转hTERT基因的牛胎儿成纤维细胞作为SCNT核供体的研究
摘要
体细胞核移植技术作为一种生产转基因动物的方法有其不可代替的优越性,只要供体细胞筛选严格,可以保证100%的克隆后代表达外源基因。为了保证核移植效率,一般取低世代数(少于30 PDs)的动物体细胞作为核供体。但由于一些转基因方法,如基因打靶等,虽然可以将外源珍贵基因定点整合到阳性细胞基因组中,但效率较低,阳性细胞需要经历正负双重筛选阶段,而且为获得高效表达外源基因的阳性细胞还需经历复筛阶段,因此阳性细胞在体外培养需要经历较长的时间,导致最终获得的阳性细胞生长活力下降,不能满足核移植试验的要求。
用于生产转基因动物的核供体细胞至少应保持旺盛的长势至45 PDs。而早期胎儿来源的细胞与其它动物体细胞一样,随着分裂次数的增加,染色体末端端粒结构进行性缩短,当端粒缩短到一定阈值时,便丧失了保护染色体序列完整性的功能,导致染色体稳定性下降,编码区序列丢失,进而发生融合或降解,最终导致细胞衰老死亡。因此,如何延长核供体细胞在体外培养的寿命,甚至使其永生化成为生产转基因动物的一个根本需求。
研究发现,所有的配子细胞、干细胞以及近90%的癌细胞均表达端粒酶。这些细胞能够以端粒酶携带的RNA序列为模板,不断合成染色体末端端粒DNA序列,从而弥补细胞在分裂中端粒结构进行性缩短现象,延长细胞的寿命。
至1998年,Bodnar等首先通过重建细胞端粒酶活性延长了正常人类细胞的寿命以后,不同动物来源的多种类型细胞均可在端粒酶的诱导下延长体外培养的寿命。但没有关于牛胎儿成纤维细胞(Bovine Fetal Fibroblast Cell,BFF)的报道。研究证明端粒酶逆转录酶(Telomerase Reverse Transcriptase,TERT)是端粒酶活性的限速成分,外源性人端粒酶逆转录酶(Human Telomerase Reverse Transcriptase, hTERT)基因导入多种正常体细胞后,能够激活端粒酶活性,使细胞的寿命延长,甚至永生化。
本试验为获得一株生长性状稳定且具有优良遗传背景的荷斯坦奶牛成纤维细胞系,将含有hTERT基因的真核表达载体pCI-neo-hTERT通过脂质体介导法导入荷斯坦牛胎儿成纤维细胞。对转染后获得的阳性细胞进行周期检测,凋亡检测和倍体分析,并首次将表达hTERT基因的牛胎儿成纤维细胞作为体细胞核移植(Somatic Cell Nuclear Transfer, SCNT)供体细胞进行核移植试验。获得以下结果:
(1)本实验利用手术法采集了妊娠46日龄荷斯坦奶牛胎儿,并分离、纯化了胎儿成纤维细胞。利用Lipofectimain 2000 Reagent将携带hTERT基因的真核表达载体pCI-neo-hTERT导入第5代胎儿成纤维细胞中,通过G418初筛和复筛,获得了表达hTERT基因的牛胎儿成纤维细胞,该细胞在体外培养至第50代时依然具有旺盛的生长活力;
(2)对阳性细胞进行了周期检测、凋亡检测和倍性分析检测,结果显示:阳性第30代细胞与正常第30代细胞相比,处于S期细胞由19.59%上升至53.52%,正常第50代细胞开始出现凋亡现象,处于S期细胞仅占8.15%,而阳性第50代细胞仍保持较高活力(S=57.00%);正常第5代细胞与阳性第50代细胞凋亡率分别为:4.2%和4.5%,差异不显著,而阳性第50代细胞凋亡率为30.2%,与前两者差异极显著;阳性第50代胎儿成纤维细胞与正常第5代胎儿成纤维细胞具有同样的染色体核型。
(3)试验中以阳性第50代细胞、正常第5代细胞和正常第50代细胞分别作为核供体进行了核移植试验,对克隆胚胎发育结果统计显示:以阳性第50代细胞和正常第5代细胞作为核供体获得的克隆胚胎囊胚率分别为21.33%(16/75)和24.10%(20/83),而以正常第50代细胞作为核供体获得的克隆胚胎囊胚率只有6.93%(7/101),统计分析显示,以正常第50代细胞作为核供体的克隆胚胎囊胚率显著低于前两者(P<0.01)。
本试验结果表明:hTERT基因导入牛胎儿成纤维细胞后,可以延长细胞在体外培养的寿命并具有稳定的生物学特性,以其作为核移植供体的克隆胚具有正常的发育潜能,为生产转基因克隆动物提供了一株良好的核供体细胞。
Technology of somatic cell nuclear transfer (SCNT) is an excellent method in producing transgenic animals and each transgenic animal produced by SCNT expressed external gene if the donor cell was screened strictly. In order to improve the efficiency of SCNT, the somatic cells with low population doublings (less than 30) are often chosen as the donor cells. Some kinds of advanced transgenic methods, such as gene targeting, could introduce the external interesting gene to a special site of genome exactly. However, the efficiency is low and often cost a long time to obtain a positive cell line for the positive cells expressing exogenous gene highly need to be screened by three main stages: the positive screening stage, the negative screening stage and the re-screening stage. The viability of positive cells will become lower with the process of selecting, so as to reduce the efficiency of SCNT.
The donor cell used to produce transgenic animals should keep its excellent vigor to 45 population doublings at least. The bovine fetal fibroblast cells (BFF) are same as other kinds of somatic cells. The sequences of telomere at the end of each chromosome are shortened gradually during proliferation and if the lost sequences reach some threshold value, the telomere would lose its ability to keep the chromosome steadily. Then the code sequences (CDs) of some functional gene at the end of chromosome would also lose and the chromosome itself would be amalgamation or degradation easily. And finally, the cells are going to be consenescence and death. So prolonging the donor cells’lifetime cultured in vitro or enduring the donor cells to be immortalization are becoming a kind of essential requirement when producing trangenetic animals.
All of the germ cells, the stem cells and nearly 90% of the carcinoma cell are positive of telomerase [6]. These cells can synthesis telomere DNA sequence at the end of chromosome spontaneously using its own RNA sequence as the template, so the lost telomere DNA sequence during cell proliferation would be remedy gradually, and the lifetime of these cells would be extened also.
Since Bodnar (1998) extended the lifetime of normal human cells by introduction of telomerase, a great deal of cells derived from different animals having been extended their lifetime already. But there is on report about BFF in this field. Many studies showed the activity of telomerase is controlled by telomerase reverse transcriptase (TERT). Introduction of external hTERT gene could enhance the activity of telomerase, extend the lifespan and even endure the normal cells into immortalization.
To work out a reliable system for transgenic animal studies, it is necessary to establish a BFF cell line with extended lifespan, excellent background in genetic and consistent functions and characteristics. In this study, the plasmid pCI-neo-hTERT was transfected into BFF by Lipofection 2000 Reagent. After screening, the cell cycles, cell apoptosis and the karyotype of the positive cells were detected by FCM, and the SCNT embryo cloned from hTERT transfected cells were also studied originally. Experimental results obtained as follows:
1. Bovine fetal fibroblast cells were isolated and purified from a 46 days Hostan fetal obtained by operation. The vector pCI-neo-hTERT was transfected into 5th generation of BFF by Lipofection 2000 Reagent. After screening, the positive cells were expanded and cultured to passage 50 in vitro still with excellent vigor.
