重组逆转录病毒-精原干细胞介导的转基因研究
|
摘要
转基因动物技术作为生命科学领域中的一项重要的研究手段，广泛应用于医药、
农业及生物学领域。自1980年Gordon等首次将显微注射技术用于小鼠受精卵的基因
导入以来，人们对于转基因技术的研究与探索从未停止，因此，在显微注射技术不断发
展和完善的同时，还相继建立了胚胎干细胞介导的基因转移，重组逆转录病毒介导的基
因转移和精子载体法等转基因技术。但这些转基因技术普遍存在着操作复杂、投入较高、
外源基因在受体细胞基因组上的整合率较低等不足之处。为了克服这些不足之处，本研
究在多年跟踪国际转基因动物研究前沿的基础上，从选择高效的基因转移的介导方式和
理想的受体细胞两个方面同时着手，进行了基因转移新技术的研究。
在众多的真核细胞基因转移技术中，动物病毒载体系统以其简单的基因组结构、比
较清楚的分子生物学背景、易于改造和操作、转染率高等特点而最为引人注目，迄今已
有数种病毒被改造为基因转移载体。其中，逆转录病毒载体-包装细胞系统被认为是
在具有分裂功能的细胞中进行基因转移和表达最为成功的真核细胞基因转移技术。因此
本研究选择重组逆转录病毒作为基因转移的介导方式。将3.7kb的LacZ基因片段分别
插入到骨架为Moloney小鼠白血病病毒（MoMLV）、含有标记基因Neo~R的逆转录病毒
载体pLXSN、pLNCX的5'ETR和CMV Promoter的下游，构建了含有LacZ的重组
逆转录病毒载体pLNCL、pLLSN。运用脂质体、磷酸钙两种介质，以包装效率高、安
全性好的包装细胞系PA317对pLNCX、pLXSN、pLLSN和pLNCL进行包装，得到了
PA-1、PA-2、PA-3和PA-4四株产毒细胞。透射电镜下观察经差速离心法浓缩后的病毒
悬液，观察到了具有逆转录病毒形态特征（球形，有囊膜，直径平均约为100nm）的重
组病毒粒子。
精原干细胞属于未分化细胞，具有自增殖能力，可产生不断更新的干细胞及可定向分
化的精原细胞，每次分裂形成的两个细胞，一个通过初级精母细胞、次级精母细胞、精子
细胞等过程发育为精子，一个仍为干细胞，保持原始未分化状态和继续分裂增殖的能力。
如果重组逆转录病毒介导的外源基因转染入精原干细胞，外源基因就会整合入精原干细
胞基因组。由精原干细胞发育成熟的精子，就能够通过整合的方式携带外源基因，外源基
因也会随干细胞增殖而复制并在干细胞中得到长期保留。这就克服了以受精卵作为目的
基因载体时需要不断采卵和反复注射带来的巨额成本和繁重的工作强度等不足之处，从
而提高转基因动物的生产效率。
由于以精原干细胞作为外源基因转染的靶细胞以建立转基因动物的研究具有极其
重要的理论意义和实际应用前景。1997年，Kim JH等和本室赖良学等分别进行了脂质
体包裹外源基因，曲细精管内转染精原干细胞的研究，并且各自得到了附睾精子和子代
鼠整合检测的阳性PCR结果。Kim JH等还在睾丸组织切片中检测到了外源基因的表
达。
中文摘要
一
基于本研究以小鼠曲细精管内精原干细胞作为9懈塌因转染的靶细胞以建立转基因
小鼠的目时为了 烙的重斑议臼色录病毒体内感染精原干细胞的效果，进行了含有
ixc、NeoR基因的重组逆转录病毒体夕陋澡NIH3Th细胞的研究。将含有aH基因的
浓缩病毒渤叨材摘滦NllB℃细胞，经多聚甲醛固定、Xwi染色，观察臣了蓝染细胞
说明sxc基因获得表达。同时，将来自四株产毒细胞的浓缩病毒悬液分另u体夕h孜染
NIH3Th细胞以测喉空逾跺嫡度，通过比较，含有标记基因Nr、由产毒细胞系PAZ
所产生的浓缩重组逆转录病毒的感染滴度最高，过臣了3X106cllvilll。
在＄e了含有标记基因 Ne、感搁傲颤丝了 3 X 106 dilllTll的重组逆转录病毒的
前提下经过注射条件和参数的多次优化，将pM台盼蓝、Poo按
一定比例配制域注白比狡，单似收拄时ldsl6日龄公鼠寒丸曲细精管以体内感染精原干细
胞。注射后4（hi，公圃倚溉进行精液品质（精子活率、精子顶体完整率、精于畸
形率三项指标）的匀觎o并利用根据M炉基因序列设计的三对引物对精子基因组DNA
进干了IWI；1和套式ICIt检狈。实验结果表明，注射液对公鼠精液品质无不良影响，不影
响公鼠繁殖力。10 ff｝BfftZ；fR7so（品中，有8份为PCR和套式PCR附仕，说明Nd基
因已经坐尧淮公鼠精子基因组中，从而首次成功地实现了M炉基因在公鼠生殖系细胞
的转移。注射后45d，与母鼠交配，共获得子代鼠48只。采取鼠尾，提取基因组，利
用根据Nej基因序歹uM的一对引物对子代鼠基因组DNA进行PCR检测，阳性鼠9
只。经 Southern blotting检狈，阳性鼠 4只，其中 3只母鼠，l只公鼠。将这 4 ／q FO代
小鼠与4F＄：FH因小鼠交配，共获得F；代小鼠15只。应用PCR方法（引物同FO代小
鼠进行检测中使用的5！物）对这些F；代小励锁0，结果F；代1号母鼠听生5只
子鼠中有 4只为阳胜。获得的转基因小鼠阳性率达到了 83％（4／48）。从而首次建立
了重组逆转录病毒介导，以公鼠曲细精管内精原干细胞为9陇塌因转染的靶细胞的基因
转移技术。并利用该技术成功的?
Cunent gene transfer methods in transgenic animals include embiyo microzrijection,
embiyonic stem cell mediated and retrovims mediated They have been videIy applied to
construct transgeriic large animal as well as mice. However, when these methods were used to
construct transgenic animals, there are some shortcomings, for example, time consuming and
high cost
In order to overcome these shortcomings we bad developed a new method to construct
transgenic mice. Two important considerations had been taken into account, i.e. the gene transfer
efficiency and the target cell. Recombinant retrovirus is selected as gene transfer method and
spermtogonial stem cells were used as target cells.
Recombinant retroviral vectors (pLLSN and pLNCL) cartying LazZ were constnLcted.
The two vectors, together with pLNCX and pLXSN canying NeoR were transfected into PA3 17
for packaging by means of Lipofectin and calcium phosphate precipitation respectively. After
G41 8 selection at a concentration of O.3g/L for 2 weeks, G41 8-resistant PA317 colonies were
obtained and amplified, and four G41 8-resistant PA3 17 cell lines (PA-I, PA-2, PA-3, PA-4) were
obtainecL The supernatant of the cell culture was harvested, concentrated and observed under
transmission electron microscope, the mature vinis particles possessed the typically
morphological feature of retrovirus.
The concentrated viral supemantant fim PA-3 and PA-4 was used to infect Nll{3T3. The
infected NIH3T3 cells were fixed by O.5(wlv) parafoima]dehyde and stained by X-gal, those
cells that expressed LacZ became blue. The result showed LzcZ gene had been expressed on
NIH3T3 cell. At the same time, Nll-13T3 cells were infected to measure the viral titer of
recombinant retrovirus, Results showed that the titer of concentrated viral supematant from
PA-2 was the highest, and was 3 X lO6cfu/ml.
84
In order to study the gene transfer mediated by recombinant retrovims- spermatogonial
stem cells, recombinant reUuvirus from PA-2, canying NeoR, together with typan blue and
Polybrene, were injected into contorted seminiferous tubule of 10 mice (14-16 day) to infect
spermatogonial stem cells in vivo. After 40 days, sperms were harvested from injected mice. Its
qualities( living mtio. apical body intact ratio and deformation ratio) were examined, the data
were collected and statistically analyzed. PCR was used to examine the integration of NeoR
the genome of sperm. The results showed that injection solution doesn抰 have conlraiy effect on
the development of sperm. NeaR gene were confirmed to be integrated into the genome of
mature sperms from eight mice.
Female mice were mated with the eight male mice respectively, and forty-eight ofipring
were produced. PCR and Southern blotting were used to screen NeaR gene from the genomic
DNA samples extracted from the forty-eight olpring tails. Four of them including one male
and three females were demonstrated to be lransgenic. Four founders were further mated with
the nontransgenic mice and fifceen offspring were produced and PCR were used to screen NeOR
gene integration. Four out of five offspring from one female founder were transgenic mice.
Theinteonratewas8.3%(4/48),andwasalmostastheeashaven
reported. But gene transfer mediated by retrovirus-spemitogonial stem cell is much easier to
manipulate than other methods and it saves time. So, gene transfer mediated by recombinant
retrovinis - sperrnatogonial stem cells appears to be a promising way to construct transgenic
animals, such as mice as well as large animals.
