外源基因在鸡早期胚胎内整合及表达的研究
摘要
本研究以LacZ基因为报告基因，构建了两种载体质粒：p-CMV-β-Gal和p-OV-β-Gal，分别利用显微注射法和精子介导法将这两种载体质粒转移到鸡早期胚胎内，对外源基因的整合及表达情况进行检测分析。同时，对添加抗菌素溶液是否有利于鸡胚的正常发育进行了探讨。结果如下：
1、在处理过的受精蛋中添加一定量的抗菌素(青、链霉素)溶液对于提高鸡胚抵御外部环境污染能力、避免鸡胚早期死亡的效果并不显著，但在实际试验过程中发现添加的抗菌素溶液能起到补充水分的作用，保证鸡胚的正常发育。
2、利用显微注射法转移载体质粒(p-CMV-β-Gal)的过程破坏了受精蛋的结构，对鸡胚的发育造成影响，表现在鸡胚在早期孵化阶段大批死亡，死亡率高达58.07％(367／632)，但这种方法能够将载体质粒转移到鸡早期胚胎内，外源基因整合阳性率为21.43％(15／70)，在部分鸡胚中能检测到外源基因的表达，阳性率为9.52％(4／42)。
3、利用精子介导法能将载体质粒(p-OV-β-Gal)转移到鸡早期胚胎内，外源基因整合阳性率为5.91％(11／186)，表达阳性率为16.94％(41／242)。
以上试验结果显示：显微注射法和精子介导法均能将载体质粒转移到鸡早期胚胎中，外源基因能被整合到宿主基因组中，有些能进一步得到表达。说明这两种方法均可作为制备转基因鸡的研究方法。
To explore the possibility of generation of transgenic chicken, two vectors (p-CMV- β -Gal and p-OV- 3 -Gal) of LacZ gene as report gene were transmitted into the chicken's early embryo by two methods: Microinjection Method and Sperm-mediated Method, then the LacZ gene's integration and expression were monitored. At the same time, the effect of bacteriophage was discussed. The results were as showed:
1. After working the egg, a litter of bacteriophage liquor was appended into the egg. Its effect of enhancing the immunity of the embryo and avoiding the embryo dying early was not remarkable. But during the course of the experiment, another effect was discovered: The appended liquor could supply the wet that quickly volatilized in the course of hatching.
2. During the course of microinjecting, the structure of the egg was broken. It
resulted in that the growth of the embryo was affected and many embryos died early, the death percentage was 58.07%(367/632). In fact, the vector (p-CMV-β-Gal) could be transmitted into the chicken's early embryo by Microinjection Method, and the percentage of LacZ gene's integration and expression respectively were 21.43%(15/7o)
and 9.52 %(4/42)-
3. The vector (p-OV- β -Gal) could be transmitted into the chicken's early embryo by sperm-mediated method, the percentage of LacZ gene's integration and expression respectively were 5.91 %(n/,86) and 16.94%(41/242).
From the results of the experiments, it could be concluded that two vectors could be transmitted into the chicken's early embryo by microinjection method or sperm-mediated method, and the LacZ gene could be integrated and expressed. It was showed that the two methods could be used for generation of the transgenic chicken.
1、在处理过的受精蛋中添加一定量的抗菌素(青、链霉素)溶液对于提高鸡胚抵御外部环境污染能力、避免鸡胚早期死亡的效果并不显著，但在实际试验过程中发现添加的抗菌素溶液能起到补充水分的作用，保证鸡胚的正常发育。
2、利用显微注射法转移载体质粒(p-CMV-β-Gal)的过程破坏了受精蛋的结构，对鸡胚的发育造成影响，表现在鸡胚在早期孵化阶段大批死亡，死亡率高达58.07％(367／632)，但这种方法能够将载体质粒转移到鸡早期胚胎内，外源基因整合阳性率为21.43％(15／70)，在部分鸡胚中能检测到外源基因的表达，阳性率为9.52％(4／42)。
3、利用精子介导法能将载体质粒(p-OV-β-Gal)转移到鸡早期胚胎内，外源基因整合阳性率为5.91％(11／186)，表达阳性率为16.94％(41／242)。
以上试验结果显示：显微注射法和精子介导法均能将载体质粒转移到鸡早期胚胎中，外源基因能被整合到宿主基因组中，有些能进一步得到表达。说明这两种方法均可作为制备转基因鸡的研究方法。
To explore the possibility of generation of transgenic chicken, two vectors (p-CMV- β -Gal and p-OV- 3 -Gal) of LacZ gene as report gene were transmitted into the chicken's early embryo by two methods: Microinjection Method and Sperm-mediated Method, then the LacZ gene's integration and expression were monitored. At the same time, the effect of bacteriophage was discussed. The results were as showed:
1. After working the egg, a litter of bacteriophage liquor was appended into the egg. Its effect of enhancing the immunity of the embryo and avoiding the embryo dying early was not remarkable. But during the course of the experiment, another effect was discovered: The appended liquor could supply the wet that quickly volatilized in the course of hatching.