2. The cell cycles, cell apoptosis and the karyotype of the positive cells were detected by FCM. The experimental results obtained as follows:
(a) Compared with 30th generation of negative cells, the percentage of S stage of generation 30 positive cells increased from 19.59% to 53.52%; 50th generation of the positive cells still have the ability of proliferation with the S stage of 57.00% but in negative cells of passage 50, the S stage is only account for 8.15% and cell apoptosis phenomena could be detected;
(b) The apoptosis rate of 5th generation of negative cells and positive cells of passage 50 are 4.2% and 4.5%, respectively. The discrepancy is not significant (P>0.05). The apoptosis rate of negative cells of passage 50 is 30.2%. The discrepancy between negative cells and positive cells in passage 50 is significant (P<0.01).
(c) Both the 5th generation of negative cells and 50th generation of positive cells have normal karyotype.
3. In this study, three groups of BFF were used as donor cells to produce SCNT embryos. And the embryo development was observed and analyzed by statistic software SAS 6.12. The blastocyst rate in group positive 50 (P50) and negative 5 (N5) are 21.33% (16/75) and 24.10% (20/83), respectively. While the blastocyst rate in group N50 is just 6.93% (7/101). The discrepancy between group N50 and P50 is significant (P<0.01).
The findings above demonstrated that the hTERT gene was transfected into BFF successfully and the lifespan of positive cells was extended. The positive cells cultured in vitro remained normal biological characteristics and the cloned embryos derived from them have excellent developmental potential.
用于生产转基因动物的核供体细胞至少应保持旺盛的长势至45 PDs。而早期胎儿来源的细胞与其它动物体细胞一样,随着分裂次数的增加,染色体末端端粒结构进行性缩短,当端粒缩短到一定阈值时,便丧失了保护染色体序列完整性的功能,导致染色体稳定性下降,编码区序列丢失,进而发生融合或降解,最终导致细胞衰老死亡。因此,如何延长核供体细胞在体外培养的寿命,甚至使其永生化成为生产转基因动物的一个根本需求。
研究发现,所有的配子细胞、干细胞以及近90%的癌细胞均表达端粒酶。这些细胞能够以端粒酶携带的RNA序列为模板,不断合成染色体末端端粒DNA序列,从而弥补细胞在分裂中端粒结构进行性缩短现象,延长细胞的寿命。
至1998年,Bodnar等首先通过重建细胞端粒酶活性延长了正常人类细胞的寿命以后,不同动物来源的多种类型细胞均可在端粒酶的诱导下延长体外培养的寿命。但没有关于牛胎儿成纤维细胞(Bovine Fetal Fibroblast Cell,BFF)的报道。研究证明端粒酶逆转录酶(Telomerase Reverse Transcriptase,TERT)是端粒酶活性的限速成分,外源性人端粒酶逆转录酶(Human Telomerase Reverse Transcriptase, hTERT)基因导入多种正常体细胞后,能够激活端粒酶活性,使细胞的寿命延长,甚至永生化。
本试验为获得一株生长性状稳定且具有优良遗传背景的荷斯坦奶牛成纤维细胞系,将含有hTERT基因的真核表达载体pCI-neo-hTERT通过脂质体介导法导入荷斯坦牛胎儿成纤维细胞。对转染后获得的阳性细胞进行周期检测,凋亡检测和倍体分析,并首次将表达hTERT基因的牛胎儿成纤维细胞作为体细胞核移植(Somatic Cell Nuclear Transfer, SCNT)供体细胞进行核移植试验。获得以下结果:
(1)本实验利用手术法采集了妊娠46日龄荷斯坦奶牛胎儿,并分离、纯化了胎儿成纤维细胞。利用Lipofectimain 2000 Reagent将携带hTERT基因的真核表达载体pCI-neo-hTERT导入第5代胎儿成纤维细胞中,通过G418初筛和复筛,获得了表达hTERT基因的牛胎儿成纤维细胞,该细胞在体外培养至第50代时依然具有旺盛的生长活力;
(2)对阳性细胞进行了周期检测、凋亡检测和倍性分析检测,结果显示:阳性第30代细胞与正常第30代细胞相比,处于S期细胞由19.59%上升至53.52%,正常第50代细胞开始出现凋亡现象,处于S期细胞仅占8.15%,而阳性第50代细胞仍保持较高活力(S=57.00%);正常第5代细胞与阳性第50代细胞凋亡率分别为:4.2%和4.5%,差异不显著,而阳性第50代细胞凋亡率为30.2%,与前两者差异极显著;阳性第50代胎儿成纤维细胞与正常第5代胎儿成纤维细胞具有同样的染色体核型。
(3)试验中以阳性第50代细胞、正常第5代细胞和正常第50代细胞分别作为核供体进行了核移植试验,对克隆胚胎发育结果统计显示:以阳性第50代细胞和正常第5代细胞作为核供体获得的克隆胚胎囊胚率分别为21.33%(16/75)和24.10%(20/83),而以正常第50代细胞作为核供体获得的克隆胚胎囊胚率只有6.93%(7/101),统计分析显示,以正常第50代细胞作为核供体的克隆胚胎囊胚率显著低于前两者(P<0.01)。
本试验结果表明:hTERT基因导入牛胎儿成纤维细胞后,可以延长细胞在体外培养的寿命并具有稳定的生物学特性,以其作为核移植供体的克隆胚具有正常的发育潜能,为生产转基因克隆动物提供了一株良好的核供体细胞。
Technology of somatic cell nuclear transfer (SCNT) is an excellent method in producing transgenic animals and each transgenic animal produced by SCNT expressed external gene if the donor cell was screened strictly. In order to improve the efficiency of SCNT, the somatic cells with low population doublings (less than 30) are often chosen as the donor cells. Some kinds of advanced transgenic methods, such as gene targeting, could introduce the external interesting gene to a special site of genome exactly. However, the efficiency is low and often cost a long time to obtain a positive cell line for the positive cells expressing exogenous gene highly need to be screened by three main stages: the positive screening stage, the negative screening stage and the re-screening stage. The viability of positive cells will become lower with the process of selecting, so as to reduce the efficiency of SCNT.
The donor cell used to produce transgenic animals should keep its excellent vigor to 45 population doublings at least. The bovine fetal fibroblast cells (BFF) are same as other kinds of somatic cells. The sequences of telomere at the end of each chromosome are shortened gradually during proliferation and if the lost sequences reach some threshold value, the telomere would lose its ability to keep the chromosome steadily. Then the code sequences (CDs) of some functional gene at the end of chromosome would also lose and the chromosome itself would be amalgamation or degradation easily. And finally, the cells are going to be consenescence and death. So prolonging the donor cells’lifetime cultured in vitro or enduring the donor cells to be immortalization are becoming a kind of essential requirement when producing trangenetic animals.
All of the germ cells, the stem cells and nearly 90% of the carcinoma cell are positive of telomerase [6]. These cells can synthesis telomere DNA sequence at the end of chromosome spontaneously using its own RNA sequence as the template, so the lost telomere DNA sequence during cell proliferation would be remedy gradually, and the lifetime of these cells would be extened also.
