Production of homeobox A10 gene transgenic pigs by somatic cell nuclear transfer
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摘要
Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.
Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.
Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.
引文
Andersson K L,Bussani C,Fambrini M,Polverino V,Taddei G L,Gemzell-Danielsson K,Scarselli G.2014.DNA methylation of HOXA10 in eutopic and ectopic endometrium.Human Reproduction,29,1906-1911.
Bagot C N,Troy P J,Taylor H S.2000.Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation.Gene Therapy,7,1378-1384.
Blitek A,Kaczmarek M M,Kiewisz J,Ziecik A J.2010.Endometrial and conceptus expression of HoxA10,transforming growth factor beta1,leukemia inhibitory factor,and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus.Domestic Animal Endocrinology,38,222-234.
Campbell K H,Ritchie W A,Wilmut I.1993.Nuclearcytoplasmic interactions during the first cell cycle of nuclear transfer reconstructed bovine embryos:Implications for deoxyribonucleic acid replication and development.Biology of Reproduction,5,933-942.
Dalman A,Eftekhari-Yazdi P,Valojerdi M R,Shahverdi A,Gourabi H,Janzamin E,Fakheri R,Sadeghian F,Hasani F.2010.Synchronizing cell cycle of goat fibroblasts by serum starvation causes apoptosis.Reproduction in Domestic Animals,45,doi:10.1111/j.1439-0531.2009.01520.x
Denning C,Dickinson P,Burl S,Wylie D,Fletcher J,Clark A J.2001.Gene targeting in primary fetal fibroblasts from sheep and pig.Cloning and Stem Cells,3,221-231.
Ekici A B,Strissel P L,Oppelt P G,Renner S P,Brucker S,Beckmann M W,Strick R.2013.HOXA10 and HOXA13sequence variations in human female genital malformations including congenital absence of the uterus and vagina.Gene,518,267-272.
Goto Y,Hirayama M,Takeda K,Tukamoto N,Sakata O,Kaeriyama H,Geshi M.2013.Effect of synchronization of donor cells in early G1-phase using shake-off method on developmental potential of somatic cell nuclear transfer embryos in cattle.Animal Science Journal,84,592-599.
Hasegawa S,Harada K,Morokoshi Y,Tsukamoto S,Furukawa T,Saga T.2013.Growth retardation and hair loss in transgenic mice overexpressing human H-ferritin gene.Transgenic Research,22,651-658.
Ingham D J,Beer S,Money S,Hansen G.2001.Quantitative real-time PCR assay for determining transgene copy number in transformed plants.Bio Techniques,31,132-134.
Jiang Y,Yan G,Zhang H,Shan H,Kong C,Yan Q,Xue B,Diao Z,Hu Y,Sun H.2014.Activation of matrix metalloproteinase-26 by HOXA10 promotes embryo adhesion in vitro.Biochemical and Biophysical Research Communications,445,622-628.
Johnson R K,Nielsen M K,Casey D S.1999.Responses in ovulation rate,embryonal survival,and litter traits in swine to 14 generations of selection to increase litter size.Journal of Animal Science,77,541-557.
Kondo M,Hornung U,Nanda I,Imai S,Sasaki T,Shimizu A,Asakawa S,Hori H,Schmid M,Shimizu N,Schartl M.2006.Genomic organization of the sex-determining and adjacent regions of the sex chromosomes of medaka.Genome Research,16,815-826.
Lai L,Prather R S.2003.Production of cloned pigs by using somatic cells as donors.Cloning&Stem Cells,5,233-241.
Lambert J F,Benoit B O,Colvin G A,Carlson J,Delville Y,Quesenberry P J.2000.Quick sex determination of mouse fetuses.Journal of Neuroscience Methods,95,127-132.
Li S,Chen X,Fang Z,Shi J,Sheng H Z.2006.Rabbits generated from fibroblasts through nuclear transfer.Reproduction,131,1085-1090.
