T, Tanaka K, Sekihara H, Shimotohno K, and Kato N. 1998. Lactoferrin markedly inhibits hepatitis C virus infection in cultured human hepatocytes. Biochem Biophys Res Commun 245: 549-53.
28. Ivarie R. 2003. Avian transgenesis: progress towards the promise. Trends Biotechnol 21: 14-9.
29. Iyer S, and Lonnerdal B. 1993. Lactoferrin, lactoferrin receptors and iron metabolism. Eur J Clin Nutr 47: 232-41.
30. Jeltsch JM, Hen R, Maroteaux L, Garnier JM, and Chambon P. 1987. Sequence of the chicken ovotransferrin gene. Nucleic Acids Res 15: 7643-5.
31. Jung-Testas I, Garcia T, and Baulieu EE. 1986. Steroid hormones induce cell proliferation and specific protein synthesis in primary chick oviduct cultures. J Steroid Biochem 24: 273-9.
32. Kato S, Tora L, Yamauchi J, Masushige S, Bellard M, and Chambon P. 1992. A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5'-TGACC-3' motifs acting synergistically. Cell 68: 731-42.
33. Kaye JS, Bellard M, Dretzen G, Bellard F, and Chambon P. 1984. A close association between sites of DNase I hypersensitivity and sites of enhanced cleavage by micrococcal nuclease in the 5'-flanking region of the actively transcribed ovalbumin gene. Embo J 3: 1137-44.
34. Kaye JS, Pratt-Kaye S, Bellard M, Dretzen G, Bellard F, and Chambon P. 1986. Steroid hormone dependence of four DNase I-hypersensitive regions located within the 7000-bp 5'-flanking segment of the ovalbumin gene. Embo J 5: 277-85.
35. Kim WS, Tanaka T, Kumura H, and Shimazaki K. 2002. Lactoferrin-binding proteins in Bifidobacterium bifidum. Biochem Cell Biol 80: 91-4.
36. Kumura H, Hiramatsu Y, Ukai Y, Mikawa K, and Shimazaki K. 1998. Expression of human lactoferrin in transfected rat mammary epithelial cells. Adv Exp Med Biol 443: 85-9.
37. Lee DC, McKnight GS, and Palmiter RD. 1978. The action of estrogen and progesterone on the expression of the transferrin gene. A comparison of the response in chick liver and oviduct. J Biol Chem 253: 3494-503.
38. Levay PF, and Viljoen M. 1995. Lactoferrin: a general review. Haematologica 80: 252-67.
39. Li Y, Behnam J, and Simkiss K. 1995. Ballistic transfection of avian primordial germ cell in ovo. Transgenic Res 4: 26-9.
40. Love J, Gribbin C, Mather C, and Sang H. 1994. Transgenic birds by DNA microinjection. Biotechnology (N Y) 12: 60-3.
41. Machnicki M, Zimecki M, and Zagulski T. 1993. Lactoferrin regulates the release of tumour necrosis factor alpha and interleukin 6 in vivo. Int J Exp Pathol 74: 433-9.
42. Marchetti M, Pisani S, Antonini G, Valenti P, Seganti L, and Orsi N. 1998. Metal complexes of bovine lactoferrin inhibit in vitro replication of herpes simplex virus type 1 and 2. Biometals 11: 89-94.
43. Masson PL, and Heremans JF. 1971. Lactoferrin in milk from different species. Comp Biochem Physiol B 39: 119-29.
44. McKnight GS, Lee DC, Hemmaplardh D, Finch CA, and Palmiter RD. 1980. Transferrin gene expression. Effects of nutritional iron deficiency. J Biol Chem 255: 144-7.
45. McKnight GS, Lee DC, and Palmiter RD. 1980. Transferrin gene expression. Regulation of mRNA transcription in chick liver by steroid hormones and iron deficiency. J Biol Chem 255: 148-53.
46. McKnight GS, and Palmiter RD. 1979. Transcriptional regulation of the ovalbumin and conalbumin genes by steroid hormones in chick oviduct. J Biol Chem 254: 9050-8.