Since Bodnar (1998) extended the lifetime of normal human cells by introduction of telomerase, a great deal of cells derived from different animals having been extended their lifetime already. But there is on report about BFF in this field. Many studies showed the activity of telomerase is controlled by telomerase reverse transcriptase (TERT). Introduction of external hTERT gene could enhance the activity of telomerase, extend the lifespan and even endure the normal cells into immortalization.
To work out a reliable system for transgenic animal studies, it is necessary to establish a BFF cell line with extended lifespan, excellent background in genetic and consistent functions and characteristics. In this study, the plasmid pCI-neo-hTERT was transfected into BFF by Lipofection 2000 Reagent. After screening, the cell cycles, cell apoptosis and the karyotype of the positive cells were detected by FCM, and the SCNT embryo cloned from hTERT transfected cells were also studied originally. Experimental results obtained as follows:
1. Bovine fetal fibroblast cells were isolated and purified from a 46 days Hostan fetal obtained by operation. The vector pCI-neo-hTERT was transfected into 5th generation of BFF by Lipofection 2000 Reagent. After screening, the positive cells were expanded and cultured to passage 50 in vitro still with excellent vigor.
2. The cell cycles, cell apoptosis and the karyotype of the positive cells were detected by FCM. The experimental results obtained as follows:
(a) Compared with 30th generation of negative cells, the percentage of S stage of generation 30 positive cells increased from 19.59% to 53.52%; 50th generation of the positive cells still have the ability of proliferation with the S stage of 57.00% but in negative cells of passage 50, the S stage is only account for 8.15% and cell apoptosis phenomena could be detected;
(b) The apoptosis rate of 5th generation of negative cells and positive cells of passage 50 are 4.2% and 4.5%, respectively. The discrepancy is not significant (P>0.05). The apoptosis rate of negative cells of passage 50 is 30.2%. The discrepancy between negative cells and positive cells in passage 50 is significant (P<0.01).
(c) Both the 5th generation of negative cells and 50th generation of positive cells have normal karyotype.
3. In this study, three groups of BFF were used as donor cells to produce SCNT embryos. And the embryo development was observed and analyzed by statistic software SAS 6.12. The blastocyst rate in group positive 50 (P50) and negative 5 (N5) are 21.33% (16/75) and 24.10% (20/83), respectively. While the blastocyst rate in group N50 is just 6.93% (7/101). The discrepancy between group N50 and P50 is significant (P<0.01).
The findings above demonstrated that the hTERT gene was transfected into BFF successfully and the lifespan of positive cells was extended. The positive cells cultured in vitro remained normal biological characteristics and the cloned embryos derived from them have excellent developmental potential.
引文
[1] Hayflick L, Moorhead P S. The serial cultivation of human diploid cells trains[J]. Exp Cell Res, 1961, 25: 585-621.
[2] Stein GH, Namba M, Corsaro CM. Relationship of finite proliferative lifespan, senescence and quiescence in human cells [J]. J Cell Physiol, 1985, 122(3):343-349.
[3] Lustig AJ.Crisis intervention: therole of telomerase[J]. Proc Natl Acad Sci USA, 1999, 96(7):3339.
[4] Elango N, Thomas JW, Yi SV, et al. Variable molecular clocks in hominoids [J]. Evolution, 2006, 103:1370-1375.
[5] Helder MN, Wisman GB, van der Zee GJ. Telomerase and telomeres: from basic biology to cancer treatment [J]. Cancer Invest, 2002, 20(1): 82-101.
[6] Zhenyu J, Lenhard R. Telomere dysfunction and stem cell ageing[J]. Biochimie, 2008, 90(1):24-32.
[7] Cukusi? A, Skrobot Vidacek N, Sopta M, Rubelj I. Telomerase regulation at the crossroads of cell fate[J]. Cytogenet Genome Research. 2008, 122 (3-4):263-272.
[8] Fossel M. Cell senescence in human aging and disease [J]. Ann NY Acad Sci, 2002, 959: 14-23.
[9] Avilion AA, Piatyszek MA, Gupta J. Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues [J]. Cancer Research, 2006, l 56(3): 645-650.
[10] Elizabeth H. Blackburn. Structure and function of telomere[J]. Nature, 1991, 350: 569-573.
[11] Costa A, Daidone MG, Daprai L, Villa R, Cantu S, Pilotti S, Mariani L, Gronchi A, Henson JD, Reddel RR, Zaffaroni N Telomere maintenance mechanisms in liposarcomas: association with histologic subtypes and disease progression[J]. Cancer Res, 2006, 66: 8918-8924.
[12] Johnson JE, Gettings EJ, Schwalm J, Pei J, Testa JR, Litwin S, Mehren M, Broccoli D Whole-genome profiling in liposarcomas reveals genetic alterations common to specific telomere maintenance mechanisms[J]. Cancer Res, 2007, 67: 9221-9228.
[13] Toshio O. Apoptosis signal-regulating kinase mediates cellular senescence induced by high glucose in endothelial[J]. Cells Diabetes, 2006, 55(6):423-431.
[14] Joseph AB, Ying Z, Jerry WS, et al. Telomere position effect in human cells [J]. Science, 2001, 292: 2075-2077.
[15] Cerone MA, Londono JA, Autexier C. Telomerase inhibition enhances the response to anticancer drug treatment in human breast cancer cells[J]. Mol Cancer Ther, 2006, 5: 1669-1675.
[16] Steven AJ, Elaine RP, Thomas RC. Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase[J]. Nature Structural & Molecular Biology, 2006, 13:218-225.
[17] Shay JW, Van Der Haegen BA, Ying Y. The frequency of immortalization of human fibroblasts and mammary epithelial cells transfected with SV40 large T-antigen[J]. Exp Cell Res, 1993, 209(1):45-52.
[18] Nakamura TM, Morin GB, Chapman KB. Telomerase catalytic subunit homologs from fission yeast and human[J]. Science, 1997, 277: 955-959.
[19] Sheila A. Stewart, lttai Ben-porath, et al. Erosion of the telomeric single-strand overange at replicative senescence [J]. Nature Genetics, 2003, 33: 492-496.
[20] Patricia S. Metastasis suppressors alter the signal transduction of cancer [J]. Cells, 2003, 3:55-63.
[21] Fabrizio d'Adda di Fagagna, Hande MP, Tong WM, et al. Functions of poly (ADP-ribose) polymerase in controlling telomere length and chromosomal stability[J]. Nature Genetics, 1999, 23: 76-80.
[22] Hug N, Lingner J. Telomere length homeostasis[J]. Chromosoma, 2006, 115(6):413-425.
[23] Helen V, Hilda AP. Molecular characterization of inter-telomere and intra-telomere mutations in human ALT cells [J]. Nature Genetics, 2002, 30: 301-305.
[24] Joseph CP, Lucy GA, Trygve OT. Activity, function, and gene regularion of the catalytic subunit of thelomerase (hTERT) [J]. Elsevier Gene, 2001, 269: 1-12.
[25] Wylliel FS, Jones CJ. Telomerase prevents the accelerated cell ageing of Werner syndrome fibroblasts[J]. Nature Genetics, 2000, 24: 16-17.
[26] Srinivasan A, Clellan AJ, Brasiskyte D. The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain [J]. Mol Cell Biol, 1997, 17 (8): 4761-4773.
[27] Li NF, Broad S, Lu YJ, Yang JS, Watson R, Hagemann T, Wilbanks G, Jacobs I, Balkwill F, Dafou D, Gayther SA. Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression[J]. Cell Prolif, 2007, 40(5):780-94.