农业及生物学领域。自1980年Gordon等首次将显微注射技术用于小鼠受精卵的基因
导入以来，人们对于转基因技术的研究与探索从未停止，因此，在显微注射技术不断发
展和完善的同时，还相继建立了胚胎干细胞介导的基因转移，重组逆转录病毒介导的基
因转移和精子载体法等转基因技术。但这些转基因技术普遍存在着操作复杂、投入较高、
外源基因在受体细胞基因组上的整合率较低等不足之处。为了克服这些不足之处，本研
究在多年跟踪国际转基因动物研究前沿的基础上，从选择高效的基因转移的介导方式和
理想的受体细胞两个方面同时着手，进行了基因转移新技术的研究。
在众多的真核细胞基因转移技术中，动物病毒载体系统以其简单的基因组结构、比
较清楚的分子生物学背景、易于改造和操作、转染率高等特点而最为引人注目，迄今已
有数种病毒被改造为基因转移载体。其中，逆转录病毒载体-包装细胞系统被认为是
在具有分裂功能的细胞中进行基因转移和表达最为成功的真核细胞基因转移技术。因此
本研究选择重组逆转录病毒作为基因转移的介导方式。将3.7kb的LacZ基因片段分别
插入到骨架为Moloney小鼠白血病病毒（MoMLV）、含有标记基因Neo~R的逆转录病毒
载体pLXSN、pLNCX的5'ETR和CMV Promoter的下游，构建了含有LacZ的重组
逆转录病毒载体pLNCL、pLLSN。运用脂质体、磷酸钙两种介质，以包装效率高、安
全性好的包装细胞系PA317对pLNCX、pLXSN、pLLSN和pLNCL进行包装，得到了
PA-1、PA-2、PA-3和PA-4四株产毒细胞。透射电镜下观察经差速离心法浓缩后的病毒
悬液，观察到了具有逆转录病毒形态特征（球形，有囊膜，直径平均约为100nm）的重
组病毒粒子。
精原干细胞属于未分化细胞，具有自增殖能力，可产生不断更新的干细胞及可定向分
化的精原细胞，每次分裂形成的两个细胞，一个通过初级精母细胞、次级精母细胞、精子
细胞等过程发育为精子，一个仍为干细胞，保持原始未分化状态和继续分裂增殖的能力。
如果重组逆转录病毒介导的外源基因转染入精原干细胞，外源基因就会整合入精原干细
胞基因组。由精原干细胞发育成熟的精子，就能够通过整合的方式携带外源基因，外源基
因也会随干细胞增殖而复制并在干细胞中得到长期保留。这就克服了以受精卵作为目的
基因载体时需要不断采卵和反复注射带来的巨额成本和繁重的工作强度等不足之处，从
而提高转基因动物的生产效率。
由于以精原干细胞作为外源基因转染的靶细胞以建立转基因动物的研究具有极其
重要的理论意义和实际应用前景。1997年，Kim JH等和本室赖良学等分别进行了脂质
体包裹外源基因，曲细精管内转染精原干细胞的研究，并且各自得到了附睾精子和子代
鼠整合检测的阳性PCR结果。Kim JH等还在睾丸组织切片中检测到了外源基因的表
达。
中文摘要
一
基于本研究以小鼠曲细精管内精原干细胞作为9懈塌因转染的靶细胞以建立转基因
小鼠的目时为了 烙的重斑议臼色录病毒体内感染精原干细胞的效果，进行了含有
ixc、NeoR基因的重组逆转录病毒体夕陋澡NIH3Th细胞的研究。将含有aH基因的
浓缩病毒渤叨材摘滦NllB℃细胞，经多聚甲醛固定、Xwi染色，观察臣了蓝染细胞
说明sxc基因获得表达。同时，将来自四株产毒细胞的浓缩病毒悬液分另u体夕h孜染
NIH3Th细胞以测喉空逾跺嫡度，通过比较，含有标记基因Nr、由产毒细胞系PAZ
所产生的浓缩重组逆转录病毒的感染滴度最高，过臣了3X106cllvilll。
在＄e了含有标记基因 Ne、感搁傲颤丝了 3 X 106 dilllTll的重组逆转录病毒的
前提下经过注射条件和参数的多次优化，将pM台盼蓝、Poo按
一定比例配制域注白比狡，单似收拄时ldsl6日龄公鼠寒丸曲细精管以体内感染精原干细
胞。注射后4（hi，公圃倚溉进行精液品质（精子活率、精子顶体完整率、精于畸
形率三项指标）的匀觎o并利用根据M炉基因序列设计的三对引物对精子基因组DNA
进干了IWI；1和套式ICIt检狈。实验结果表明，注射液对公鼠精液品质无不良影响，不影
响公鼠繁殖力。10 ff｝BfftZ；fR7so（品中，有8份为PCR和套式PCR附仕，说明Nd基
因已经坐尧淮公鼠精子基因组中，从而首次成功地实现了M炉基因在公鼠生殖系细胞
的转移。注射后45d，与母鼠交配，共获得子代鼠48只。采取鼠尾，提取基因组，利
用根据Nej基因序歹uM的一对引物对子代鼠基因组DNA进行PCR检测，阳性鼠9
只。经 Southern blotting检狈，阳性鼠 4只，其中 3只母鼠，l只公鼠。将这 4 ／q FO代
小鼠与4F＄：FH因小鼠交配，共获得F；代小鼠15只。应用PCR方法（引物同FO代小
鼠进行检测中使用的5！物）对这些F；代小励锁0，结果F；代1号母鼠听生5只
子鼠中有 4只为阳胜。获得的转基因小鼠阳性率达到了 83％（4／48）。从而首次建立
了重组逆转录病毒介导，以公鼠曲细精管内精原干细胞为9陇塌因转染的靶细胞的基因
转移技术。并利用该技术成功的?
Cunent gene transfer methods in transgenic animals include embiyo microzrijection,
embiyonic stem cell mediated and retrovims mediated They have been videIy applied to
construct transgeriic large animal as well as mice. However, when these methods were used to
construct transgenic animals, there are some shortcomings, for example, time consuming and
high cost
In order to overcome these shortcomings we bad developed a new method to construct
transgenic mice. Two important considerations had been taken into account, i.e. the gene transfer
efficiency and the target cell. Recombinant retrovirus is selected as gene transfer method and
spermtogonial stem cells were used as target cells.
Recombinant retroviral vectors (pLLSN and pLNCL) cartying LazZ were constnLcted.
The two vectors, together with pLNCX and pLXSN canying NeoR were transfected into PA3 17
for packaging by means of Lipofectin and calcium phosphate precipitation respectively. After
G41 8 selection at a concentration of O.3g/L for 2 weeks, G41 8-resistant PA317 colonies were
obtained and amplified, and four G41 8-resistant PA3 17 cell lines (PA-I, PA-2, PA-3, PA-4) were
obtainecL The supernatant of the cell culture was harvested, concentrated and observed under
transmission electron microscope, the mature vinis particles possessed the typically
morphological feature of retrovirus.
The concentrated viral supemantant fim PA-3 and PA-4 was used to infect Nll{3T3. The
infected NIH3T3 cells were fixed by O.5(wlv) parafoima]dehyde and stained by X-gal, those
cells that expressed LacZ became blue. The result showed LzcZ gene had been expressed on
NIH3T3 cell. At the same time, Nll-13T3 cells were infected to measure the viral titer of
recombinant retrovirus, Results showed that the titer of concentrated viral supematant from
PA-2 was the highest, and was 3 X lO6cfu/ml.
84
In order to study the gene transfer mediated by recombinant retrovims- spermatogonial
stem cells, recombinant reUuvirus from PA-2, canying NeoR, together with typan blue and
Polybrene, were injected into contorted seminiferous tubule of 10 mice (14-16 day) to infect
spermatogonial stem cells in vivo. After 40 days, sperms were harvested from injected mice. Its
qualities( living mtio. apical body intact ratio and deformation ratio) were examined, the data
were collected and statistically analyzed. PCR was used to examine the integration of NeoR
the genome of sperm. The results showed that injection solution doesn抰 have conlraiy effect on
the development of sperm. NeaR gene were confirmed to be integrated into the genome of
mature sperms from eight mice.
Female mice were mated with the eight male mice respectively, and forty-eight ofipring
were produced. PCR and Southern blotting were used to screen NeaR gene from the genomic
DNA samples extracted from the forty-eight olpring tails. Four of them including one male
and three females were demonstrated to be lransgenic. Four founders were further mated with
the nontransgenic mice and fifceen offspring were produced and PCR were used to screen NeOR
gene integration. Four out of five offspring from one female founder were transgenic mice.
Theinteonratewas8.3%(4/48),andwasalmostastheeashaven
reported. But gene transfer mediated by retrovirus-spemitogonial stem cell is much easier to
manipulate than other methods and it saves time. So, gene transfer mediated by recombinant
retrovinis - sperrnatogonial stem cells appears to be a promising way to construct transgenic
animals, such as mice as well as large animals.
引文
1. James H, Schreiber, Jennifer A, et al. Recombinant retrovirus containing novel reporter genes. Biotechniques, 1993, 14: 818.
2．刘子夜，齐义鹏，王业富．用于基因治疗的病毒载体．中国病毒学，1998，13：101．
3. Kevin, Karen Moore and Robert Wall. Transgene vectors go retro. Nature Biotechnology, 1999, 17:25.
4．陈诗书，汤雪明主编．医学细胞与分子生物学．上海：上海医科大学出版社，1999．
5．殷霞，刘景华主编．动物病毒学．第二版．北京：科学出版社，1997。
3．侯云德著．分子病毒学．北京，学苑出版社，1990．
4. Holland J, Spindler K, Horodyski F, Nichol S and Vandepol. Rapid evolution of RNA virus genomes. Science, 1982, 215:1577-1585.
