Factors in?uencing the somatic cell nuclear transfer ef?ciency in pigs
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摘要
Using a data set from our laboratory, we assessed the effects of several factors on pig cloning ef?ciency. The results demonstrated that cells at high con?uence( > 90%) used as donor cell resulted in higher pregnancy rate, delivery rate and overall cloning ef?ciency(number of live offspring born per reconstructed embryo transferred to recipients) compared with the cells at 60% to79% con?uence and 80% to 89% con?uence. Cells with four, ?ve and six passages compromised the pregnancy and delivery rates compared with ?rst passage cells. The number of blastocysts transferred by somatic cell nuclear transfer(SCNT) did not signi?cantly affect the cloning ef?ciency, but transfer of blastocyst derived from in vitro culture 5 d after SCNT achieved a signi?cantly higher pregnancy rate compared with one to two cell SCNT embryos from overnight culture. The highest pregnancy rate, delivery rate and the largest litter size were obtained when Bama Miniature pig ?broblasts were used as donor cells and Landrace/Yorkshire hybrid gilts were used as recipients. Recipients treated with chemicals for estrus synchronization had higher pregnancy rates compared with untreated recipients. Our data might be helpful for improving SCNT ef?ciency in pigs.
Using a data set from our laboratory, we assessed the effects of several factors on pig cloning ef?ciency. The results demonstrated that cells at high con?uence( > 90%) used as donor cell resulted in higher pregnancy rate, delivery rate and overall cloning ef?ciency(number of live offspring born per reconstructed embryo transferred to recipients) compared with the cells at 60% to79% con?uence and 80% to 89% con?uence. Cells with four, ?ve and six passages compromised the pregnancy and delivery rates compared with ?rst passage cells. The number of blastocysts transferred by somatic cell nuclear transfer(SCNT) did not signi?cantly affect the cloning ef?ciency, but transfer of blastocyst derived from in vitro culture 5 d after SCNT achieved a signi?cantly higher pregnancy rate compared with one to two cell SCNT embryos from overnight culture. The highest pregnancy rate, delivery rate and the largest litter size were obtained when Bama Miniature pig ?broblasts were used as donor cells and Landrace/Yorkshire hybrid gilts were used as recipients. Recipients treated with chemicals for estrus synchronization had higher pregnancy rates compared with untreated recipients. Our data might be helpful for improving SCNT ef?ciency in pigs.
Using a data set from our laboratory, we assessed the effects of several factors on pig cloning ef?ciency. The results demonstrated that cells at high con?uence( > 90%) used as donor cell resulted in higher pregnancy rate, delivery rate and overall cloning ef?ciency(number of live offspring born per reconstructed embryo transferred to recipients) compared with the cells at 60% to79% con?uence and 80% to 89% con?uence. Cells with four, ?ve and six passages compromised the pregnancy and delivery rates compared with ?rst passage cells. The number of blastocysts transferred by somatic cell nuclear transfer(SCNT) did not signi?cantly affect the cloning ef?ciency, but transfer of blastocyst derived from in vitro culture 5 d after SCNT achieved a signi?cantly higher pregnancy rate compared with one to two cell SCNT embryos from overnight culture. The highest pregnancy rate, delivery rate and the largest litter size were obtained when Bama Miniature pig ?broblasts were used as donor cells and Landrace/Yorkshire hybrid gilts were used as recipients. Recipients treated with chemicals for estrus synchronization had higher pregnancy rates compared with untreated recipients. Our data might be helpful for improving SCNT ef?ciency in pigs.