[28] Endo A, Kano Y, Kihara K. Alteration in the retinoblastoma gene associated with immortalization of human fibroblasts treated with 60Co gammarays [J]. J Cancer Res Clinoncol, 1993, 119(9):522-526.
[29] Sugimoto M, Ide T, Goto M, et al. Reconsideration of senescence, immortalization and telomere maintenance of Epstein-Barr virus-transformed human B-lymphoblastoid cell lines [J]. Mech Ageing Dev, 1999, 107(1):51-60.
[30] Lei KJ, Gluzman Y, Pan CJ. Immortalization of virus-free human placental cells that express tissue specific functions[J]. Mol Endocrinol, 1992, 6(5):703-712.
[31] Christian BJ, Loretz LJ, Oberley TD, et al. Characterization of human uroepithelial cells immortalized in vitro by simian virus 40 [J]. Cancer Res, 1987, 47(22):6066-6073.
[32] He YL, Wu YH, He XN, Liu FJ, He XY, Zhang Y. An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer[J]. Theriogenology, 2009, 71(9):1417-1424.
[33] Steinert S, Shay JW, Wright WE. Transient expression of human telomerase extends the lifespan of normal human fibroblasts [J]. Biochem Biophys Res Commun, 2000, 273(3): 1095-1098.
[34] Ausubel FM, Brent R, Kingston RE. Short protocols in molecular biology [M]. 3rd ed. John Wiley&Sons, Inc., New York, 1995: 274-326.
[35] Miquel C, Gaghoux P, Durand C, et al. Establishment and characterization of cell line LSVS that retains the altered adhesive properties of human functional epidermolysis bullosa keratmocyte [J]. Exp Cell Res, 1996, 224(2): 279-290.
[36] Ray FA. Meyne J, and Kraemer PM. SV40 T antigen induced chromosomal changes reflect a process that is both clastogenic and aneuploidogenic and is ongoing throughout neoplastil progression of human fibroblasts [J]. Mutat Res, 1992, 284(2): 265-273.
[37] Souttou B, Hamelin R, and Crepin M. EGF2 as an autocrine growth factor for immortal human breast epithelial cells [J]. Cell Growth Differ, 1994, 5(6): 615-623.
[38] Feng J, Funk WD, Wang SS. The RNA component of human telomerase[J]. Science, 1995, 269:1236-1239.
[39] Bhattacharyya A, Blackburn EH. A functional telomerase RNA swap in vivo reveals the importance ofnontemplate RNA domains [J]. Proc Natl Acad Sci USA, 1997, 94:2823-2830.
[40] McCormick-Graham M, Haynes WJ, Romero DP. Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase [J]. EMBO J, 1997, 16:3233-3237.
[41] Mckenzie KE, Umbricht CB, Sukumar S. Applications of telomerase research in the fight against cancer[J]. Mol Med Today, 1999, 5:114-122.
[42] Beattie T, Zhou W, Robinson M. Reconstitution of telomerase activity in vitro [J]. Curr Biol, 1998, 8:177-182.
[43] Tahara H, Yasui W, Tahara E. Immunohistochemical detection of human telomerase catalytic component, hTERT, in human colorectal tumor and non tumor tissue sections [J]. Oncogene, 1999, 18:1561-1567.
[44] Lai SR, Phipps SM, Liu L, et al. Epigenetic control of telomerase and modes of telomere maintenance in aging and abnormal systems [J]. Front Biosci, 2005, 10: 1779-1796.
[45] Danielle Robertson, Li L, Stephen Fisher, et al. Characterization of Growth and Differentiation in a Telomerase-Immortalized Human Corneal Epithelial Cell Line[J]. Investigative Ophthalmology and Visual Science, 2005, 46: 470-478.
[46] Toyohiko Y, Keisuke F, Osamu Y, Mizuo H, Junichi M, Yukihiro T, Hidenobu K, Hidenori I, Toshio O. Apoptosis signal-regulating kinase mediates cellular senescence induced by high glucose in endothelial[J]. Cells. Diabetes, 2006, 55(6):423-431.
[47] Van SB, Lange TD. Control of telomere length by the human telomeric protein TRF1[J]. Nature, 1997, 385:740-742.
[48] Hande MP. DNA repair factors and telomere-chromosome integrity in mammalian cells [J]. Cytogenet Genome Res, 2004, 104(1-4):116-122.
[49] Shin KH, Kang MK, et al. Introduction of human telomerase reverse transcriptase to normal human fibroblasts enhances DNA repair capacity [J]. Clin Cancer Res, 2004; 10(7):2551-2560.
[50] Pirzio LM, Freulet-Marrière MA, et al. Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation [J]. Cytogenet Genome Res, 2004, 104(1-4):87-94.
[51] Sharma GG, Gupta A. hTERT associates with human telomeres and enhances genomic stability and DNA repair [J]. Oncogene 2003, 22:131-146.
[52] Sara B, Valentina C, Dino A, Daniele C, Rosella S. The current role of telomerase in the diagnosis of bladder cancer[J]. Indian J Urol, 2009, 25(1): 40-46.
[53] Saretzki G, Ludwig A, et al. Ribozyme-mediated telomerase inhibition induces immediate cell loss but not telomere shortening in ovariancancer cells [J]. Cancer Gene Ther, 2001, 8: 827-834.
[54] Hao ZM, Luo JY, et al, Intensive Inhibition of hTERT Expression by a Ribozyme Induces Rapid Apoptosis of Cancer Cells through a Telomere Length-Independent Pathway [J]. Cancer Biol Ther, 2005, 4(10):1098-1103.
[55] Folini M, Pennati M, et al. Targeting human telomerase by antisense oligonucleotides and ribozymes[J]. Curr Med Chem Anti-Canc Agents, 2002, 2(5): 605-612.
[56] Xu DW, Wang Q, et al. Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells [J]. Oncogene, 2000, 19: 5123-5133.
[57] Cao Y, Li H, et al., TERT regulates cell survival independent of telomerase enzymatic activity [J].Oncogene, 2002, 21:3130-3138.
[58] Rahman R, Latonen, et al. hTERT antagonizes p53-induced apoptosis independently of telomerase activity [J]. Oncogene, 2005, 24:1320-1327.
[59] Smith LL, Coller HA, et al. Telomerase modulates expression of growth-controlling genes and enhances cell proliferation [J]. Nat Cell Biol. 2003, 5: 474-479.
[60] Li S, Rosenberg JE, Donjacour AA, et al. Rapid inhibition of cancer cell growth induced by lentiviral delivery and expression of mutant- template telomerase RNA and anti-telomerase shortinterfering RNA [J]. Cancer Res, 2004, 64: 4833- 4840.
[61] K. Anno, A. Hayashi, T. Takahashi, Y. Mitsui, T. Ide and H. Tahara, Telomerase activation induces elongation of the telomeric single-stranded overhang, but does not prevent chromosome aberrations in human vascular endothelial cells[J]. Biochem Biophys Res Commun, 2007, 353:926-932.
[62] Li S, Crothers J, Haqq CM, et al. Cellular and gene expression responses involved in the rapid growth inhibition of human cancer cells by RNA interference- mediated depletion of telomerase RNA [J]. J Biol Chem. 2005, 280: 23709-23717.
[63] Stephen N, Gary P. Telomere maintenance as a target for anticancer drug discovery [J]. Nature Drug Discovery, 2002, 1:383-393.