8. Clark SP and Mak TW. Fluidity of a retrovirus genome. J Virol, 1984, 50: 759-765.
9. Hansen J, Schulze T, Mellert W, Modeling K. Identification and characterization of HW specific RNase H by monoclonal antibody. EMBO J, 1988, 7: 239-243.
10. Johnson M S, McClure M A, Feng D-F, Gray J, Doolittle RF. Computer analysis of retroviruse genomes. Science, 1986, 215:1577-1585.
11. Thayer R M, Power M D, Bryant M, Gardner M B, Barr P J, Luciw P A. Sequence relationships of type D retroviruses which cause simian acquired immunodeficiency syndrom. Virol, 1987, 157: 317-329.
12. Temin R M. Evolution and modes of transmission of RNA tumor viruses. Am J Pathol, 1975, 81: 590-605.
13. Yokayama S, Chung L and Gojobori T. Molecular evolution of the human immunodeficiency and related viruses. Mol Bio Evol, 1988, 5: 237-251.
14. Lineal M and Blair D. (1984) Genetics of Retroviruses. In: Weiss R. Teich N. Varmus H. Coffin J(eds) Molecular Biology of Tumor Virus: RNA Tumor Viruses. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York. pp. 649-783.
15. Coffin JM, et al. 1995, Sixth report on virus classification and nomenclature. 193-203.
16. Wain Haberson S, Alizon M, Montagnier. Relationship of AIDS to other retrovirus. Nature, 1985, 733-743.
17. Brown D W, Blais BP, Robinson HL Long terminal repeat (LTR) sequences, env, and a region near the 5LTR influence the pathogenic potential of recombinations between Rous-associated virus type 0 and 1. J Virol, 1988, 62:3431-3437.
18. Chatis PA, Holland CA, Hartley JW, Rowe WP, Hopkins N. Role for the 3'end of the genome in determining disease specificity of Friend and Moloney murine leukemia virus.
Proc Natl Acad Sci USA, 1983,80:4408-4411．
19． Charts PA, Holland CA, Silver JE, Frederick TN, Hopkins N, Hartley JW. A 3'end fragment encompassing the transcriptional enhancer of nondefective Friend virus confer erythroleukemogenicity on Moloney leukemia virus. J Virol, 1984,52:248-254．
20． DeFranco D and Yamamoto K. The two different factors act separately or together specify functionally distinct activities at a single transcriptional enhancer. Mol Cell Bio, 1986, 993-1001．
21． DesGranseiller L and Jolicoceur P. The tandem direct repeats within the long terminal murine leukemia viruses are the primary determinant of their leukemogenic potential. J Vuol, 1986,56:945-952．
22． DesGranseiller L,Rassert E, Jolicoceur P. The mortropism of murine leukemia virus is conferred the long terminal repeat. Proc Natl Acad Sci USA, 1983,80:42034207．
23． Gorman CM, Merlino GT, Willingham MC, Pastan I, Howard BH. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Nad Acad Sci USA, 1982,79:6777-6781．
24． Graves BJ, Eisenberg SP, Coen DM, McKnight SL. Alerlnate utilization of two regulatory domains within the moloney murine sarcoma virus long terminal repeat. Mol Cell Biol, 1985a, 5:1959-1968．
25． Graves BJ, Eisenberg SP, Coen DM, McKnight SL. Delineation of transcriptional control signals within the moloney murine sarcoma virus long terminal repeat Mol Cell Bio, 1985b, 5:1948-1958．
26． Shimotono K, Mizutani S, Temin HM. Sequence of retrovims provirus resembles that of bacterial transposable elements. Nature, 1980,285:550-554．
27． Hughes SH, Mutschler A, Bishop JM, Varmus HE. A rous sarcoma virus provirus is flanking short direct repeats of a cellulare DNA sequence present in only one copy prior to integration. Natl Acad Sci USA, 1981,78:4299-4303．
28． Hughes SH, Shank PR, Spector DH, Kung HJ, Bishop JM, Varmus HE, Vogt PK, Breitman. Proviruses of avian sarcoma viruses are terminally redundant, co-extensive united linear DNA and integrated at many sites. Cell, 1978,15:1397-1410．
29． Colicelli J and Goff SP. Mutants and pseudo revertants of moloney murine leukemia virus with alterations at the integration site. Cell, 1985,42: 573-580．
30． Colicelli J and Goff SP. Sequence and spacing requirements of a retrovirus integration site. J MolBio, 1988a,199:47-59．
31． Coffin J M. Retrovridae and their replication, in: Virology 2nd ed. Fields B N, Knipe D M. eds. New York: Raven Press, 1990,1437-1500．
32． Varmus HE, Brown P (1989) Retroviruses In: Howe M, BERG D (eds) Mobile DNA.
ASM Press,Washingdon DC, pp53-109．
33． Varmus HE and Swanstrom R. (1982) Replication of retroviruses. In: Weiss R, Teich N, Varmus H, Coffin J (eds) RNA tumo viruses. Cold Spring Harbor Laboratory, NY, pp369-512．
34． Panganiban and Temia The terminal nucleotides of retrovirus DNA are required for integration but not virus production. Nature, 1983,306:155-160．
35． Mann R, Mulligan R C and Baltimore D. Construction of a retrovirus packaging mutant and it use to produce helper tree defective retrovirus. Cell (Cambridge, Mass) 1983, 33: 153-159．
36． Swanstrom R and Vogt PK. (1990) Retroviruses Strategies of Replication. Springer p54-56． Berlin Hedelberg, New York.
37． 张玉静主编，分子遗传学，北京：科学出版社.2000．
38． Huber B E, Richards C A, Krenitsky T A. Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma:An innovative approach for cancer therapy. Proc Natl Acad Sci USA, 1991,88:8039-8043．
39． Mulligan R C. The basic science of gene therapy. Science, 1993,260:926-932．
40． Lim B , F Apperley , S H Orion and D A Williams, Long term expression of human adenosine deaminase in mice transplanted with retrovirus infected hematopoietic stem cells. Proc Natl Acad Sci USA, 1989,8892-8896．
41． Cone R G and Mulligan R C. High-efficiency gene transfer into mammalian cells: Generation of helper-tree recombination retrovirusses with broad mammalian host range. Proc Natl Acad Sci USA, 1984,81(20) : 6349-6353．
42． Markowitz D, Goff S and Bank A. A safe packaging line for gene transfer: Separating viral genes on two different plasmid. J Virol, 1988a, 62:1120-1124．
43． Markowitz D, Goff S and B ank A. Construction and use of a safe and efficient amphotropic packaging cell line. Virology, 1988b, 167:400-406．
44． Eglitis MA. Positive selectable markers for use with mammalian cells in culture. Hum Gene Ther, 1991,2:195-201．
45． Miller A D and Rossman G J. Improved retroviral vectors for gene transfer and expression. Biotechnique, 1989,7:980-990．
46． Miller A D and Baltimore G J. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol, 1986,6:2895-2902．
47． Stylianos Andreadis, Thomas Lavery, Howard E, Davis, et al. Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection. J Virol, 2000,74:1258-1266．
48． Miller AD, Bender MA, Harris EA, Kaleko M, Gelinas RE. Design of retrovirus vectors for
transfer and expression of the human beta-globin gene. J Virol, 1988,62(11) : 4337-4345．
49． Mann R, Mulligan R C, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell, 1983,33(1) : 153-159．
50． Wilson J M, D M Jefferson, J R Chowdhury, P M Novikoff, D E Johnston, R C Mulligan. Retrovirus-mediated transduction of adult hepatocytes. Proc Natl Acad Sci USA, 1988． 85: 4421-4425．
51． Miller A D.Retrovirus packaging cells. Hum Gene Ther, 1990,1(1) :5-14．
52． Lynch CM and Miller A D. Production of high-titer help virus-free retroviral vectors by cocultivation of packaging cells with different host ranges. J Virol, 1991,65:3887-3890．
53． 金宁一，动物病毒载体研究进展，未发表
54． Salmons B and Gungbug WH. Targeting of retroviral vectors for gene therapy. Hum Gene Ther, 1993,4:129-141．
55． Young J A, Bates P, Willert K, Varmus H E. Efficient incorporation of human CD4 protein into avian leukosis virus particles-Science, 1990,250:1421-1423．
56． Dalgleish A G, P C L Beverley, P R Clapham, D H Crawford, M F Greaves and R A Weiss. The CD4 (T4) antigen is an essential component of the receptor for the ADDS retroviius. Nature (London) 1984,312: 736-766．
57． Klatzmann D E, Champagne S, Chamaret J, Gruest D, Guetard T, Hercend J C, Gluckman and Montagnier. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV Nature (London), 1984,312:767-768．
58． Sattentau Q J, P R Clapham, R A Weiss, P C L Beverley, L Montagnier, M F Alhabi, J C Gluckman, D Klatzmann. The human and simian immunodificiency viruses HIV-1, HIV-2 and SIV interact with similar epitopes on their cellular receptor, CD4 molecule. AIDS, 1988, 2:101-105．
59． Weiss R A. Viral glycoproteins and cellular receptors involved in retrovirus infection. P.1-108． In J A Levy (ed). The Retroviridae. Vol 2． Plenum Press. New York.