引文
1.Lai L,Prather R S.Creating genetically modified pigs by using nuclear transfer.Reproductive Biology and Endocrinology,2003,1(1):82
2.Ahn K S,Kim Y J,Kim M,Lee B H,Heo S Y,Kang M J,Kang Y K,Lee J W,Lee K K,Kim J H,Nho W G,Hwang S S,Woo J S,Park JK,Park S B,Shim H.Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts.Theriogenology,2011,75(5):933-939
3.Fan N,Chen J,Shang Z,Dou H,Ji G,Zou Q,Wu L,He L,Wang F,Liu K,Liu N,Han J,Zhou Q,Pan D,Yang D,Zhao B,Ouyang Z,Liu Z,Zhao Y,Lin L,Zhong C,Wang Q,Wang S,Xu Y,Luan J,Liang Y,Yang Z,Li J,Lu C,Vajta G,Li Z,Ouyang H,Wang H,Wang Y,Yang Y,Liu Z,Wei H,Luan Z,Esteban M A,Deng H,Yang H,Pei D,Li N,Pei G,Liu L,Du Y,Xiao L,Lai L.Piglets cloned from induced pluripotent stem cells.Cell Research,2013,23(1):162-166
4.Lai L,Kolber-Simonds D,Park K W,Cheong H T,Greenstein J L,Im G S,Samuel M,Bonk A,Rieke A,Day B N,Murphy C N,Carter D B,Hawley R J,Prather R S.Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.Science,2002,295(5557):1089-1092
5.Ramsoondar J J,Macháty Z,Costa C,Williams B L,Fodor W L,Bondioli K R.Production of alpha 1,3-galactosyltransferaseknockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase.Biology of Reproduction,2003,69(2):437-445
6.Betthauser J,Forsberg E,Augenstein M,Childs L,Eilertsen K,Enos J,Forsythe T,Golueke P,Jurgella G,Koppang R,Lesmeister T,Mallon K,Mell G,Misica P,Pace M,Pfister-Genskow M,Strelchenko N,Voelker G,Watt S,Thompson S,Bishop M.Production of cloned pigs from in vitro systems.Nature Biotechnology,2000,18(10):1055-1059
7.Onishi A,Iwamoto M,Akita T,Mikawa S,Takeda K,Awata T,Hanada H,Perry A C.Pig cloning by microinjection of fetal fibroblast nuclei.Science,2000,289(5482):1188-1190
8.Polejaeva I A,Chen S H,Vaught T D,Page R L,Mullins J,Ball S,Dai Y,Boone J,Walker S,Ayares D L,Colman A,Campbell K H.Cloned pigs produced by nuclear transfer from adult somatic cells.Nature,2000,407(6800):86-90
9.Klymiuk N,Aigner B,Brem G,Wolf E.Genetic modification of pigs as organ donors for xenotransplantation.Molecular Reproduction and Development,2010,77(3):209-221
10.Prather R S,Shen M,Dai Y.Genetically modified pigs for medicine and agriculture.Biotechnology&Genetic Engineering Reviews,2008,25:245-265
11.Schmidt M,Kragh P M,Li J,Du Y,Lin L,Liu Y,B?gh I B,Winther K D,Vajta G,Callesen H.Pregnancies and piglets from large white sow recipients after two transfer methods of cloned and transgenic embryos of different pig breeds.Theriogenology,2010,74(7):1233-1240
12.Vajta G,Callesen H.Establishment of an efficient somatic cell nuclear transfer system for production of transgenic pigs.Theriogenology,2012,77(7):1263-1274
13.Whitworth K M,Prather R S.Somatic cell nuclear transfer efficiency:how can it be improved through nuclear remodeling and reprogramming?Molecular Reproduction and Development,2010,77(12):1001-1015
14.Yang X,Smith S L,Tian X C,Lewin H A,Renard J P,Wakayama T.Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning.Nature Genetics,2007,39(3):295-302
15.Zhao J,Whyte J,Prather R S.Effect of epigenetic regulation during swine embryogenesis and on cloning by nuclear transfer.Cell and Tissue Research,2010,341(1):13-21