[64] S. Puttmann, V. Senner, S. Braune, B. Hillmann, R. Exeler and C.H. Rickert et al., Establishment of a benign meningioma cell line by hTERT-mediated immortalization[J]. Lab Invest, 2005, 85: 1163-1171.
[65] Homayoun V Samuel B. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative lifespan [J]. Current Biology, 1998, 8(5):279-282.
[66] Wang J, Lin YX, Susan Allan. Myc activates telomerase [J]. Research Communication, 1998, 12(12): 1769-1774.
[67] Michel M, Ouellette, Lisa D, Woodring E, et al. The establishment of telomerase immortalized cell lines representing human chromosome instability syndromes [J]. Human Molecular Genetics, 2000, 9(3): 403-411.
[68] Erik Hooijberg, Janneke J. Ruizendaal, Peter J. F. Snijders, et al. Immortalization of Human CD8+ T Cell Clones by Ectopic Expression of Telomerase Reverse Transcriptase [J]. The Journal of Immunology, 2000, 165: 4239-4245.
[69] Brittney-Shea Herbert, Woodring E Wright and Jerry W Shay. pl6IN4Ka inactivation is not required to immortalize human mammary epithelial cells [J]. Oncogene, 2002, 21: 7897-7900.
[70] Veitonmaki N, Niina Veitonm?ki, Jonas Fuxe, et al. Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16INK4a/pRb [J]. FASEB J, 2006, 17 (6): 764-780.
[71] Fiedler W, Reinicke D, Aurich H. In Vitro Immortalisation of Porcine epatocytes by Transfection with the Gene for the Human Catalytic Subunit of Telomerase Reverse ranscriptase (HTERT)[J]. Journal of Hepatology, 2004, 40(1): 102.
[72] Ira Kogan, Naomi Goldfinger, Michael Milyavsky. hTERT-immortalized prostate epithelial and stromal-derived cells: an authentic in vitro model for differentiation and carcinogenesis [J]. Cancer Research, 2006, 66: 3531-3540.
[73] Kapanadze B, Morris E, Simth E, Trojanowska M. Establishment and characterization of sclerodermafibroblast clonal cell lines by introduction of the hTERT gene [J]. Cell Mol Med. 2009, 11, [Epub ahead of print].
[74] He YL, Wu YH, He XN, Liu FJ, He XY, Zhang Y. An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer [J]. Theriogenology. 2009, 71(9):1417-24.
[75] Tian XC, Xu J, Yang XZ. Normal telomere lengths found in cloned cattle [J]. Nature Genetics, 2000, 26: 272-273.
[76] Shao G, Balajee AS, Hei TK, Zhao Y. p16INK4a downregulation is involved in immortalization of primary human prostate epithelial cells induced by telomerase[J]. Mol Carcinog, 2008, 47:775-783.
[77] Hana B, Orit CB. Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells[J]. Cancer research, 2001, 61: 5529-5536.
[78] Palmiter RD, Brinster RL, et al. Dramatic growth of mice that develop from eggs microinjected with metallothione in growth hormone fusiongenes [J]. Nature, 1982, 300: 611-615.
[79] Magin TM, McWhir J, Melton DW. A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency[J]. Nucleic Acids Res, 1992, 20: 3795-3796.
[80] Schnieke AE, Kind AJ, Ritchie WA. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts[J]. Science, 1997, 278: 2130-2133.
[81] Perry AC, Wakayama T, Kishikawa H. Mammalian transgenesis by intracytoplasmic sperm injection [J]. Science, 1999, 284: 1180-1183.
[82] Kerr DE, Plaut K, BramLey AJ, et al. Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice [J]. Nat Biotech, 2001, 19: 66-70.
[83] Kang JX, Wang JD, Wu L, et al. Fat-1 mice convert n-6 to n-3 fatty acid [J]. Nature, 2004, 427: 504-505.
[84] Lai L, Kang JX, Li R, Wang J, Witt WT, et al.Generation of cloned transgenic pigs rich in omega-3 fatty acids [J]. Nature Biotechnology, 2006, 24 (4): 435-436.
[85] Smith K, Spadafora C. Sperm-mediated gene transfer: applications and implications[J]. Bioessays, 2005, 27(5):551-62.
[86] Hossein MS, Jeong YW, Park SW, Kim JJ, Lee E, Ko KH, Kim HS, Kim YW, Hyun SH, Shin T, Hawthorne L, Hwang WS Cloning missy: obtaining multiple offspring of a specific canine genotype by somatic cell nuclear transfer[J]. Cloning Stem Cells, 2009, 11(1):123-30.
[87] Peng CA. Analysis of gene transfer rate with immobilized retroviral vectors. Ann N Y Acad Sci, 2009, 1161:26-33.
[88] Liang JZ, Li R, Zhang JY, Zheng H, Luo RC. Retrovirus-mediated SHP-1 gene expression in human breast cancer MDA-MB-231 cells[J]. Nan Fang Yi Ke Da Xue Xue Bao, 2009, 29(5):902-5.
[89] Kim JH, Jung-Ha HS, Lee HT, Chung KS. Development of a positive method for male stem cell mediated gene transfer in mouse and pig[J]. Molecular Reproduction and Development, 1997, 46: 515-526
[90] Shen W, Li L, Pan Q, Min L, Dong H and Deng J. Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method [J]. Molecular Reproduction and Development, 2006, 73: 589-594.
[91] Yang SY, Wang JG, Cui HX, Sun SG, Li Q, Gu L, Hong HP, Ciu PP and Liu WQ. Efficientgeneration of transgenic mice by direct intraovarian injection of plasmid DNA [J].Biochemical and Biophysical Research Communications, 2007, 358 (1):266-271.
[92] Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells [J]. Cell, 1980, (2): 479-488.
[93] Smithies O, Gregg RG, Boggs SS, et al. Insertion of DNA sequences into the human chromosal beta-globin locus by homologous recombination [J]. Nature, 1985, 317: 230-234.
[94] Fire A,Xu S,MontgomeryMK. Potent and specific genetic inter-ferenceby double-strand RNA in coenorhabditis elegans[J].Nature, 1998, 391(6669), 744-745.
[95] Cogoni C, Macino G. Post transcriptional gene silencing across kingdoms [J]. Curr Opin Genet Dev, 2000, 10 (6):638-643.
[96] Acosta J, Carpio Y, Borroto I, González O and Estrada MP.Myostatin gene silenced by RNAi show a zebrafish giant phenotype [J]. Journal of Biotechnology, 2005, 119 (4): 324-331.
[97] Dann CT, Alvarado AL, Hammer RE and Garbers DL. Heritableand stable gene knockdown in rats[J] .Proceedings of National Academy of Science of the USA, 2006, 103 (30):11246-11251.
[98] Brunetti D, Perota A, Lagutina I, Colleoni S, Duchi R, Calabrese F, Seveso M, Cozzi E, Lazzari G, Lucchini F, Galli C Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts[J]. Cloning Stem Cells, 2008, 10(4):409-19.
[99] Matsunari H, Onodera M, Tada N, Mochizuki H, Karasawa S, Haruyama E, Nakayama N, Saito H, Ueno S, Kurome M, Miyawaki A, Nagashima H. Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange[J]. Cloning Stem Cells, 2008, 10(3): 313-23.
[100] Pursel VG, Pinkert CA, Miller KF, et al. Genetic engineering of livestock [J]. Science, 1989, 244: 1281-1288.