60． Hatzoglou M, Lamers W, Bosch F, et al. Hepatic gene transfer in Animals using retroviruses containing the promoter from the gene for phosphoenopyruvate carboxykinase. J BioChem, 1990,265:17285-17293．
61． Roux P, et al. A versatile and potentially general approach to the targeting of specific cell types by retrovimses. Proc Natl Cell Biol, 11:3070-3074．
62． Brinster R L, J M Allen, R R Behringer, R E Gellinas and R D Palmiter. Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA, 1988,85:836-840．
63． Buchman A R and P Berg. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol, 1988,8:4395-4405．
64． Choi T, M Huang, C Gorman and R A Jaenisch. A generic intron increase gene expression
intransgenic mice. Mol Cell Biol,1991,11:3070-3074．
65． Huang M T and C M Gorman. Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA. Nucleaic Acids Res, 1990, 18:937-947．
66． Lachmi B and L Karriainen. Control of protein synthesis in Semliki Forest virus-infected cells. J Virol, 1977,22:142-149．
67． Palmiter R D, E P Aandgren, M R Avarbock, D D Allen and L Blister. Heterologous introns can enhance expression of transgenic in mice. Proc Natl Acad Sci USA, 1991,88: 478-482．
68． Byun J, S H Kim, J M Kim, S S Yu, P D Robbins, J Yim and S Kim. Analysis of (he relative level of gene expression different retroviral vectors used for gene therapy. Gene Ther, 1996,3:780-788．
69． Krall W J, D C Skehon, X J Yu, I Revirere, P lehn, R C Mulligan and D B Kohn. Increased levels of spliced RNA account for augmented expression from the MFG retroviral vector in hematopoietic cells.Gene Ther, 1996,3:37-48．
70． Reviere L, KBrose and RC Mulligan. Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipient engrafted with genetically modified cells. Proc.Natl.Natl.Acad Sci, 1995,92:6733-6737．
71． Rabson A B and B J Graves. 1997． Synthesis and processing of viral RNA.p.205-261． In J.M.Coffin. S.Hughes and H.Varmus (eds). Retroviuses. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
72． Dzierzak E A, T Papayannopoulou and R C Mulligan. Lineagespecific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature, 1988,331:35-41．
73． Karlsson S, T Papayannopoulou, S G Schweiger, G Stamatoyannopoulos and W.Nienhuis. Retorviral-mediated transfer of genomic globin genes leads to regulated production of RNA and protein. Proc Nafl Acad Sci USA, 1987,84:2411-2415．
74． Miller A D, M A Bender, E A Harris, M Kaleko and R E Gellinas. Design of retrovirus vectors gor transfer and expression of the human beta-globin gene. J Virol, 1988,62:4337-4345．
75． Li KJ and H Garoff. Production of infectious recombinatm Moloney murine leukemia virus particles in BHK cells using Semiliki Forest virus-derived RNA expressing vector. Proc Natl Acad Sci USA, 1996,93:11658-11663．
76． Li K J and H Garoff. Packaging of intron-containg genes into retrovirus vectors by alphavirus vectors. Proc Natl Acad Sci USA, 1998,95:3650-3654．
77． Wengler G. Protein synthesis in BHK-21 cells infected with Semliki Forest virus. J Virol,
1975,17:10-19．
78． Said I.Ismail, Susan M, Kingsman Alan, J Kingsman and Mark Uden. Split-intron retroviral vectors: enhanced expression with improved safety 2000,74:2365-2371．
79． Miler A D M and G J Rosman. Improved retroviral vectors for gene transfer and expression. Biotechniques, 1989,7:980-982,984-986,989-990．
80． SoneokaY,P M Cannon,E E Ramsdale,J C Griffiths,G Romano,SM Kingsman and A J Kingsman, A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res, 1995,23:628-633．
81． Mayeda A and Y Ohshima. Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro. Mol Cell Biol,1988,8:4484-4491．
82． Telesnitsky A and S P Gogg. 1997． Reverse transcriptase and generation of retroviral DNA, p. 121-160． In J M Coffin, S Hughes and H Varmus (ed), Retroviruses. Cold Spring Harbor Laoratory Press. Plainview. N.Y.
83． Pinol-Roma S and Gdreyfuss. Shuttling of pre-mRNA binding protein between nucleus and cytoplasm. Nature, 1992,355:730-732．
84． Niwa M, S D Rose and S M Berget In vitro polyadenylation is stimulated by the presence of an upstream intron. Genes Dev, 1990,7:2194-2205．
85． Pandey N B, N Choddhoy, T J Liu and W F Marzluff. Introns in histone genes alter the distribution of 3' ends.Nucleic AcidsRes, 1990,18:3161-3170．
86． Lachmi B and L Kaariainen. Control of protein synthesis in Semiki Forest virus-infected cells. J Virol, 1997,22:142-149．
87． Emerman M, Temin HM. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell, 1984,39 (3 Pt 2) : 449-67．
88． Dzierzak E A, Papayannopoulou T and Mulligan R C. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature, 1988,331:35-41．
89． Theodore Friedmann. Progress toward human gene therapy. Science, 1989,244:1275-1281．
90． Shin C C, Stoye J P, Coffin JM. Highly preferred targets for retrovirus integration. Cell, 1988,53(4) : 531-537．
91． Vijaya S, Steffen D L, Robinson H L. Acceptor sites for retroviral integrations map near DNase I-hypersensitive sites in chromatin. J Virol, 1986,60:683-692．
92． Yee J K, Moores J C, Jolly D J, Wolff J A, Respess JG, Friedmann T. Gene expression from transcriptionally disabled retroviral vectors. Proc Natl Acad Sci USA, 1987, 84: 5197-5201．
93． Yu SF, von Ruden T, KantoffPW, Garber C, Seiberg M, Ruther U, Anderson WF,
EF, Gilboa E. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc Natl Acad Sci USA. 1986,83:3194-3198．
94． Lirmey E, Neill SD, Prestridge DS. Retroviral vector gene expression in F9 embryonal carcinoma cells. Virol, 1987,61:3248-3253．
95． Okuyama T, Huber RM, Bowling W, Peariine R, Kennedy SC, Flye MW, Ponder KP. Liver-directed gene therapy: a retroviral vector with a complete LTR and the ApoE enhancer-alpha 1-antitrypsin promoter dramatically increases expression of human alpha 1-antitrypsin in vivo. Hum Gene Ther. 19%, 7:637-645．
96． Cometta K.et al. Safety issues related to retroviral-mediated gene transfer in humans. Humman Gene Therapy, 1991,2:5-14．
97． Cometta K, Moen RC, Culver K, Morgan RA, McLachlin JR, Sturm S, Selegue J, London W, Blaese RM, Anderson WF. Amphotropic murine leukemia retrovirus is not an acute pathogen for primates. Hum Gene Ther, 1990,1:15-30．
98． 伍志坚，逆转录病毒载体用于基因治疗的安全性. Letters in Biotechnology, 1994,5: 80-81．
99． Temin HM. Retroviruses and cancer. Mol Carcino, 1990,3:183-184．
100． Selten G, Cuypers HT, Berns A. Proviral activation of the putative oncogene Pim-1 in MuLV induced T-cell lymphomas. EMBO J, 1985,4:1793-1798．
101． van Lohuizen M, Verbeek S, Krimpenfort P, Domen J, Saris C, Radaszkiewicz T, Berns A. Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors. Cell, 1989,56:673-682．
102． Courtney M G, Schmidt L J, Getz M J. Organization and expression of endogenous virus-like (VL30) DNA sequences in nontransformed and chemically transformed mice embryo cells in culture. Cancer Res, 1982,42:569-576．
103． OnoM, Cole MD, White AT, Huang RC. Sequence organization of cloned intracistemal A particle genes. Cell, 1980,21:465-473．
104． Hatzoglou M, Hodgson CP, Mularo F, Hanson RW. Efficient packaging of a specific VL30 retroelement by psi 2 cells which produce MoMLV recombinant retroviruses. Hum Gene Ther, 1990,1:385-397．
105． Bender MA, Palmer TD, Gelinas RE, Miller AD. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region, J Virol, 1987, 61: 1639-1646．
106． Markowitz D, Goff S, Bank A. A safe packaging line for gene transfer separating viral genes on two different plasmids. J Virol, 1988,62:1120-1124．
107． Markowitz D, Goff S, Bank A. Construction of a safe and efficient retrovirus packaging cell line. Adv Exp Med Biol, 1988,241:35-40．
108. Markowitz D G, Goff S P, Bank A. Safe and efficient ecotropic and amphotropic packaging lines for use in gene transfer experiments. Trans Assoc Am Physicians, 1988, 101: 212-218.
109. Jaenisch R and Mintz B. Simian virus SV40 DNA sequence in DNA of healthy adult mice derived from preimplantation blastocytes injected with viral DNA. Proc Ncad Sci USA, 1974, 74: 1250-1254.
110. Palmiter RD, Brinster RE, Hammer ME, et al. Dramatic growth of mice that develop from eggs microinjected with a metallothionein-growth hormone fusion gene. Nature, 1982, 300. 611-615.
111. Schnieke A E, Kind A J, Ritchie W A, et al. Human factor Ⅸ transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science, 1997, 278. 2130-2133.
112. Gordon A, Scangos GA, Plotkin DJ, et al. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA, 1980, 77: 7380-7384.
113. Jaenisch R. Transgenic animals. Science, 1988,240: 1468-1474.
114. Brackett BG, Barabska W, Sawicki W, et al. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Natl Acad Sci USA, 1971,68. 353-357.