Lin R,Wu D,Zhao C,Chen S,Xiao Q,Li X,Zhao S.2016.Inducible overexpression of porcine homeobox A10 in the endometrium of transgenic mice.Journal of Integrative Agriculture,15,1338-1344.
Lin R,Zhao C,Jing L,Zhao S.2015.Identification of LncRNAs/mRNAs related to endometrium function regulated by Homeobox A10 in Ishikawa cells.Cell Biology International,39,842-851.
Linville R C,Pomp D,Johnson R K,Rothschild M F.2001.Candidate gene analysis for loci affecting litter size and ovulation rate in swine.Journal of Animal Science,79,60-67.
Liu W,Wu L H,Yue M,Nashun B,Tang H,Chen Y,Chen B,Yuan J,Xiao D,Gu W.2017.Generation of DKK1 transgenic Tibet minipigs by somatic cell nuclear transfer(SCNT).Oncotarget,8,74331-74339.
Marchal R,Vigneron C,Perreau C,Bali-Papp A,Mermillod P.2002.Effect of follicular size on meiotic and developmental competence of porcine oocytes.Theriogenology,57,1523-1532.
McCreath K J,Howcroft J,Campbell K H,Colman A,Schnieke A E,Kind A J.2000.Production of gene-targeted sheep by nuclear transfer from cultured somatic cells.Nature,405,1066-1069.
Onishi A,Iwamoto M,Akita T,Mikawa S,Takeda K,Awata T,Hanada H,Perry A C.2000.Pig cloning by microinjection of fetal fibroblast nuclei.Science,289,1188-1190.
Ponchel F,Toomes C,Bransfield K,Leong F T,Douglas S H,Field S L,Bell S M,Combaret V,Puisieux A,Mighell A J,Robinson P A,Inglehearn C F,Isaacs J D,Markham A F.2003.Real-time PCR based on SYBR-Green I fluorescence:an alternative to the TaqMan assay for a relative quantification of gene rearrangements,gene amplifications and micro gene deletions.BMC Biotechnology,3,doi:10.1186/1472-6750-3-18
Satokata I,Benson G,Maas R.1995.Sexually dimorphic sterility phenotypes in Hoxa10-deficient mice.Nature,374,460-463.
Taylor H S.2000.The role of HOX genes in the development and function of the female reproductive tract.Seminars in Reproductive Medicine,18,81-90.
Taylor H S,Bagot C,Kardana A,Olive D,Arici A.1999.HOXgene expression is altered in the endometrium of women with endometriosis.Human Reproduction,14,1328-1331.
Taylor H S,Vanden Heuvel G B,Igarashi P.1997.A conserved Hox axis in the mouse and human female reproductive system:Late establishment and persistent adult expression of the Hoxa cluster genes.Biology of Reproduction,57,1338-1345.
Team R.2013.R:A language and environment for statistical computing.Computing,1,12-21.
Wu D,Song D,Li X,Yu M,Li C,Zhao S.2013.Molecular characterization and identification of the E2/P4 response element in the porcine HOXA10 gene.Molecular and Cellular Biochemistry,374,213-222.
Wu Z,Xu Z,Zou X,Zeng F,Shi J,Liu D,Urschitz J,Moisyadi S,Li Z.2013.Pig transgenesis by piggy Bac transposition in combination with somatic cell nuclear transfer.Transgenic Research,22,1107-1118.
Xu B,Geerts D,Bu Z,Ai J,Jin L,Li Y,Zhang H,Zhu G.2014.Regulation of endometrial receptivity by the highly expressed HOXA9,HOXA11 and HOXD10 HOX-class homeobox genes.Human Reproduction,29,781-790.
Zhu L H,Sun L H,Hu Y L,Jiang Y,Liu H Y,Shen X Y,Jin X Y,Zhen X,Sun H X,Yan G J.2013.PCAF impairs endometrial receptivity and embryo implantation by down-regulating beta3-integrin expression via HOXA10 acetylation.The Journal of Clinical Endocrinology and Metabolism,98,4417-4428.