[101] Damak S, Su H, Jay NP, et al. Improved wool production in transgenic sheep expressing insulin-like growth factor1[J]. Biotechnology, 1996, 14: 185-188.
[102] Kang JX, Wang JD, Wu L, et al. Fat-1 mice convertω-6 toω-3 fatty acid[J]. Nature, 2004, 427: 504-505.
[103] Berm G. Production of transgenic and possible application to pig breeding[J]. Soc Anim Prod, 1992, 12: 15-31.
[104] Wadih A, Renata P, Erkki R, et a1. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse mode1[J]. Science, 1998, 279:377-380.
[134] Wei X, Luo Y, Hyde DR. Molecular cloning of three zebrafish lin7 genes and their expression patterns in the retina[J]. Exp Eye Res. 2006, 82:122-31
[105] Pan X, Wan H, Chia W, Tong Y, Gong Z. Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system[J]. Transgenic Res. 2005, 14:217-23.
[106] Collery RF, Cederlund ML, Smyth VA, Kennedy BN. Applying transgenic zebrafish technology to study the retina[J]. Adv Exp Med Biol, 2006, 572:201-207.
[107] Amsterdam A, Hopkins N. Mutagenesis strategies in zebrafish for identifying genes involved in development and disease[J]. Trends Genet, 2006, 22:473-478.
[2] Stein GH, Namba M, Corsaro CM. Relationship of finite proliferative lifespan, senescence and quiescence in human cells [J]. J Cell Physiol, 1985, 122(3):343-349.
[3] Lustig AJ.Crisis intervention: therole of telomerase[J]. Proc Natl Acad Sci USA, 1999, 96(7):3339.
[4] Elango N, Thomas JW, Yi SV, et al. Variable molecular clocks in hominoids [J]. Evolution, 2006, 103:1370-1375.
[5] Helder MN, Wisman GB, van der Zee GJ. Telomerase and telomeres: from basic biology to cancer treatment [J]. Cancer Invest, 2002, 20(1): 82-101.
[6] Zhenyu J, Lenhard R. Telomere dysfunction and stem cell ageing[J]. Biochimie, 2008, 90(1):24-32.
[7] Cukusi? A, Skrobot Vidacek N, Sopta M, Rubelj I. Telomerase regulation at the crossroads of cell fate[J]. Cytogenet Genome Research. 2008, 122 (3-4):263-272.
[8] Fossel M. Cell senescence in human aging and disease [J]. Ann NY Acad Sci, 2002, 959: 14-23.
[9] Avilion AA, Piatyszek MA, Gupta J. Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues [J]. Cancer Research, 2006, l 56(3): 645-650.
[10] Elizabeth H. Blackburn. Structure and function of telomere[J]. Nature, 1991, 350: 569-573.
[11] Costa A, Daidone MG, Daprai L, Villa R, Cantu S, Pilotti S, Mariani L, Gronchi A, Henson JD, Reddel RR, Zaffaroni N Telomere maintenance mechanisms in liposarcomas: association with histologic subtypes and disease progression[J]. Cancer Res, 2006, 66: 8918-8924.
[12] Johnson JE, Gettings EJ, Schwalm J, Pei J, Testa JR, Litwin S, Mehren M, Broccoli D Whole-genome profiling in liposarcomas reveals genetic alterations common to specific telomere maintenance mechanisms[J]. Cancer Res, 2007, 67: 9221-9228.
[13] Toshio O. Apoptosis signal-regulating kinase mediates cellular senescence induced by high glucose in endothelial[J]. Cells Diabetes, 2006, 55(6):423-431.
[14] Joseph AB, Ying Z, Jerry WS, et al. Telomere position effect in human cells [J]. Science, 2001, 292: 2075-2077.
[15] Cerone MA, Londono JA, Autexier C. Telomerase inhibition enhances the response to anticancer drug treatment in human breast cancer cells[J]. Mol Cancer Ther, 2006, 5: 1669-1675.
[16] Steven AJ, Elaine RP, Thomas RC. Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase[J]. Nature Structural & Molecular Biology, 2006, 13:218-225.
[17] Shay JW, Van Der Haegen BA, Ying Y. The frequency of immortalization of human fibroblasts and mammary epithelial cells transfected with SV40 large T-antigen[J]. Exp Cell Res, 1993, 209(1):45-52.
[18] Nakamura TM, Morin GB, Chapman KB. Telomerase catalytic subunit homologs from fission yeast and human[J]. Science, 1997, 277: 955-959.
[19] Sheila A. Stewart, lttai Ben-porath, et al. Erosion of the telomeric single-strand overange at replicative senescence [J]. Nature Genetics, 2003, 33: 492-496.
[20] Patricia S. Metastasis suppressors alter the signal transduction of cancer [J]. Cells, 2003, 3:55-63.
[21] Fabrizio d'Adda di Fagagna, Hande MP, Tong WM, et al. Functions of poly (ADP-ribose) polymerase in controlling telomere length and chromosomal stability[J]. Nature Genetics, 1999, 23: 76-80.
[22] Hug N, Lingner J. Telomere length homeostasis[J]. Chromosoma, 2006, 115(6):413-425.
[23] Helen V, Hilda AP. Molecular characterization of inter-telomere and intra-telomere mutations in human ALT cells [J]. Nature Genetics, 2002, 30: 301-305.
[24] Joseph CP, Lucy GA, Trygve OT. Activity, function, and gene regularion of the catalytic subunit of thelomerase (hTERT) [J]. Elsevier Gene, 2001, 269: 1-12.
[25] Wylliel FS, Jones CJ. Telomerase prevents the accelerated cell ageing of Werner syndrome fibroblasts[J]. Nature Genetics, 2000, 24: 16-17.
[26] Srinivasan A, Clellan AJ, Brasiskyte D. The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain [J]. Mol Cell Biol, 1997, 17 (8): 4761-4773.
[27] Li NF, Broad S, Lu YJ, Yang JS, Watson R, Hagemann T, Wilbanks G, Jacobs I, Balkwill F, Dafou D, Gayther SA. Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression[J]. Cell Prolif, 2007, 40(5):780-94.
[28] Endo A, Kano Y, Kihara K. Alteration in the retinoblastoma gene associated with immortalization of human fibroblasts treated with 60Co gammarays [J]. J Cancer Res Clinoncol, 1993, 119(9):522-526.
[29] Sugimoto M, Ide T, Goto M, et al. Reconsideration of senescence, immortalization and telomere maintenance of Epstein-Barr virus-transformed human B-lymphoblastoid cell lines [J]. Mech Ageing Dev, 1999, 107(1):51-60.
[30] Lei KJ, Gluzman Y, Pan CJ. Immortalization of virus-free human placental cells that express tissue specific functions[J]. Mol Endocrinol, 1992, 6(5):703-712.
[31] Christian BJ, Loretz LJ, Oberley TD, et al. Characterization of human uroepithelial cells immortalized in vitro by simian virus 40 [J]. Cancer Res, 1987, 47(22):6066-6073.
[32] He YL, Wu YH, He XN, Liu FJ, He XY, Zhang Y. An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer[J]. Theriogenology, 2009, 71(9):1417-1424.
[33] Steinert S, Shay JW, Wright WE. Transient expression of human telomerase extends the lifespan of normal human fibroblasts [J]. Biochem Biophys Res Commun, 2000, 273(3): 1095-1098.