2. During the course of microinjecting, the structure of the egg was broken. It
resulted in that the growth of the embryo was affected and many embryos died early, the death percentage was 58.07%(367/632). In fact, the vector (p-CMV-β-Gal) could be transmitted into the chicken's early embryo by Microinjection Method, and the percentage of LacZ gene's integration and expression respectively were 21.43%(15/7o)
and 9.52 %(4/42)-
3. The vector (p-OV- β -Gal) could be transmitted into the chicken's early embryo by sperm-mediated method, the percentage of LacZ gene's integration and expression respectively were 5.91 %(n/,86) and 16.94%(41/242).
From the results of the experiments, it could be concluded that two vectors could be transmitted into the chicken's early embryo by microinjection method or sperm-mediated method, and the LacZ gene could be integrated and expressed. It was showed that the two methods could be used for generation of the transgenic chicken.
引文
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2. Jaenish R, Mintz B. Simian Virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA[J]. Proc Nalt Acad Sci USA. 1974,71:1250～1254.
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5. Brem G, Brening B, Goodman H M, et al. Production of transgenic mice, rabbits and pigs by microinjection into pronulei. Zuchthygiene. 1985,20:251～252.
6. Clark A J, Bessos H, Bishop J O, et al. Expression of human anti-hemophilic factor Ⅸ in the milk of transgenic sheep. Biotechnology. 1989,7:487～492.
7. Hammer R E, Brinster R L, Rosenfeld M G, et al. Production of transgenic rabbits, sheep and pigs by microinjection. Nature. 1985(b),315:680～683.
8. Knight K L, Polet H S, Kazdin D S, et al. Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immumoglobulin heavy chain enhancer. Proc Natl Acad Sci USA. 1988,85:3130-3134.
9. Murray J D, Nancarrow C D, Marshall, et al. Production of transgenic merino sheep by microinjection of ovine metallothionein-ovine growth hormone fusion genes. Reprod Fertil Dev. 1989,1:147～155.
10. Rexroad C E, Hammer R E, Bolt D J, et al. Production of transgenic sheep with growth-regulating genes. Mol Repr Dev. 1989,1:164～169.
11. Salter D W, et al. Gene insertion into the chicken germ line by retroviruses. Poltry Science. 1986,65:1445～1450.
12．李碧春，钱菊汾．禽类转基因生产及其应用的研究现状和进展(待续)．动物科学与动物医学．2001，18(1)：13～15．
13．侯庆文，陈光明，张文烨，等．转基因动物制作、鉴定和应用研究新进展．动物科学与动物医学．2001，19(5)：24～26．
14．卢一凡．邓继先，肖成祖．等．转基因动物乳腺定位表达重组蛋白质的研究现状(上)．高技术通讯．1998，1：54～57．
15．谭晓红，杨晓，程萱，等．核移植技术生产乳腺生物反应器的研究进展．生物工程进展．2000，20(6)：46～49．
16．谭毅．韦克．转基因动物表达生产人体活性蛋白的研究进展．四川动物．2000，19(1)：19～21．
17．郭继彤，张肇英．外源基因在转基因动物乳腺中的特异性表达研究．内蒙古大学学报(自然
科学版)．1998，29(6)：853～858．
18．王元兴，郎介金．动物繁殖学．江苏科学技术出版社．1993．
19．蒋唏余．鸡胚胎发育图谱．科学出版社．1983．
20．沙金．胚盘细胞移植法制各鸡—鸭嵌合体及禽类转基因的研究．中国农业大学硕士学位论文．2001．
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