115. Arezzo E Sea urchin sperm as a vector of foreign genetic information. Cell Biol Int Rep, 1989, 13: 391-404.
116．于健康，阎维，张玉康等．精子介导鱼类基因转移和聚合酶链反应法检测技术．动物学报，1994，40：96-98
117. Lavitrano M, Camaioni A, Fazio VM, et al. Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell, 1989, 57: 17.
118. Zhu Z, et al. Biological effects of human hormore gene microinjection into fertilized eggs of loach. Kexue Tongbao, 1986, 31: 988-989.
119．朱作言等．转基因鱼模型的建立．中国科学(B辑)，1989，2：147-155．
120．卢强．抗菌肽Cecropin B基因突变体原核表达及转基因鱼的建立[博士论文]．长春：解放军军需大学．2000．
121. Tanaka K, Wada T, Koga O, Nishio Y, Hertelendy F. Chick production by in vitro fertilization of the fowl ovum. J Reprod Fertil, 1994, 100(2): 447-449.
122. Fassati A, Dunckley MG, Dickson G. Retroviral vectors. Mol Cell Biol Hum Dis Ser, 1995, 5: 1-19.
123. Takeuchi Y, Pizzato M. Retrovirus vectors. Adv Exp Med Biol, 2000,465:23-35.
124. Salter DW, Smith EJ, Hughes SH, Wright SE, Crittenden LB. Transgenic chickens: insertion of retroviral genes into the chicken germ line. Virology. 1987, 157(1): 236-240.
125. Crittenden L B and Salter D W. Expression of retroviral genes in transgenic chickens. J Reprod Fertil Suppl, 1990,41: 163-171.
126. Chan A W S, Homan E J, Ballon L U, et al. Transgenic cattle produced by reversetranscribed gene transfer in ooctyes. Proc Natl Acad USA, 1998, 95:14028-14033.
127. Tarkowki A K, Witkowska A and Nowicka J. Expeimental partheongenesis in the mouse. Nature, 1979, 226: 162-165.
128. BrinsterKL. Embryo development J Anim Sci, 1974, 38: 1003-1012.
129. Evans E P and Kaufman M E. Establishment in culture of pluripotential from mice embryos. Nature, 1981, 292: 154-156.
130. Martin GR. Isolation of a pluripotential cell line from early mouse embryos cultured in medium conditioned by teratocarinoma stem cells. Proc Acad Sci USA, 1981, 78: 7634-7638.
131. Wager E F, Keller G, Gilboa E, et al. In: Cold Spring Harbor Symposia Biology. Cold Spring Harbor Laboratory, 1985, 50: 619.
132. Robertson E, Bradley A, Kuehn M, et al. Germ line transmission of gene introduced into cultured pluripotential cell by retroviral vector. Nature, 1986, 323: 445-448.
133. Rottmann OJ, Stratowa C, Homstein M, Hughes J. Tissue specific expression of hepatitis B surface antigen in mice following liposome--mediated gene transfer into blastocysts. Zentralbl Veterinarmed A, 1985, 32: 676-682.
134. Perry A C F, Wakayama T, Kishikama H, et al. Mammalian transgenesis by intracytoplasmic sperm injection. Science, 1999, 284(5422): 1180-1183.
135．周荣家，张思促，赵连三．小鼠精子捕获外源DNA的研院．科学通报，1992，37：1246-1247．
136. Nakanishi A and Iritani A, Gene transfer in the chicken by sperm-mediated methods. Reprod Dev, 1993, 36: 258-261.
137. Horan R et al, Association of foreign DNA with porcine spermtozoa, Arch Androl, 1991, 26: 83-92.
138. Bachiller D, Schellander K, Peli J, Ruther U. Liposome-mediated DNA uptake by sperm cells. Mol Reprod Dev. 1991, 30(3): 194-200.
139．郭志儒，金宁一．2000年动物克隆及相关领域研究进展．中国兽医学报，2001，3：208-212
140．克隆研究大事记．科学时报，2001-4-16．
141. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A, Campbell KH. Human factor Ⅸ transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science. 1997, 19, 278(5346): 2130-2133.
142. Wilmut I, Schnieke A E, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature, 1997, 385: 810-813.
143. Polejaeva I A, Chen S H, Vaught T D, et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 2000, 407: 86-89.
144. Onishi A, Iwamoto M, Takeda K, et al. Pig cloning by microinjection of fetal fibroblast nuclei. Science, 2000, 289: 1188-1189.
145. Cibeli J B, Stice S L, Golueke P J, et al. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science, 1998, 280: 1256-1258.
146. PPL develops new and improved techniques to create transgenic animals. Biotechnol News, 1999.08.05(6).
147．田小利，陈兰英．转基因动物研究中存在的问题．生物工程进展，1995，15：41-45．
148. Theodre friedmann. Progress toward human gene therapy. Science, 1989, 244: 1275-1281.
149. Crystal R G. Transfer of genes to humans: early lessons and obstacles to success. Science, 1995, 270: 404-410.
150. Russell S T. Lymphokine gene transfer for cancer. Immunology Today, 1990, 11: 196-200.
151. Kotani H, P B I Newton, S Zhang, Y L, et al. Improved methods of retroviral vector transduction and production of gene therapy. Human gene therapy, 1994, 5: 19-28.
152．J 萨姆布鲁克，E F 弗里奇，T 曼尼阿蒂斯．分子克隆实验指南．第2版．金冬雁．黎孟枫译．北京：科学出版社，1992，786-792．
153．卢圣栋主编．现代分子生物学实验技术．北京：高等教育出版社，1993．
154．F 奥斯伯，R 布伦特，R E 金斯顿等．精编分子生物学实验指南．严子颖，王海林译．北京：科学出版社，1998，312-328．
155. Sita Reddy, Helen Raybum, Harald von Melchner et al. Fluorescence-activated sorting of totipolent embryonic stem cells expressing developmentally regulated LacZ fussion genes. Proc Natl Acad Sci USA, 1992, 89:6721.
156．章金刚．猪瘟病毒反义基因片段体外效应研究[博士论文]．长春：解放军农牧大学，1994．
157. Stylianos andreadis, Thomas lavery, Howard E Davis, et al,. Toward a more accurate quantitation of the activity of recombinant retrovimses: alternatives to titer and multiplicity of infection. J Virol, 2000, 74: 1258-1266.
158. Morgan J R, J M Ledoux, R G Snow, et al. Retrovirus infection: effect of time and target cell number. J Virol. 1995, 69: 6994-7000.
159. Stephen J Russell, Robert E Haekins and Greg Winter. Retroviral vectors displaying functional antibody fragments. Nucleic Acids Research, 1993, 21: 1081-1085.
160. Cynthia A Chambers, Joonsoo Kang and Nobumichi Hozumi. Long term expression of IL-4 in vivo using retroviral-mediated gene transfer. J Virol, 1992, 149: 2895-2905.
161. Mitchill L Drumm, Heidi A Pope, William H Cliff, et al. Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer. Cell, 62: 1227-1233.
162. Jahner D, et al. Proc Natl Acad Sci USA. 1985, 82: 6927-6931.
163．扈荣良．小鼠体内狂犬病病毒糖蛋白基因转移研究[博士学位论文]．长春．解放军农牧大学，1995．
164．赖良学，张学明，安靓等．精原干细胞体内转染途径建立转基因小鼠的可能性研究．生物技术通讯，1997，8：103-104．
165. Lai L X, Huang W M, Wang K, et al. Generation of transgenic mice expressing human tissue-type plasminogen activator in mammary gland by transfecting spermatogonia in vivo. In: Annual Anthology of University of Agriculture and Animal Science, PLA. Changchun: Published by University of Agriculture and Animal Science of PLA. 1997, 11-16.
166．赖良学．乳腺表达人组织性纤溶酶源激活剂的转基因小鼠建立．解放军农牧大学博士后报告，1997．
167. Kim J H, Jung Ha H S, Lee H T, et al. Development of a positive method for male stem cell - mediated gene transfer in mouse and pig, Mol Reprod Dev, 1997, 46 (4): 515-526.
168．孙敬方，朱德生，郝光荣等．实验动物学技术．北京：科学技术文献出版社，1993，28-29．
169．米娜瓦．阿木提，热孜亚．买买提．西蒙塔尔和黑白花种公牛精子顶体完整率、畸形率和活力测定．新疆农业科学．1994，4：185-186．
170．孙淑琴，稀释液中添加V_B12注射液对牛细管冷冻精液品质的影响[硕士学位论文]．延吉：延边大学，2000．
171．陈飞，黄丽娟，张罗漫．实用医学统计学．解放军农牧大学自编教材．94-99．
172．C W迪芬巴赫，GS德维克期勒，PCR技术实验指南。黄培堂，俞炜源，陈添弥等译．北京：科学出版社，1998，23-40．
173．张学明，文兴豪，赖良学等．性成熟前小鼠生精细胞的发育过程．中国兽医学报，2000，3：293-298．
174. Gordon J W, Scangos G A, Plotkin D J, Barbosa J A, Ruddle H. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA, 1980,77: 7380-7384.
175．乔贵林，周轶琳，赵君等．应用曲细精管注射法建立人白细胞抗原B27(HLA-B27)转基因鼠．中国兽医学报，1999．19：192．
176．田小利，陈兰英，扈荣良主编，转基因动物原理、技术与应用．吉林：吉林科学技术出版社，1994．
2．刘子夜，齐义鹏，王业富．用于基因治疗的病毒载体．中国病毒学，1998，13：101．
3. Kevin, Karen Moore and Robert Wall. Transgene vectors go retro. Nature Biotechnology, 1999, 17:25.
4．陈诗书，汤雪明主编．医学细胞与分子生物学．上海：上海医科大学出版社，1999．
5．殷霞，刘景华主编．动物病毒学．第二版．北京：科学出版社，1997。
3．侯云德著．分子病毒学．北京，学苑出版社，1990．
4. Holland J, Spindler K, Horodyski F, Nichol S and Vandepol. Rapid evolution of RNA virus genomes. Science, 1982, 215:1577-1585.