16.Yin X J,Tani T,Yonemura I,Kawakami M,Miyamoto K,Hasegawa R,Kato Y,Tsunoda Y.Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes.Biology of Reproduction,2002,67(2):442-446
17.Kühholzer B,Hawley R J,Lai L,Kolber-Simonds D,Prather R S.Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs.Biology of Reproduction,2001,64(6):1695-1698
18.Nakayama A,Sato M,Shinohara M,Matsubara S,Yokomine T,Akasaka E,Yoshida M,Takao S.Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.Cloning and Stem Cells,2007,9(4):523-534
19.Vajta G,Zhang Y,Macháty Z.Somatic cell nuclear transfer in pigs:recent achievements and future possibilities.Reproduction,Fertility,and Development,2007,19(2):403-423
20.Koo O J,Park H J,Kwon D K,Kang J T,Jang G,Lee B C.Effect of recipient breed on delivery rate of cloned miniature pig.Zygote,2009,17(3):203-207
21.Kurome M,Geistlinger L,Kessler B,Zakhartchenko V,Klymiuk N,Wuensch A,Richter A,Baehr A,Kraehe K,Burkhardt K,Flisikowski K,Flisikowska T,Merkl C,Landmann M,Durkovic M,Tschukes A,Kraner S,Schindelhauer D,Petri T,Kind A,Nagashima H,Schnieke A,Zimmer R,Wolf E.Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer:multi-factorial analysis of a large data set.BMCBiotechnology,2013,13(1):43
22.Kaeoket K.Study on the oestrous synchronization in gilts by using progestin altrenogest and h CG:its effect on the follicular development,ovulation time and subsequent reproductive performance.Reproduction in Domestic Animals,2008,43(1):127-129
23.Campbell K H,Loi P,Otaegui P J,Wilmut I.Cell cycle coordination in embryo cloning by nuclear transfer.Reviews of Reproduction,1996,1(1):40-46
24.Boquest A C,Day B N,Prather R S.Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells.Biology of Reproduction,1999,60(4):1013-1019
25.Mc Elroy S L,Kim J H,Kim S,Jeong Y W,Lee E G,Park S M,Hossein M S,Koo O J,Abul Hashem M D,Jang G,Kang S K,Lee B C,Hwang W S.Effects of culture conditions and nuclear transfer protocols on blastocyst formation and m RNA expression in preimplantation porcine embryos.Theriogenology,2008,69(4):416-425
26.Miyoshi K,Inoue S,Himaki T,Mikawa S,Yoshida M.Birth of cloned miniature pigs derived from somatic cell nuclear transferred embryos activated by ultrasound treatment.Molecular Reproduction and Development,2007,74(12):1568-1574
27.Wakai T,Sugimura S,Yamanaka K,Kawahara M,Sasada H,Tanaka H,Ando A,Kobayashi E,Sato E.Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries.Cloning and Stem Cells,2008,10(2):249-262
28.Walker S C,Shin T,Zaunbrecher G M,Romano J E,Johnson G A,Bazer F W,Piedrahita J A.A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes.Cloning and Stem Cells,2002,4(2):105-112
29.Cibelli J B,Stice S L,Golueke P J,Kane J J,Jerry J,Blackwell C,Ponce de León F A,Robl J M.Cloned transgenic calves produced from nonquiescent fetal fibroblasts.Science,1998,280(5367):1256-1258
30.Wilmut I,Schnieke A E,Mc Whir J,Kind A J,Campbell K H.Viable offspring derived from fetal and adult mammalian cells.Nature,1997,385(6619):810-813
31.Enright B P,Jeong B S,Yang X,Tian X C.Epigenetic characteristics of bovine donor cells for nuclear transfer:levels of histone acetylation.Biology of Reproduction,2003,69(5):1525-1530
32.Condic M L.Alternative sources of pluripotent stem cells:altered nuclear transfer.Cell Proliferation,2008,41(S1):7-19