[34] Ausubel FM, Brent R, Kingston RE. Short protocols in molecular biology [M]. 3rd ed. John Wiley&Sons, Inc., New York, 1995: 274-326.
[35] Miquel C, Gaghoux P, Durand C, et al. Establishment and characterization of cell line LSVS that retains the altered adhesive properties of human functional epidermolysis bullosa keratmocyte [J]. Exp Cell Res, 1996, 224(2): 279-290.
[36] Ray FA. Meyne J, and Kraemer PM. SV40 T antigen induced chromosomal changes reflect a process that is both clastogenic and aneuploidogenic and is ongoing throughout neoplastil progression of human fibroblasts [J]. Mutat Res, 1992, 284(2): 265-273.
[37] Souttou B, Hamelin R, and Crepin M. EGF2 as an autocrine growth factor for immortal human breast epithelial cells [J]. Cell Growth Differ, 1994, 5(6): 615-623.
[38] Feng J, Funk WD, Wang SS. The RNA component of human telomerase[J]. Science, 1995, 269:1236-1239.
[39] Bhattacharyya A, Blackburn EH. A functional telomerase RNA swap in vivo reveals the importance ofnontemplate RNA domains [J]. Proc Natl Acad Sci USA, 1997, 94:2823-2830.
[40] McCormick-Graham M, Haynes WJ, Romero DP. Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase [J]. EMBO J, 1997, 16:3233-3237.
[41] Mckenzie KE, Umbricht CB, Sukumar S. Applications of telomerase research in the fight against cancer[J]. Mol Med Today, 1999, 5:114-122.
[42] Beattie T, Zhou W, Robinson M. Reconstitution of telomerase activity in vitro [J]. Curr Biol, 1998, 8:177-182.
[43] Tahara H, Yasui W, Tahara E. Immunohistochemical detection of human telomerase catalytic component, hTERT, in human colorectal tumor and non tumor tissue sections [J]. Oncogene, 1999, 18:1561-1567.
[44] Lai SR, Phipps SM, Liu L, et al. Epigenetic control of telomerase and modes of telomere maintenance in aging and abnormal systems [J]. Front Biosci, 2005, 10: 1779-1796.
[45] Danielle Robertson, Li L, Stephen Fisher, et al. Characterization of Growth and Differentiation in a Telomerase-Immortalized Human Corneal Epithelial Cell Line[J]. Investigative Ophthalmology and Visual Science, 2005, 46: 470-478.
[46] Toyohiko Y, Keisuke F, Osamu Y, Mizuo H, Junichi M, Yukihiro T, Hidenobu K, Hidenori I, Toshio O. Apoptosis signal-regulating kinase mediates cellular senescence induced by high glucose in endothelial[J]. Cells. Diabetes, 2006, 55(6):423-431.
[47] Van SB, Lange TD. Control of telomere length by the human telomeric protein TRF1[J]. Nature, 1997, 385:740-742.
[48] Hande MP. DNA repair factors and telomere-chromosome integrity in mammalian cells [J]. Cytogenet Genome Res, 2004, 104(1-4):116-122.
[49] Shin KH, Kang MK, et al. Introduction of human telomerase reverse transcriptase to normal human fibroblasts enhances DNA repair capacity [J]. Clin Cancer Res, 2004; 10(7):2551-2560.
[50] Pirzio LM, Freulet-Marrière MA, et al. Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation [J]. Cytogenet Genome Res, 2004, 104(1-4):87-94.
[51] Sharma GG, Gupta A. hTERT associates with human telomeres and enhances genomic stability and DNA repair [J]. Oncogene 2003, 22:131-146.
[52] Sara B, Valentina C, Dino A, Daniele C, Rosella S. The current role of telomerase in the diagnosis of bladder cancer[J]. Indian J Urol, 2009, 25(1): 40-46.
[53] Saretzki G, Ludwig A, et al. Ribozyme-mediated telomerase inhibition induces immediate cell loss but not telomere shortening in ovariancancer cells [J]. Cancer Gene Ther, 2001, 8: 827-834.
[54] Hao ZM, Luo JY, et al, Intensive Inhibition of hTERT Expression by a Ribozyme Induces Rapid Apoptosis of Cancer Cells through a Telomere Length-Independent Pathway [J]. Cancer Biol Ther, 2005, 4(10):1098-1103.
[55] Folini M, Pennati M, et al. Targeting human telomerase by antisense oligonucleotides and ribozymes[J]. Curr Med Chem Anti-Canc Agents, 2002, 2(5): 605-612.
[56] Xu DW, Wang Q, et al. Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells [J]. Oncogene, 2000, 19: 5123-5133.
[57] Cao Y, Li H, et al., TERT regulates cell survival independent of telomerase enzymatic activity [J].Oncogene, 2002, 21:3130-3138.
[58] Rahman R, Latonen, et al. hTERT antagonizes p53-induced apoptosis independently of telomerase activity [J]. Oncogene, 2005, 24:1320-1327.
[59] Smith LL, Coller HA, et al. Telomerase modulates expression of growth-controlling genes and enhances cell proliferation [J]. Nat Cell Biol. 2003, 5: 474-479.
[60] Li S, Rosenberg JE, Donjacour AA, et al. Rapid inhibition of cancer cell growth induced by lentiviral delivery and expression of mutant- template telomerase RNA and anti-telomerase shortinterfering RNA [J]. Cancer Res, 2004, 64: 4833- 4840.
[61] K. Anno, A. Hayashi, T. Takahashi, Y. Mitsui, T. Ide and H. Tahara, Telomerase activation induces elongation of the telomeric single-stranded overhang, but does not prevent chromosome aberrations in human vascular endothelial cells[J]. Biochem Biophys Res Commun, 2007, 353:926-932.
[62] Li S, Crothers J, Haqq CM, et al. Cellular and gene expression responses involved in the rapid growth inhibition of human cancer cells by RNA interference- mediated depletion of telomerase RNA [J]. J Biol Chem. 2005, 280: 23709-23717.
[63] Stephen N, Gary P. Telomere maintenance as a target for anticancer drug discovery [J]. Nature Drug Discovery, 2002, 1:383-393.
[64] S. Puttmann, V. Senner, S. Braune, B. Hillmann, R. Exeler and C.H. Rickert et al., Establishment of a benign meningioma cell line by hTERT-mediated immortalization[J]. Lab Invest, 2005, 85: 1163-1171.
[65] Homayoun V Samuel B. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative lifespan [J]. Current Biology, 1998, 8(5):279-282.
[66] Wang J, Lin YX, Susan Allan. Myc activates telomerase [J]. Research Communication, 1998, 12(12): 1769-1774.
[67] Michel M, Ouellette, Lisa D, Woodring E, et al. The establishment of telomerase immortalized cell lines representing human chromosome instability syndromes [J]. Human Molecular Genetics, 2000, 9(3): 403-411.
[68] Erik Hooijberg, Janneke J. Ruizendaal, Peter J. F. Snijders, et al. Immortalization of Human CD8+ T Cell Clones by Ectopic Expression of Telomerase Reverse Transcriptase [J]. The Journal of Immunology, 2000, 165: 4239-4245.
[69] Brittney-Shea Herbert, Woodring E Wright and Jerry W Shay. pl6IN4Ka inactivation is not required to immortalize human mammary epithelial cells [J]. Oncogene, 2002, 21: 7897-7900.