Zou Y,Li Z,Zou Y,Hao H,Li N,Li Q.2018.An FBXO40,knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects.Biochemical&Biophysical Research Communications,498,940-945.
Bagot C N,Troy P J,Taylor H S.2000.Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation.Gene Therapy,7,1378-1384.
Blitek A,Kaczmarek M M,Kiewisz J,Ziecik A J.2010.Endometrial and conceptus expression of HoxA10,transforming growth factor beta1,leukemia inhibitory factor,and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus.Domestic Animal Endocrinology,38,222-234.
Campbell K H,Ritchie W A,Wilmut I.1993.Nuclearcytoplasmic interactions during the first cell cycle of nuclear transfer reconstructed bovine embryos:Implications for deoxyribonucleic acid replication and development.Biology of Reproduction,5,933-942.
Dalman A,Eftekhari-Yazdi P,Valojerdi M R,Shahverdi A,Gourabi H,Janzamin E,Fakheri R,Sadeghian F,Hasani F.2010.Synchronizing cell cycle of goat fibroblasts by serum starvation causes apoptosis.Reproduction in Domestic Animals,45,doi:10.1111/j.1439-0531.2009.01520.x
Denning C,Dickinson P,Burl S,Wylie D,Fletcher J,Clark A J.2001.Gene targeting in primary fetal fibroblasts from sheep and pig.Cloning and Stem Cells,3,221-231.
Ekici A B,Strissel P L,Oppelt P G,Renner S P,Brucker S,Beckmann M W,Strick R.2013.HOXA10 and HOXA13sequence variations in human female genital malformations including congenital absence of the uterus and vagina.Gene,518,267-272.
Goto Y,Hirayama M,Takeda K,Tukamoto N,Sakata O,Kaeriyama H,Geshi M.2013.Effect of synchronization of donor cells in early G1-phase using shake-off method on developmental potential of somatic cell nuclear transfer embryos in cattle.Animal Science Journal,84,592-599.
Hasegawa S,Harada K,Morokoshi Y,Tsukamoto S,Furukawa T,Saga T.2013.Growth retardation and hair loss in transgenic mice overexpressing human H-ferritin gene.Transgenic Research,22,651-658.
Ingham D J,Beer S,Money S,Hansen G.2001.Quantitative real-time PCR assay for determining transgene copy number in transformed plants.Bio Techniques,31,132-134.
Jiang Y,Yan G,Zhang H,Shan H,Kong C,Yan Q,Xue B,Diao Z,Hu Y,Sun H.2014.Activation of matrix metalloproteinase-26 by HOXA10 promotes embryo adhesion in vitro.Biochemical and Biophysical Research Communications,445,622-628.
Johnson R K,Nielsen M K,Casey D S.1999.Responses in ovulation rate,embryonal survival,and litter traits in swine to 14 generations of selection to increase litter size.Journal of Animal Science,77,541-557.
Kondo M,Hornung U,Nanda I,Imai S,Sasaki T,Shimizu A,Asakawa S,Hori H,Schmid M,Shimizu N,Schartl M.2006.Genomic organization of the sex-determining and adjacent regions of the sex chromosomes of medaka.Genome Research,16,815-826.
Lai L,Prather R S.2003.Production of cloned pigs by using somatic cells as donors.Cloning&Stem Cells,5,233-241.
Lambert J F,Benoit B O,Colvin G A,Carlson J,Delville Y,Quesenberry P J.2000.Quick sex determination of mouse fetuses.Journal of Neuroscience Methods,95,127-132.
Li S,Chen X,Fang Z,Shi J,Sheng H Z.2006.Rabbits generated from fibroblasts through nuclear transfer.Reproduction,131,1085-1090.
Lin R,Wu D,Zhao C,Chen S,Xiao Q,Li X,Zhao S.2016.Inducible overexpression of porcine homeobox A10 in the endometrium of transgenic mice.Journal of Integrative Agriculture,15,1338-1344.