33.Deshmukh R S,?strup O,?strup E,Vejlsted M,Niemann H,LucasHahn A,Petersen B,Li J,Callesen H,Hyttel P.DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization,parthenogenetic activation and somatic cell nuclear transfer.Epigenetics,2011,6(2):177-187
34.Bork S,Pfister S,Witt H,Horn P,Korn B,Ho A D,Wagner W.DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells.Aging Cell,2010,9(1):54-63
35.Noer A,S?rensen A L,Boquest A C,Collas P.Stable Cp Ghypomethylation of adipogenic promoters in freshly isolated,cultured,and differentiated mesenchymal stem cells from adipose tissue.Molecular Biology of the Cell,2006,17(8):3543-3556
36.Wagner M,Schmelz K,D?rken B,Tamm I.Epigenetic and genetic analysis of the survivin promoter in acute myeloid leukemia.Leukemia Research,2008,32(7):1054-1060
37.Petersen B,Lucas-Hahn A,Oropeza M,Hornen N,Lemme E,Hassel P,Queisser A L,Niemann H.Development and validation of a highly efficient protocol of porcine somatic cloning using preovulatory embryo transfer in peripubertal gilts.Cloning and Stem Cells,2008,10(3):355-362
38.Rim C H,Fu Z,Bao L,Chen H,Zhang D,Luo Q,Ri H C,Huang H,Luan Z,Zhang Y,Cui C,Xiao L,Jong U M.The effect of the number of transferred embryos,the interval between nuclear transfer and embryo transfer,and the transfer pattern on pig cloning efficiency.Animal Reproduction Science,2013,143(1-4):91-96
39.Maheshwari A,Hamilton M,Bhattacharya S.Should we be promoting embryo transfer at blastocyst stage?Reproductive Biomedicine Online,2016,32(2):142-146
40.Kolibianakis E M,Devroey P.Blastocyst culture:facts and fiction.Reproductive Biomedicine Online,2002,5(3):285-293
2.Ahn K S,Kim Y J,Kim M,Lee B H,Heo S Y,Kang M J,Kang Y K,Lee J W,Lee K K,Kim J H,Nho W G,Hwang S S,Woo J S,Park JK,Park S B,Shim H.Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts.Theriogenology,2011,75(5):933-939
3.Fan N,Chen J,Shang Z,Dou H,Ji G,Zou Q,Wu L,He L,Wang F,Liu K,Liu N,Han J,Zhou Q,Pan D,Yang D,Zhao B,Ouyang Z,Liu Z,Zhao Y,Lin L,Zhong C,Wang Q,Wang S,Xu Y,Luan J,Liang Y,Yang Z,Li J,Lu C,Vajta G,Li Z,Ouyang H,Wang H,Wang Y,Yang Y,Liu Z,Wei H,Luan Z,Esteban M A,Deng H,Yang H,Pei D,Li N,Pei G,Liu L,Du Y,Xiao L,Lai L.Piglets cloned from induced pluripotent stem cells.Cell Research,2013,23(1):162-166
4.Lai L,Kolber-Simonds D,Park K W,Cheong H T,Greenstein J L,Im G S,Samuel M,Bonk A,Rieke A,Day B N,Murphy C N,Carter D B,Hawley R J,Prather R S.Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.Science,2002,295(5557):1089-1092
5.Ramsoondar J J,Macháty Z,Costa C,Williams B L,Fodor W L,Bondioli K R.Production of alpha 1,3-galactosyltransferaseknockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase.Biology of Reproduction,2003,69(2):437-445
6.Betthauser J,Forsberg E,Augenstein M,Childs L,Eilertsen K,Enos J,Forsythe T,Golueke P,Jurgella G,Koppang R,Lesmeister T,Mallon K,Mell G,Misica P,Pace M,Pfister-Genskow M,Strelchenko N,Voelker G,Watt S,Thompson S,Bishop M.Production of cloned pigs from in vitro systems.Nature Biotechnology,2000,18(10):1055-1059
7.Onishi A,Iwamoto M,Akita T,Mikawa S,Takeda K,Awata T,Hanada H,Perry A C.Pig cloning by microinjection of fetal fibroblast nuclei.Science,2000,289(5482):1188-1190