47. Mikogami T, Heyman M, Spik G, and Desjeux JF. 1994. Apical-to-basolateral transepithelial transport of human lactoferrin in the intestinal cell line HT-29cl.19A. Am J Physiol 267: G308-15.
48. Mukai M, Torikata C, Iri H, Mikata A, Sakamoto T, Hanaoka H, Shinohara C, Baba N, Kanaya K, and Kageyama K. 1984. Alveolar soft part sarcoma. An elaboration of a three-dimensional configuration of the crystalloids by digital image processing. Am J Pathol 116: 398-406.
49. Mulvihill ER, and Palmiter RD. 1977. Relationship of nuclear estrogen receptor levels to induction of ovalbumin and conalbumin mRNA in chick oviduct. J Biol Chem 252: 2060-8.
50. Naito M, Sakurai M, and Kuwana T. 1998. Expression of exogenous DNA in the gonads of chimaeric chicken embryos produced by transfer of primordial germ cell transfected in vitro and subsequent fate of the introduced DNA. J Reprod Fertil 113: 137-43.
51. Nguyen-Huu MC, Barrett KJ, Giesecke K, Wurtz T, Sippel AE, and Schutz G. 1978. Transcription of the chicken ovalbumin and conalbumin gene during early secondary induction with estrogens. Hoppe Seylers Z Physiol Chem 359: 1307-13.
52. Nordstrom LA, Dean DM, and Sanders MM. 1993. A complex array of double-stranded and single-stranded DNA-binding proteins mediates induction of the ovalbumin gene by steroid hormones. J Biol Chem 268: 13193-202.
53. Ochiai H, Park HM, Nakamura A, Sasaki R, Okumura JI, and Muramatsu T. 1998. Synthesis of human erythropoietin in vivo in the oviduct of laying hens by localized in vivo gene transfer using electroporation. Poult Sci 77: 299-302.
54. Oka T, and Schimke RT. 1969. Interaction of estrogen and progesterone in chick oviduct development. II. Effects of estrogen and progesterone on tubular gland cell function. J Cell Biol 43: 123-37.
55. O'Malley BW, Roop DR, Lai EC, Nordstrom JL, Catterall JF, Swaneck GE, Colbert DA, Tsai MJ, Dugaiczyk A, and Woo SL. 1979. The ovalbumin gene: organization, structure, transcription, and regulation. Recent Prog Horm Res 35: 1-46.
56. Pain B, Clark ME, Shen M, Nakazawa H, Sakurai M, Samarut J, and Etches RJ. 1996. Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities. Development 122: 2339-48.
57. Pain B, Chenevier P, and Samarut J. 1999. Chicken embryonic stem cells and transgenic strategies. Cells Tissues Organs 165: 212-9.
58. Palmiter RD. 1972. Regulation of protein synthesis in chick oviduct. I. Independent regulation of ovalbumin, conalbumin, ovomucoid, and lysozyme induction. J Biol Chem 247: 6450-61.
59. Palmiter RD, and Carey NH. 1974. Rapid inactivation of ovalbumin messenger ribonucleic acid after acute withdrawal of estrogen. Proc Natl Acad Sci U S A 71: 2357-61.
60. Palmiter RD, Mulvihill ER, Shepherd JH, and McKnight GS. 1981. Steroid hormone regulation of ovalbumin and conalbumin gene transcription. A model based upon multiple regulatory sites and intermediary proteins. J Biol Chem 256: 7910-6.
61. Pastorcic M, Wang H, Elbrecht A, Tsai SY, Tsai MJ, and O'Malley BW. 1986. Control of transcription initiation in vitro requires binding of a transcription factor to the distal promoter of the ovalbumin gene. Mol Cell Biol 6: 2784-91.
62. Perrin F, Cochet M, Gerlinger P, Cami B, LePennec JP, and Chambon P. 1979. The chicken conalbumin gene: studies of the organization of cloned DNAs. Nucleic Acids Res 6: 2731-48.