8. Clark SP and Mak TW. Fluidity of a retrovirus genome. J Virol, 1984, 50: 759-765.
9. Hansen J, Schulze T, Mellert W, Modeling K. Identification and characterization of HW specific RNase H by monoclonal antibody. EMBO J, 1988, 7: 239-243.
10. Johnson M S, McClure M A, Feng D-F, Gray J, Doolittle RF. Computer analysis of retroviruse genomes. Science, 1986, 215:1577-1585.
11. Thayer R M, Power M D, Bryant M, Gardner M B, Barr P J, Luciw P A. Sequence relationships of type D retroviruses which cause simian acquired immunodeficiency syndrom. Virol, 1987, 157: 317-329.
12. Temin R M. Evolution and modes of transmission of RNA tumor viruses. Am J Pathol, 1975, 81: 590-605.
13. Yokayama S, Chung L and Gojobori T. Molecular evolution of the human immunodeficiency and related viruses. Mol Bio Evol, 1988, 5: 237-251.
14. Lineal M and Blair D. (1984) Genetics of Retroviruses. In: Weiss R. Teich N. Varmus H. Coffin J(eds) Molecular Biology of Tumor Virus: RNA Tumor Viruses. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York. pp. 649-783.
15. Coffin JM, et al. 1995, Sixth report on virus classification and nomenclature. 193-203.
16. Wain Haberson S, Alizon M, Montagnier. Relationship of AIDS to other retrovirus. Nature, 1985, 733-743.
17. Brown D W, Blais BP, Robinson HL Long terminal repeat (LTR) sequences, env, and a region near the 5LTR influence the pathogenic potential of recombinations between Rous-associated virus type 0 and 1. J Virol, 1988, 62:3431-3437.
18. Chatis PA, Holland CA, Hartley JW, Rowe WP, Hopkins N. Role for the 3'end of the genome in determining disease specificity of Friend and Moloney murine leukemia virus.
Proc Natl Acad Sci USA, 1983,80:4408-4411．
19． Charts PA, Holland CA, Silver JE, Frederick TN, Hopkins N, Hartley JW. A 3'end fragment encompassing the transcriptional enhancer of nondefective Friend virus confer erythroleukemogenicity on Moloney leukemia virus. J Virol, 1984,52:248-254．
20． DeFranco D and Yamamoto K. The two different factors act separately or together specify functionally distinct activities at a single transcriptional enhancer. Mol Cell Bio, 1986, 993-1001．
21． DesGranseiller L and Jolicoceur P. The tandem direct repeats within the long terminal murine leukemia viruses are the primary determinant of their leukemogenic potential. J Vuol, 1986,56:945-952．
22． DesGranseiller L,Rassert E, Jolicoceur P. The mortropism of murine leukemia virus is conferred the long terminal repeat. Proc Natl Acad Sci USA, 1983,80:42034207．
23． Gorman CM, Merlino GT, Willingham MC, Pastan I, Howard BH. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Nad Acad Sci USA, 1982,79:6777-6781．
24． Graves BJ, Eisenberg SP, Coen DM, McKnight SL. Alerlnate utilization of two regulatory domains within the moloney murine sarcoma virus long terminal repeat. Mol Cell Biol, 1985a, 5:1959-1968．
25． Graves BJ, Eisenberg SP, Coen DM, McKnight SL. Delineation of transcriptional control signals within the moloney murine sarcoma virus long terminal repeat Mol Cell Bio, 1985b, 5:1948-1958．
26． Shimotono K, Mizutani S, Temin HM. Sequence of retrovims provirus resembles that of bacterial transposable elements. Nature, 1980,285:550-554．
27． Hughes SH, Mutschler A, Bishop JM, Varmus HE. A rous sarcoma virus provirus is flanking short direct repeats of a cellulare DNA sequence present in only one copy prior to integration. Natl Acad Sci USA, 1981,78:4299-4303．
28． Hughes SH, Shank PR, Spector DH, Kung HJ, Bishop JM, Varmus HE, Vogt PK, Breitman. Proviruses of avian sarcoma viruses are terminally redundant, co-extensive united linear DNA and integrated at many sites. Cell, 1978,15:1397-1410．
29． Colicelli J and Goff SP. Mutants and pseudo revertants of moloney murine leukemia virus with alterations at the integration site. Cell, 1985,42: 573-580．
30． Colicelli J and Goff SP. Sequence and spacing requirements of a retrovirus integration site. J MolBio, 1988a,199:47-59．
31． Coffin J M. Retrovridae and their replication, in: Virology 2nd ed. Fields B N, Knipe D M. eds. New York: Raven Press, 1990,1437-1500．
32． Varmus HE, Brown P (1989) Retroviruses In: Howe M, BERG D (eds) Mobile DNA.
ASM Press,Washingdon DC, pp53-109．
33． Varmus HE and Swanstrom R. (1982) Replication of retroviruses. In: Weiss R, Teich N, Varmus H, Coffin J (eds) RNA tumo viruses. Cold Spring Harbor Laboratory, NY, pp369-512．
34． Panganiban and Temia The terminal nucleotides of retrovirus DNA are required for integration but not virus production. Nature, 1983,306:155-160．
35． Mann R, Mulligan R C and Baltimore D. Construction of a retrovirus packaging mutant and it use to produce helper tree defective retrovirus. Cell (Cambridge, Mass) 1983, 33: 153-159．
36． Swanstrom R and Vogt PK. (1990) Retroviruses Strategies of Replication. Springer p54-56． Berlin Hedelberg, New York.
37． 张玉静主编，分子遗传学，北京：科学出版社.2000．
38． Huber B E, Richards C A, Krenitsky T A. Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma:An innovative approach for cancer therapy. Proc Natl Acad Sci USA, 1991,88:8039-8043．
39． Mulligan R C. The basic science of gene therapy. Science, 1993,260:926-932．
40． Lim B , F Apperley , S H Orion and D A Williams, Long term expression of human adenosine deaminase in mice transplanted with retrovirus infected hematopoietic stem cells. Proc Natl Acad Sci USA, 1989,8892-8896．
41． Cone R G and Mulligan R C. High-efficiency gene transfer into mammalian cells: Generation of helper-tree recombination retrovirusses with broad mammalian host range. Proc Natl Acad Sci USA, 1984,81(20) : 6349-6353．
42． Markowitz D, Goff S and Bank A. A safe packaging line for gene transfer: Separating viral genes on two different plasmid. J Virol, 1988a, 62:1120-1124．
43． Markowitz D, Goff S and B ank A. Construction and use of a safe and efficient amphotropic packaging cell line. Virology, 1988b, 167:400-406．
44． Eglitis MA. Positive selectable markers for use with mammalian cells in culture. Hum Gene Ther, 1991,2:195-201．
45． Miller A D and Rossman G J. Improved retroviral vectors for gene transfer and expression. Biotechnique, 1989,7:980-990．
46． Miller A D and Baltimore G J. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol, 1986,6:2895-2902．
47． Stylianos Andreadis, Thomas Lavery, Howard E, Davis, et al. Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection. J Virol, 2000,74:1258-1266．
48． Miller AD, Bender MA, Harris EA, Kaleko M, Gelinas RE. Design of retrovirus vectors for
transfer and expression of the human beta-globin gene. J Virol, 1988,62(11) : 4337-4345．
49． Mann R, Mulligan R C, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell, 1983,33(1) : 153-159．
50． Wilson J M, D M Jefferson, J R Chowdhury, P M Novikoff, D E Johnston, R C Mulligan. Retrovirus-mediated transduction of adult hepatocytes. Proc Natl Acad Sci USA, 1988． 85: 4421-4425．
51． Miller A D.Retrovirus packaging cells. Hum Gene Ther, 1990,1(1) :5-14．
52． Lynch CM and Miller A D. Production of high-titer help virus-free retroviral vectors by cocultivation of packaging cells with different host ranges. J Virol, 1991,65:3887-3890．
53． 金宁一，动物病毒载体研究进展，未发表
54． Salmons B and Gungbug WH. Targeting of retroviral vectors for gene therapy. Hum Gene Ther, 1993,4:129-141．
55． Young J A, Bates P, Willert K, Varmus H E. Efficient incorporation of human CD4 protein into avian leukosis virus particles-Science, 1990,250:1421-1423．
56． Dalgleish A G, P C L Beverley, P R Clapham, D H Crawford, M F Greaves and R A Weiss. The CD4 (T4) antigen is an essential component of the receptor for the ADDS retroviius. Nature (London) 1984,312: 736-766．
57． Klatzmann D E, Champagne S, Chamaret J, Gruest D, Guetard T, Hercend J C, Gluckman and Montagnier. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV Nature (London), 1984,312:767-768．
58． Sattentau Q J, P R Clapham, R A Weiss, P C L Beverley, L Montagnier, M F Alhabi, J C Gluckman, D Klatzmann. The human and simian immunodificiency viruses HIV-1, HIV-2 and SIV interact with similar epitopes on their cellular receptor, CD4 molecule. AIDS, 1988, 2:101-105．
59． Weiss R A. Viral glycoproteins and cellular receptors involved in retrovirus infection. P.1-108． In J A Levy (ed). The Retroviridae. Vol 2． Plenum Press. New York.