8.Polejaeva I A,Chen S H,Vaught T D,Page R L,Mullins J,Ball S,Dai Y,Boone J,Walker S,Ayares D L,Colman A,Campbell K H.Cloned pigs produced by nuclear transfer from adult somatic cells.Nature,2000,407(6800):86-90
9.Klymiuk N,Aigner B,Brem G,Wolf E.Genetic modification of pigs as organ donors for xenotransplantation.Molecular Reproduction and Development,2010,77(3):209-221
10.Prather R S,Shen M,Dai Y.Genetically modified pigs for medicine and agriculture.Biotechnology&Genetic Engineering Reviews,2008,25:245-265
11.Schmidt M,Kragh P M,Li J,Du Y,Lin L,Liu Y,B?gh I B,Winther K D,Vajta G,Callesen H.Pregnancies and piglets from large white sow recipients after two transfer methods of cloned and transgenic embryos of different pig breeds.Theriogenology,2010,74(7):1233-1240
12.Vajta G,Callesen H.Establishment of an efficient somatic cell nuclear transfer system for production of transgenic pigs.Theriogenology,2012,77(7):1263-1274
13.Whitworth K M,Prather R S.Somatic cell nuclear transfer efficiency:how can it be improved through nuclear remodeling and reprogramming?Molecular Reproduction and Development,2010,77(12):1001-1015
14.Yang X,Smith S L,Tian X C,Lewin H A,Renard J P,Wakayama T.Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning.Nature Genetics,2007,39(3):295-302
15.Zhao J,Whyte J,Prather R S.Effect of epigenetic regulation during swine embryogenesis and on cloning by nuclear transfer.Cell and Tissue Research,2010,341(1):13-21
16.Yin X J,Tani T,Yonemura I,Kawakami M,Miyamoto K,Hasegawa R,Kato Y,Tsunoda Y.Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes.Biology of Reproduction,2002,67(2):442-446
17.Kühholzer B,Hawley R J,Lai L,Kolber-Simonds D,Prather R S.Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs.Biology of Reproduction,2001,64(6):1695-1698
18.Nakayama A,Sato M,Shinohara M,Matsubara S,Yokomine T,Akasaka E,Yoshida M,Takao S.Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.Cloning and Stem Cells,2007,9(4):523-534
19.Vajta G,Zhang Y,Macháty Z.Somatic cell nuclear transfer in pigs:recent achievements and future possibilities.Reproduction,Fertility,and Development,2007,19(2):403-423
20.Koo O J,Park H J,Kwon D K,Kang J T,Jang G,Lee B C.Effect of recipient breed on delivery rate of cloned miniature pig.Zygote,2009,17(3):203-207
21.Kurome M,Geistlinger L,Kessler B,Zakhartchenko V,Klymiuk N,Wuensch A,Richter A,Baehr A,Kraehe K,Burkhardt K,Flisikowski K,Flisikowska T,Merkl C,Landmann M,Durkovic M,Tschukes A,Kraner S,Schindelhauer D,Petri T,Kind A,Nagashima H,Schnieke A,Zimmer R,Wolf E.Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer:multi-factorial analysis of a large data set.BMCBiotechnology,2013,13(1):43
22.Kaeoket K.Study on the oestrous synchronization in gilts by using progestin altrenogest and h CG:its effect on the follicular development,ovulation time and subsequent reproductive performance.Reproduction in Domestic Animals,2008,43(1):127-129
23.Campbell K H,Loi P,Otaegui P J,Wilmut I.Cell cycle coordination in embryo cloning by nuclear transfer.Reviews of Reproduction,1996,1(1):40-46
24.Boquest A C,Day B N,Prather R S.Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells.Biology of Reproduction,1999,60(4):1013-1019