63. Perry MM. 1988. A complete culture system for the chick embryo. Nature 331: 70-2.
64. Pühler A., 1993. Genetic Engineering of Animals, VCH, New York, NY (USA)
Qiu Y, Krishnan V, Pereira FA, Tsai SY, and Tsai MJ. 1996. Chicken ovalbumin upstream promoter-transcription factors and their regulation. J Steroid Biochem Mol Biol 56: 81-5.
65. Rado TA, Wei XP, and Benz EJ, Jr. 1987. Isolation of lactoferrin cDNA from a human myeloid library and expression of mRNA during normal and leukemic myelopoiesis. Blood 70: 989-93.
66. Rainard P. 1987. Bacteriostatic activity of bovine lactoferrin in mastitic milk. Vet Microbiol 13: 159-66.
67. Sanders MM, and McKnight GS. 1985. Chicken egg white genes: multihormonal regulation in a primary cell culture system. Endocrinology 116: 398-405.
68. Sanders MM, and McKnight GS. 1986. in Molecular Genetics of Mammalian Cells (Malacinaski GM, ed). pp. 183-216. Macmillan Press, New York.
69. Sanders MM, and McKnight GS. 1988. Positive and negative regulatory elements control the steroid-responsive ovalbumin promoter. Biochemistry 27: 6550-7.
70. Sang H. 1994. Transgenic chickens--methods and potential applications. Trends Biotechnol 12: 415-20.
71. Schweers LA, Frank DE, Weigel NL, and Sanders MM. 1990. The steroid-dependent regulatory element in the ovalbumin gene does not function as a typical steroid response element. J Biol Chem 265: 7590-5.
72. Schweers LA, and Sanders MM. 1991. A protein with a binding specificity similar to NF-kappa B binds to a steroid-dependent regulatory element in the ovalbumin gene. J Biol Chem 266: 10490-7.
73. Sensenbaugh KR, and Sanders MM. 1999. Multiple promoter elements including a novel repressor site modulate expression of the chick ovalbumin gene. DNA Cell Biol 18: 147-56.
74. Vorland LH. 1999. Lactoferrin: a multifunctional glycoprotein. Apmis 107: 971-81.
75. Wakabayashi H, Abe S, Okutomi T, Tansho S, Kawase K, and Yamaguchi H. 1996. Cooperative anti-Candida effects of lactoferrin or its peptides in combination with azole antifungal agents. Microbiol Immunol 40: 821-5.
76. Ward PP, Zhou X, and Conneely OM. 1996. Cooperative interactions between the amino- and carboxyl-terminal lobes contribute to the unique iron-binding stability of lactoferrin. J Biol Chem 271: 12790-4.
77. Ward PP, Chu H, Zhou X, and Conneely OM. 1997. Expression and characterization of recombinant murine lactoferrin. Gene 204: 171-6.
78. Wasan EK, Reimer DL, and Bally MB. 1996. Plasmid DNA is protected against ultrasonic cavitation-induced damage when complexed to cationic liposomes. J Pharm Sci 85: 427-33.
79. Watanabe M, Naito M, Sasaki E, Sakurai M, Kuwana T, and Oishi T. 1994. Liposome-mediated DNA transfer into chicken primordial germ cells in vivo. Mol Reprod Dev 38: 268-74.
80. Watanabe M, Shirayoshi Y, Koshimizu U, Hashimoto S, Yonehara S, Eguchi Y, Tsujimoto Y, and Nakatsuji N. 1997. Gene transfection of mouse primordial germ cells in vitro and analysis of their survival and growth control. Exp Cell Res 230: 76-83.
81. Yamauchi K, Tomita M, Giehl TJ, and Ellison RT, 3rd. 1993. Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment. Infect Immun 61: 719-28.
82. Yu LC, and Chen YH. 1993. The developmental profile of lactoferrin in mouse epididymis. Biochem J 296 (Pt 1): 107-11.
83. 王怡玲, 2001. 鴨始基生殖細胞之非病毒轉染法。國立成功大學理學院生物科技研究所碩士論文
84. 吳明憲, 2002. 台灣獼猴乳鐵蛋白基因的構築與轉殖。國立成功大學理學院生物科技研究所碩士論文
  • 同意授權校內瀏覽/列印電子全文服務,於2009-08-11起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2009-08-11起公開。