60． Hatzoglou M, Lamers W, Bosch F, et al. Hepatic gene transfer in Animals using retroviruses containing the promoter from the gene for phosphoenopyruvate carboxykinase. J BioChem, 1990,265:17285-17293．
61． Roux P, et al. A versatile and potentially general approach to the targeting of specific cell types by retrovimses. Proc Natl Cell Biol, 11:3070-3074．
62． Brinster R L, J M Allen, R R Behringer, R E Gellinas and R D Palmiter. Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA, 1988,85:836-840．
63． Buchman A R and P Berg. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol, 1988,8:4395-4405．
64． Choi T, M Huang, C Gorman and R A Jaenisch. A generic intron increase gene expression
intransgenic mice. Mol Cell Biol,1991,11:3070-3074．
65． Huang M T and C M Gorman. Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA. Nucleaic Acids Res, 1990, 18:937-947．
66． Lachmi B and L Karriainen. Control of protein synthesis in Semliki Forest virus-infected cells. J Virol, 1977,22:142-149．
67． Palmiter R D, E P Aandgren, M R Avarbock, D D Allen and L Blister. Heterologous introns can enhance expression of transgenic in mice. Proc Natl Acad Sci USA, 1991,88: 478-482．
68． Byun J, S H Kim, J M Kim, S S Yu, P D Robbins, J Yim and S Kim. Analysis of (he relative level of gene expression different retroviral vectors used for gene therapy. Gene Ther, 1996,3:780-788．
69． Krall W J, D C Skehon, X J Yu, I Revirere, P lehn, R C Mulligan and D B Kohn. Increased levels of spliced RNA account for augmented expression from the MFG retroviral vector in hematopoietic cells.Gene Ther, 1996,3:37-48．
70． Reviere L, KBrose and RC Mulligan. Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipient engrafted with genetically modified cells. Proc.Natl.Natl.Acad Sci, 1995,92:6733-6737．
71． Rabson A B and B J Graves. 1997． Synthesis and processing of viral RNA.p.205-261． In J.M.Coffin. S.Hughes and H.Varmus (eds). Retroviuses. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
72． Dzierzak E A, T Papayannopoulou and R C Mulligan. Lineagespecific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature, 1988,331:35-41．
73． Karlsson S, T Papayannopoulou, S G Schweiger, G Stamatoyannopoulos and W.Nienhuis. Retorviral-mediated transfer of genomic globin genes leads to regulated production of RNA and protein. Proc Nafl Acad Sci USA, 1987,84:2411-2415．
74． Miller A D, M A Bender, E A Harris, M Kaleko and R E Gellinas. Design of retrovirus vectors gor transfer and expression of the human beta-globin gene. J Virol, 1988,62:4337-4345．
75． Li KJ and H Garoff. Production of infectious recombinatm Moloney murine leukemia virus particles in BHK cells using Semiliki Forest virus-derived RNA expressing vector. Proc Natl Acad Sci USA, 1996,93:11658-11663．
76． Li K J and H Garoff. Packaging of intron-containg genes into retrovirus vectors by alphavirus vectors. Proc Natl Acad Sci USA, 1998,95:3650-3654．
77． Wengler G. Protein synthesis in BHK-21 cells infected with Semliki Forest virus. J Virol,
1975,17:10-19．
78． Said I.Ismail, Susan M, Kingsman Alan, J Kingsman and Mark Uden. Split-intron retroviral vectors: enhanced expression with improved safety 2000,74:2365-2371．
79． Miler A D M and G J Rosman. Improved retroviral vectors for gene transfer and expression. Biotechniques, 1989,7:980-982,984-986,989-990．
80． SoneokaY,P M Cannon,E E Ramsdale,J C Griffiths,G Romano,SM Kingsman and A J Kingsman, A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res, 1995,23:628-633．
81． Mayeda A and Y Ohshima. Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro. Mol Cell Biol,1988,8:4484-4491．
82． Telesnitsky A and S P Gogg. 1997． Reverse transcriptase and generation of retroviral DNA, p. 121-160． In J M Coffin, S Hughes and H Varmus (ed), Retroviruses. Cold Spring Harbor Laoratory Press. Plainview. N.Y.
83． Pinol-Roma S and Gdreyfuss. Shuttling of pre-mRNA binding protein between nucleus and cytoplasm. Nature, 1992,355:730-732．
84． Niwa M, S D Rose and S M Berget In vitro polyadenylation is stimulated by the presence of an upstream intron. Genes Dev, 1990,7:2194-2205．
85． Pandey N B, N Choddhoy, T J Liu and W F Marzluff. Introns in histone genes alter the distribution of 3' ends.Nucleic AcidsRes, 1990,18:3161-3170．
86． Lachmi B and L Kaariainen. Control of protein synthesis in Semiki Forest virus-infected cells. J Virol, 1997,22:142-149．
87． Emerman M, Temin HM. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell, 1984,39 (3 Pt 2) : 449-67．
88． Dzierzak E A, Papayannopoulou T and Mulligan R C. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature, 1988,331:35-41．
89． Theodore Friedmann. Progress toward human gene therapy. Science, 1989,244:1275-1281．
90． Shin C C, Stoye J P, Coffin JM. Highly preferred targets for retrovirus integration. Cell, 1988,53(4) : 531-537．
91． Vijaya S, Steffen D L, Robinson H L. Acceptor sites for retroviral integrations map near DNase I-hypersensitive sites in chromatin. J Virol, 1986,60:683-692．
92． Yee J K, Moores J C, Jolly D J, Wolff J A, Respess JG, Friedmann T. Gene expression from transcriptionally disabled retroviral vectors. Proc Natl Acad Sci USA, 1987, 84: 5197-5201．
93． Yu SF, von Ruden T, KantoffPW, Garber C, Seiberg M, Ruther U, Anderson WF,
EF, Gilboa E. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc Natl Acad Sci USA. 1986,83:3194-3198．
94． Lirmey E, Neill SD, Prestridge DS. Retroviral vector gene expression in F9 embryonal carcinoma cells. Virol, 1987,61:3248-3253．
95． Okuyama T, Huber RM, Bowling W, Peariine R, Kennedy SC, Flye MW, Ponder KP. Liver-directed gene therapy: a retroviral vector with a complete LTR and the ApoE enhancer-alpha 1-antitrypsin promoter dramatically increases expression of human alpha 1-antitrypsin in vivo. Hum Gene Ther. 19%, 7:637-645．
96． Cometta K.et al. Safety issues related to retroviral-mediated gene transfer in humans. Humman Gene Therapy, 1991,2:5-14．
97． Cometta K, Moen RC, Culver K, Morgan RA, McLachlin JR, Sturm S, Selegue J, London W, Blaese RM, Anderson WF. Amphotropic murine leukemia retrovirus is not an acute pathogen for primates. Hum Gene Ther, 1990,1:15-30．
98． 伍志坚，逆转录病毒载体用于基因治疗的安全性. Letters in Biotechnology, 1994,5: 80-81．
99． Temin HM. Retroviruses and cancer. Mol Carcino, 1990,3:183-184．
100． Selten G, Cuypers HT, Berns A. Proviral activation of the putative oncogene Pim-1 in MuLV induced T-cell lymphomas. EMBO J, 1985,4:1793-1798．
101． van Lohuizen M, Verbeek S, Krimpenfort P, Domen J, Saris C, Radaszkiewicz T, Berns A. Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors. Cell, 1989,56:673-682．
102． Courtney M G, Schmidt L J, Getz M J. Organization and expression of endogenous virus-like (VL30) DNA sequences in nontransformed and chemically transformed mice embryo cells in culture. Cancer Res, 1982,42:569-576．
103． OnoM, Cole MD, White AT, Huang RC. Sequence organization of cloned intracistemal A particle genes. Cell, 1980,21:465-473．
104． Hatzoglou M, Hodgson CP, Mularo F, Hanson RW. Efficient packaging of a specific VL30 retroelement by psi 2 cells which produce MoMLV recombinant retroviruses. Hum Gene Ther, 1990,1:385-397．
105． Bender MA, Palmer TD, Gelinas RE, Miller AD. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region, J Virol, 1987, 61: 1639-1646．
106． Markowitz D, Goff S, Bank A. A safe packaging line for gene transfer separating viral genes on two different plasmids. J Virol, 1988,62:1120-1124．
107． Markowitz D, Goff S, Bank A. Construction of a safe and efficient retrovirus packaging cell line. Adv Exp Med Biol, 1988,241:35-40．
108. Markowitz D G, Goff S P, Bank A. Safe and efficient ecotropic and amphotropic packaging lines for use in gene transfer experiments. Trans Assoc Am Physicians, 1988, 101: 212-218.
109. Jaenisch R and Mintz B. Simian virus SV40 DNA sequence in DNA of healthy adult mice derived from preimplantation blastocytes injected with viral DNA. Proc Ncad Sci USA, 1974, 74: 1250-1254.
110. Palmiter RD, Brinster RE, Hammer ME, et al. Dramatic growth of mice that develop from eggs microinjected with a metallothionein-growth hormone fusion gene. Nature, 1982, 300. 611-615.
111. Schnieke A E, Kind A J, Ritchie W A, et al. Human factor Ⅸ transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science, 1997, 278. 2130-2133.
112. Gordon A, Scangos GA, Plotkin DJ, et al. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA, 1980, 77: 7380-7384.