25.Mc Elroy S L,Kim J H,Kim S,Jeong Y W,Lee E G,Park S M,Hossein M S,Koo O J,Abul Hashem M D,Jang G,Kang S K,Lee B C,Hwang W S.Effects of culture conditions and nuclear transfer protocols on blastocyst formation and m RNA expression in preimplantation porcine embryos.Theriogenology,2008,69(4):416-425
26.Miyoshi K,Inoue S,Himaki T,Mikawa S,Yoshida M.Birth of cloned miniature pigs derived from somatic cell nuclear transferred embryos activated by ultrasound treatment.Molecular Reproduction and Development,2007,74(12):1568-1574
27.Wakai T,Sugimura S,Yamanaka K,Kawahara M,Sasada H,Tanaka H,Ando A,Kobayashi E,Sato E.Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries.Cloning and Stem Cells,2008,10(2):249-262
28.Walker S C,Shin T,Zaunbrecher G M,Romano J E,Johnson G A,Bazer F W,Piedrahita J A.A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes.Cloning and Stem Cells,2002,4(2):105-112
29.Cibelli J B,Stice S L,Golueke P J,Kane J J,Jerry J,Blackwell C,Ponce de León F A,Robl J M.Cloned transgenic calves produced from nonquiescent fetal fibroblasts.Science,1998,280(5367):1256-1258
30.Wilmut I,Schnieke A E,Mc Whir J,Kind A J,Campbell K H.Viable offspring derived from fetal and adult mammalian cells.Nature,1997,385(6619):810-813
31.Enright B P,Jeong B S,Yang X,Tian X C.Epigenetic characteristics of bovine donor cells for nuclear transfer:levels of histone acetylation.Biology of Reproduction,2003,69(5):1525-1530
32.Condic M L.Alternative sources of pluripotent stem cells:altered nuclear transfer.Cell Proliferation,2008,41(S1):7-19
33.Deshmukh R S,?strup O,?strup E,Vejlsted M,Niemann H,LucasHahn A,Petersen B,Li J,Callesen H,Hyttel P.DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization,parthenogenetic activation and somatic cell nuclear transfer.Epigenetics,2011,6(2):177-187
34.Bork S,Pfister S,Witt H,Horn P,Korn B,Ho A D,Wagner W.DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells.Aging Cell,2010,9(1):54-63
35.Noer A,S?rensen A L,Boquest A C,Collas P.Stable Cp Ghypomethylation of adipogenic promoters in freshly isolated,cultured,and differentiated mesenchymal stem cells from adipose tissue.Molecular Biology of the Cell,2006,17(8):3543-3556
36.Wagner M,Schmelz K,D?rken B,Tamm I.Epigenetic and genetic analysis of the survivin promoter in acute myeloid leukemia.Leukemia Research,2008,32(7):1054-1060
37.Petersen B,Lucas-Hahn A,Oropeza M,Hornen N,Lemme E,Hassel P,Queisser A L,Niemann H.Development and validation of a highly efficient protocol of porcine somatic cloning using preovulatory embryo transfer in peripubertal gilts.Cloning and Stem Cells,2008,10(3):355-362
38.Rim C H,Fu Z,Bao L,Chen H,Zhang D,Luo Q,Ri H C,Huang H,Luan Z,Zhang Y,Cui C,Xiao L,Jong U M.The effect of the number of transferred embryos,the interval between nuclear transfer and embryo transfer,and the transfer pattern on pig cloning efficiency.Animal Reproduction Science,2013,143(1-4):91-96
39.Maheshwari A,Hamilton M,Bhattacharya S.Should we be promoting embryo transfer at blastocyst stage?Reproductive Biomedicine Online,2016,32(2):142-146
40.Kolibianakis E M,Devroey P.Blastocyst culture:facts and fiction.Reproductive Biomedicine Online,2002,5(3):285-293