[70] Veitonmaki N, Niina Veitonm?ki, Jonas Fuxe, et al. Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16INK4a/pRb [J]. FASEB J, 2006, 17 (6): 764-780.
[71] Fiedler W, Reinicke D, Aurich H. In Vitro Immortalisation of Porcine epatocytes by Transfection with the Gene for the Human Catalytic Subunit of Telomerase Reverse ranscriptase (HTERT)[J]. Journal of Hepatology, 2004, 40(1): 102.
[72] Ira Kogan, Naomi Goldfinger, Michael Milyavsky. hTERT-immortalized prostate epithelial and stromal-derived cells: an authentic in vitro model for differentiation and carcinogenesis [J]. Cancer Research, 2006, 66: 3531-3540.
[73] Kapanadze B, Morris E, Simth E, Trojanowska M. Establishment and characterization of sclerodermafibroblast clonal cell lines by introduction of the hTERT gene [J]. Cell Mol Med. 2009, 11, [Epub ahead of print].
[74] He YL, Wu YH, He XN, Liu FJ, He XY, Zhang Y. An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer [J]. Theriogenology. 2009, 71(9):1417-24.
[75] Tian XC, Xu J, Yang XZ. Normal telomere lengths found in cloned cattle [J]. Nature Genetics, 2000, 26: 272-273.
[76] Shao G, Balajee AS, Hei TK, Zhao Y. p16INK4a downregulation is involved in immortalization of primary human prostate epithelial cells induced by telomerase[J]. Mol Carcinog, 2008, 47:775-783.
[77] Hana B, Orit CB. Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells[J]. Cancer research, 2001, 61: 5529-5536.
[78] Palmiter RD, Brinster RL, et al. Dramatic growth of mice that develop from eggs microinjected with metallothione in growth hormone fusiongenes [J]. Nature, 1982, 300: 611-615.
[79] Magin TM, McWhir J, Melton DW. A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency[J]. Nucleic Acids Res, 1992, 20: 3795-3796.
[80] Schnieke AE, Kind AJ, Ritchie WA. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts[J]. Science, 1997, 278: 2130-2133.
[81] Perry AC, Wakayama T, Kishikawa H. Mammalian transgenesis by intracytoplasmic sperm injection [J]. Science, 1999, 284: 1180-1183.
[82] Kerr DE, Plaut K, BramLey AJ, et al. Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice [J]. Nat Biotech, 2001, 19: 66-70.
[83] Kang JX, Wang JD, Wu L, et al. Fat-1 mice convert n-6 to n-3 fatty acid [J]. Nature, 2004, 427: 504-505.
[84] Lai L, Kang JX, Li R, Wang J, Witt WT, et al.Generation of cloned transgenic pigs rich in omega-3 fatty acids [J]. Nature Biotechnology, 2006, 24 (4): 435-436.
[85] Smith K, Spadafora C. Sperm-mediated gene transfer: applications and implications[J]. Bioessays, 2005, 27(5):551-62.
[86] Hossein MS, Jeong YW, Park SW, Kim JJ, Lee E, Ko KH, Kim HS, Kim YW, Hyun SH, Shin T, Hawthorne L, Hwang WS Cloning missy: obtaining multiple offspring of a specific canine genotype by somatic cell nuclear transfer[J]. Cloning Stem Cells, 2009, 11(1):123-30.
[87] Peng CA. Analysis of gene transfer rate with immobilized retroviral vectors. Ann N Y Acad Sci, 2009, 1161:26-33.
[88] Liang JZ, Li R, Zhang JY, Zheng H, Luo RC. Retrovirus-mediated SHP-1 gene expression in human breast cancer MDA-MB-231 cells[J]. Nan Fang Yi Ke Da Xue Xue Bao, 2009, 29(5):902-5.
[89] Kim JH, Jung-Ha HS, Lee HT, Chung KS. Development of a positive method for male stem cell mediated gene transfer in mouse and pig[J]. Molecular Reproduction and Development, 1997, 46: 515-526
[90] Shen W, Li L, Pan Q, Min L, Dong H and Deng J. Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method [J]. Molecular Reproduction and Development, 2006, 73: 589-594.
[91] Yang SY, Wang JG, Cui HX, Sun SG, Li Q, Gu L, Hong HP, Ciu PP and Liu WQ. Efficientgeneration of transgenic mice by direct intraovarian injection of plasmid DNA [J].Biochemical and Biophysical Research Communications, 2007, 358 (1):266-271.
[92] Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells [J]. Cell, 1980, (2): 479-488.
[93] Smithies O, Gregg RG, Boggs SS, et al. Insertion of DNA sequences into the human chromosal beta-globin locus by homologous recombination [J]. Nature, 1985, 317: 230-234.
[94] Fire A,Xu S,MontgomeryMK. Potent and specific genetic inter-ferenceby double-strand RNA in coenorhabditis elegans[J].Nature, 1998, 391(6669), 744-745.
[95] Cogoni C, Macino G. Post transcriptional gene silencing across kingdoms [J]. Curr Opin Genet Dev, 2000, 10 (6):638-643.
[96] Acosta J, Carpio Y, Borroto I, González O and Estrada MP.Myostatin gene silenced by RNAi show a zebrafish giant phenotype [J]. Journal of Biotechnology, 2005, 119 (4): 324-331.
[97] Dann CT, Alvarado AL, Hammer RE and Garbers DL. Heritableand stable gene knockdown in rats[J] .Proceedings of National Academy of Science of the USA, 2006, 103 (30):11246-11251.
[98] Brunetti D, Perota A, Lagutina I, Colleoni S, Duchi R, Calabrese F, Seveso M, Cozzi E, Lazzari G, Lucchini F, Galli C Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts[J]. Cloning Stem Cells, 2008, 10(4):409-19.
[99] Matsunari H, Onodera M, Tada N, Mochizuki H, Karasawa S, Haruyama E, Nakayama N, Saito H, Ueno S, Kurome M, Miyawaki A, Nagashima H. Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange[J]. Cloning Stem Cells, 2008, 10(3): 313-23.
[100] Pursel VG, Pinkert CA, Miller KF, et al. Genetic engineering of livestock [J]. Science, 1989, 244: 1281-1288.
[101] Damak S, Su H, Jay NP, et al. Improved wool production in transgenic sheep expressing insulin-like growth factor1[J]. Biotechnology, 1996, 14: 185-188.
[102] Kang JX, Wang JD, Wu L, et al. Fat-1 mice convertω-6 toω-3 fatty acid[J]. Nature, 2004, 427: 504-505.
[103] Berm G. Production of transgenic and possible application to pig breeding[J]. Soc Anim Prod, 1992, 12: 15-31.
[104] Wadih A, Renata P, Erkki R, et a1. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse mode1[J]. Science, 1998, 279:377-380.
[134] Wei X, Luo Y, Hyde DR. Molecular cloning of three zebrafish lin7 genes and their expression patterns in the retina[J]. Exp Eye Res. 2006, 82:122-31
[105] Pan X, Wan H, Chia W, Tong Y, Gong Z. Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system[J]. Transgenic Res. 2005, 14:217-23.
[106] Collery RF, Cederlund ML, Smyth VA, Kennedy BN. Applying transgenic zebrafish technology to study the retina[J]. Adv Exp Med Biol, 2006, 572:201-207.
[107] Amsterdam A, Hopkins N. Mutagenesis strategies in zebrafish for identifying genes involved in development and disease[J]. Trends Genet, 2006, 22:473-478.