113. Jaenisch R. Transgenic animals. Science, 1988,240: 1468-1474.
114. Brackett BG, Barabska W, Sawicki W, et al. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Natl Acad Sci USA, 1971,68. 353-357.
115. Arezzo E Sea urchin sperm as a vector of foreign genetic information. Cell Biol Int Rep, 1989, 13: 391-404.
116．于健康，阎维，张玉康等．精子介导鱼类基因转移和聚合酶链反应法检测技术．动物学报，1994，40：96-98
117. Lavitrano M, Camaioni A, Fazio VM, et al. Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell, 1989, 57: 17.
118. Zhu Z, et al. Biological effects of human hormore gene microinjection into fertilized eggs of loach. Kexue Tongbao, 1986, 31: 988-989.
119．朱作言等．转基因鱼模型的建立．中国科学(B辑)，1989，2：147-155．
120．卢强．抗菌肽Cecropin B基因突变体原核表达及转基因鱼的建立[博士论文]．长春：解放军军需大学．2000．
121. Tanaka K, Wada T, Koga O, Nishio Y, Hertelendy F. Chick production by in vitro fertilization of the fowl ovum. J Reprod Fertil, 1994, 100(2): 447-449.
122. Fassati A, Dunckley MG, Dickson G. Retroviral vectors. Mol Cell Biol Hum Dis Ser, 1995, 5: 1-19.
123. Takeuchi Y, Pizzato M. Retrovirus vectors. Adv Exp Med Biol, 2000,465:23-35.
124. Salter DW, Smith EJ, Hughes SH, Wright SE, Crittenden LB. Transgenic chickens: insertion of retroviral genes into the chicken germ line. Virology. 1987, 157(1): 236-240.
125. Crittenden L B and Salter D W. Expression of retroviral genes in transgenic chickens. J Reprod Fertil Suppl, 1990,41: 163-171.
126. Chan A W S, Homan E J, Ballon L U, et al. Transgenic cattle produced by reversetranscribed gene transfer in ooctyes. Proc Natl Acad USA, 1998, 95:14028-14033.
127. Tarkowki A K, Witkowska A and Nowicka J. Expeimental partheongenesis in the mouse. Nature, 1979, 226: 162-165.
128. BrinsterKL. Embryo development J Anim Sci, 1974, 38: 1003-1012.
129. Evans E P and Kaufman M E. Establishment in culture of pluripotential from mice embryos. Nature, 1981, 292: 154-156.
130. Martin GR. Isolation of a pluripotential cell line from early mouse embryos cultured in medium conditioned by teratocarinoma stem cells. Proc Acad Sci USA, 1981, 78: 7634-7638.
131. Wager E F, Keller G, Gilboa E, et al. In: Cold Spring Harbor Symposia Biology. Cold Spring Harbor Laboratory, 1985, 50: 619.
132. Robertson E, Bradley A, Kuehn M, et al. Germ line transmission of gene introduced into cultured pluripotential cell by retroviral vector. Nature, 1986, 323: 445-448.
133. Rottmann OJ, Stratowa C, Homstein M, Hughes J. Tissue specific expression of hepatitis B surface antigen in mice following liposome--mediated gene transfer into blastocysts. Zentralbl Veterinarmed A, 1985, 32: 676-682.
134. Perry A C F, Wakayama T, Kishikama H, et al. Mammalian transgenesis by intracytoplasmic sperm injection. Science, 1999, 284(5422): 1180-1183.
135．周荣家，张思促，赵连三．小鼠精子捕获外源DNA的研院．科学通报，1992，37：1246-1247．
136. Nakanishi A and Iritani A, Gene transfer in the chicken by sperm-mediated methods. Reprod Dev, 1993, 36: 258-261.
137. Horan R et al, Association of foreign DNA with porcine spermtozoa, Arch Androl, 1991, 26: 83-92.
138. Bachiller D, Schellander K, Peli J, Ruther U. Liposome-mediated DNA uptake by sperm cells. Mol Reprod Dev. 1991, 30(3): 194-200.
139．郭志儒，金宁一．2000年动物克隆及相关领域研究进展．中国兽医学报，2001，3：208-212
140．克隆研究大事记．科学时报，2001-4-16．
141. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A, Campbell KH. Human factor Ⅸ transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science. 1997, 19, 278(5346): 2130-2133.
142. Wilmut I, Schnieke A E, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature, 1997, 385: 810-813.
143. Polejaeva I A, Chen S H, Vaught T D, et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 2000, 407: 86-89.
144. Onishi A, Iwamoto M, Takeda K, et al. Pig cloning by microinjection of fetal fibroblast nuclei. Science, 2000, 289: 1188-1189.
145. Cibeli J B, Stice S L, Golueke P J, et al. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science, 1998, 280: 1256-1258.
146. PPL develops new and improved techniques to create transgenic animals. Biotechnol News, 1999.08.05(6).
147．田小利，陈兰英．转基因动物研究中存在的问题．生物工程进展，1995，15：41-45．
148. Theodre friedmann. Progress toward human gene therapy. Science, 1989, 244: 1275-1281.
149. Crystal R G. Transfer of genes to humans: early lessons and obstacles to success. Science, 1995, 270: 404-410.
150. Russell S T. Lymphokine gene transfer for cancer. Immunology Today, 1990, 11: 196-200.
151. Kotani H, P B I Newton, S Zhang, Y L, et al. Improved methods of retroviral vector transduction and production of gene therapy. Human gene therapy, 1994, 5: 19-28.
152．J 萨姆布鲁克，E F 弗里奇，T 曼尼阿蒂斯．分子克隆实验指南．第2版．金冬雁．黎孟枫译．北京：科学出版社，1992，786-792．
153．卢圣栋主编．现代分子生物学实验技术．北京：高等教育出版社，1993．
154．F 奥斯伯，R 布伦特，R E 金斯顿等．精编分子生物学实验指南．严子颖，王海林译．北京：科学出版社，1998，312-328．
155. Sita Reddy, Helen Raybum, Harald von Melchner et al. Fluorescence-activated sorting of totipolent embryonic stem cells expressing developmentally regulated LacZ fussion genes. Proc Natl Acad Sci USA, 1992, 89:6721.
156．章金刚．猪瘟病毒反义基因片段体外效应研究[博士论文]．长春：解放军农牧大学，1994．
157. Stylianos andreadis, Thomas lavery, Howard E Davis, et al,. Toward a more accurate quantitation of the activity of recombinant retrovimses: alternatives to titer and multiplicity of infection. J Virol, 2000, 74: 1258-1266.
158. Morgan J R, J M Ledoux, R G Snow, et al. Retrovirus infection: effect of time and target cell number. J Virol. 1995, 69: 6994-7000.
159. Stephen J Russell, Robert E Haekins and Greg Winter. Retroviral vectors displaying functional antibody fragments. Nucleic Acids Research, 1993, 21: 1081-1085.
160. Cynthia A Chambers, Joonsoo Kang and Nobumichi Hozumi. Long term expression of IL-4 in vivo using retroviral-mediated gene transfer. J Virol, 1992, 149: 2895-2905.
161. Mitchill L Drumm, Heidi A Pope, William H Cliff, et al. Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer. Cell, 62: 1227-1233.
162. Jahner D, et al. Proc Natl Acad Sci USA. 1985, 82: 6927-6931.
163．扈荣良．小鼠体内狂犬病病毒糖蛋白基因转移研究[博士学位论文]．长春．解放军农牧大学，1995．
164．赖良学，张学明，安靓等．精原干细胞体内转染途径建立转基因小鼠的可能性研究．生物技术通讯，1997，8：103-104．
165. Lai L X, Huang W M, Wang K, et al. Generation of transgenic mice expressing human tissue-type plasminogen activator in mammary gland by transfecting spermatogonia in vivo. In: Annual Anthology of University of Agriculture and Animal Science, PLA. Changchun: Published by University of Agriculture and Animal Science of PLA. 1997, 11-16.
166．赖良学．乳腺表达人组织性纤溶酶源激活剂的转基因小鼠建立．解放军农牧大学博士后报告，1997．
167. Kim J H, Jung Ha H S, Lee H T, et al. Development of a positive method for male stem cell - mediated gene transfer in mouse and pig, Mol Reprod Dev, 1997, 46 (4): 515-526.
168．孙敬方，朱德生，郝光荣等．实验动物学技术．北京：科学技术文献出版社，1993，28-29．
169．米娜瓦．阿木提，热孜亚．买买提．西蒙塔尔和黑白花种公牛精子顶体完整率、畸形率和活力测定．新疆农业科学．1994，4：185-186．
170．孙淑琴，稀释液中添加V_B12注射液对牛细管冷冻精液品质的影响[硕士学位论文]．延吉：延边大学，2000．
171．陈飞，黄丽娟，张罗漫．实用医学统计学．解放军农牧大学自编教材．94-99．
172．C W迪芬巴赫，GS德维克期勒，PCR技术实验指南。黄培堂，俞炜源，陈添弥等译．北京：科学出版社，1998，23-40．
173．张学明，文兴豪，赖良学等．性成熟前小鼠生精细胞的发育过程．中国兽医学报，2000，3：293-298．
174. Gordon J W, Scangos G A, Plotkin D J, Barbosa J A, Ruddle H. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA, 1980,77: 7380-7384.
175．乔贵林，周轶琳，赵君等．应用曲细精管注射法建立人白细胞抗原B27(HLA-B27)转基因鼠．中国兽医学报，1999．19：192．
176．田小利，陈兰英，扈荣良主编，转基因动物原理、技术与应用．吉林：吉林科学技术出版社，1994．