Lin R,Zhao C,Jing L,Zhao S.2015.Identification of LncRNAs/mRNAs related to endometrium function regulated by Homeobox A10 in Ishikawa cells.Cell Biology International,39,842-851.
Linville R C,Pomp D,Johnson R K,Rothschild M F.2001.Candidate gene analysis for loci affecting litter size and ovulation rate in swine.Journal of Animal Science,79,60-67.
Liu W,Wu L H,Yue M,Nashun B,Tang H,Chen Y,Chen B,Yuan J,Xiao D,Gu W.2017.Generation of DKK1 transgenic Tibet minipigs by somatic cell nuclear transfer(SCNT).Oncotarget,8,74331-74339.
Marchal R,Vigneron C,Perreau C,Bali-Papp A,Mermillod P.2002.Effect of follicular size on meiotic and developmental competence of porcine oocytes.Theriogenology,57,1523-1532.
McCreath K J,Howcroft J,Campbell K H,Colman A,Schnieke A E,Kind A J.2000.Production of gene-targeted sheep by nuclear transfer from cultured somatic cells.Nature,405,1066-1069.
Onishi A,Iwamoto M,Akita T,Mikawa S,Takeda K,Awata T,Hanada H,Perry A C.2000.Pig cloning by microinjection of fetal fibroblast nuclei.Science,289,1188-1190.
Ponchel F,Toomes C,Bransfield K,Leong F T,Douglas S H,Field S L,Bell S M,Combaret V,Puisieux A,Mighell A J,Robinson P A,Inglehearn C F,Isaacs J D,Markham A F.2003.Real-time PCR based on SYBR-Green I fluorescence:an alternative to the TaqMan assay for a relative quantification of gene rearrangements,gene amplifications and micro gene deletions.BMC Biotechnology,3,doi:10.1186/1472-6750-3-18
Satokata I,Benson G,Maas R.1995.Sexually dimorphic sterility phenotypes in Hoxa10-deficient mice.Nature,374,460-463.
Taylor H S.2000.The role of HOX genes in the development and function of the female reproductive tract.Seminars in Reproductive Medicine,18,81-90.
Taylor H S,Bagot C,Kardana A,Olive D,Arici A.1999.HOXgene expression is altered in the endometrium of women with endometriosis.Human Reproduction,14,1328-1331.
Taylor H S,Vanden Heuvel G B,Igarashi P.1997.A conserved Hox axis in the mouse and human female reproductive system:Late establishment and persistent adult expression of the Hoxa cluster genes.Biology of Reproduction,57,1338-1345.
Team R.2013.R:A language and environment for statistical computing.Computing,1,12-21.
Wu D,Song D,Li X,Yu M,Li C,Zhao S.2013.Molecular characterization and identification of the E2/P4 response element in the porcine HOXA10 gene.Molecular and Cellular Biochemistry,374,213-222.
Wu Z,Xu Z,Zou X,Zeng F,Shi J,Liu D,Urschitz J,Moisyadi S,Li Z.2013.Pig transgenesis by piggy Bac transposition in combination with somatic cell nuclear transfer.Transgenic Research,22,1107-1118.
Xu B,Geerts D,Bu Z,Ai J,Jin L,Li Y,Zhang H,Zhu G.2014.Regulation of endometrial receptivity by the highly expressed HOXA9,HOXA11 and HOXD10 HOX-class homeobox genes.Human Reproduction,29,781-790.
Zhu L H,Sun L H,Hu Y L,Jiang Y,Liu H Y,Shen X Y,Jin X Y,Zhen X,Sun H X,Yan G J.2013.PCAF impairs endometrial receptivity and embryo implantation by down-regulating beta3-integrin expression via HOXA10 acetylation.The Journal of Clinical Endocrinology and Metabolism,98,4417-4428.
Zou Y,Li Z,Zou Y,Hao H,Li N,Li Q.2018.An FBXO40,knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects.Biochemical&Biophysical Research Communications,498,940-945.