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1. Rapid Irreversible Transcriptional Reprogramming in Human Stem Cells Accompanied by Discordance between Replication Timing and Chromatin Compartment

Author

Dileep, Vishnu, Wilson, Korey A., Marchal, Claire, Lyu, Xiaowen, Zhao, Peiyao A., Li, Ben, Poulet, Axel, Bartlett, Daniel A., Rivera-Mulia, Juan Carlos, Qin, Zhaohui S., Robins, Allan J., Schulz, Thomas C., Kulik, Michael J., McCord, Rachel Patton, Dekker, Job, Dalton, Stephen, Corces, Victor G., and Gilbert, David M.

Published
2019
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3. Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

Author

Wang, Linlin, Schulz, Thomas C., Sherrer, Eric S., Dauphin, Derek S., Shin, Soojung, Nelson, Angelique M., Ware, Carol B., Zhan, Mei, Song, Chao-Zhong, Chen, Xiaoji, Brimble, Sandii N., McLean, Amanda, Galeano, Maria J., Uhl, Elizabeth W., D'Amour, Kevin A., Chesnut, Jonathan D., Rao, Mahendra S., Blau, C. Anthony, and Robins, Allan J.

Published
2007
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5. Evidence for involvement of the carboxy terminus of helix 1 of growth hormone in receptor binding: use of charge reversal mutagenesis to account for calcium dependence of binding and for design of higher affinity analogues

Author

Rowlinson, Scott W., Barnard, Ross, Bastiras, Stan, Robins, Allan J., Seen, Carol, Wells, Julian R.E., Brinkworth, Ross, and Waters, Michael J.

Subjects
Somatotropin -- Receptors, Cooperative binding (Biochemistry) -- Analysis, Calcium in the body -- Analysis, Biological sciences, Chemistry
Abstract

Examination of charge reversal analogues of the porcine growth hormone (pGH) reveals the involvement of carboxy terminals of helix 1 of pGH in receptor binding. Equilibrium dialysis and Scatchard analysis of Ca2+ binding with charge reversal mutants and wild-type pGH show that Ca2+ dependence of binding between primate and nonprimate GHs depends on the positions E34 and D170. Charge reversal mutagenesis helps design higher affinity GH analogues.

Published
1994

7. A large-scale proteomic analysis of human embryonic stem cells

Author

Sherrer Eric, Brimble Sandii N, Palmarini Gail, Swistowski Andrzej, Liu Ying, Swistowska Anna, Schulz Thomas C, Robins Allan J, Rao Mahendra S, and Zeng Xianmin

Subjects
Biotechnology, TP248.13-248.65, Genetics, QH426-470
Abstract

Abstract Background Much of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs) is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells. Results Here we describe the use of two large-scale western blot assays to identify over 600 proteins expressed in undifferentiated hESCs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and/or post-translational modifications. Twenty-two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hESCs were also identified. We confirmed the expression of a subset of the identified proteins by immunofluorescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hESCs. These analyses also indicated that hESCs exhibit several features of polarized epithelia, including expression of tight junction proteins. Conclusion Our approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self-renewal.

Published
2007
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9. Human embryonic stem cells have a unique epigenetic signature

Author

Bibikova, Marina, Chudin, Eugene, Bonnie Wu, Lixin Zhou, Garcia, Eliza Wickham, Ying Liu, Soojung Shin, Plaia, Todd W., Auerbach, Jonathan M., Arking, Dan E., Gonzalez, Rodolfo, Crook, Jeremy, Davidson, Bruce, Schulz, Thomas C., Robins, Allan, Khanna, Aparna, Sartipy, Peter, Hyllner, Johan, Vanguri, Padmavathy, Savant-Bhonsale, Smita, Smith, Alan K., Chakravarti, Aravinda, Maitra, Anirban, Rao, Mahendra, Barker, David L., Loring, Jeanne F., and Jian-Bing Fan

Subjects
Cell differentiation -- Analysis, DNA -- Chemical properties, Embryonic stem cells -- Genetic aspects, Epigenetic inheritance -- Research, Methylation -- Analysis, Health
Published
2006

10. Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells

Author

Rivera-Mulia, Juan Carlos, primary, Buckley, Quinton, additional, Sasaki, Takayo, additional, Zimmerman, Jared, additional, Didier, Ruth A., additional, Nazor, Kristopher, additional, Loring, Jeanne F., additional, Lian, Zheng, additional, Weissman, Sherman, additional, Robins, Allan J., additional, Schulz, Thomas C., additional, Menendez, Laura, additional, Kulik, Michael J., additional, Dalton, Stephen, additional, Gabr, Haitham, additional, Kahveci, Tamer, additional, and Gilbert, David M., additional

Published
2015
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13. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

Author

Adewumi, Oluseun, Aflatoonian, Behrouz, Ahrlund-Richter, Lars, Amit, Michal, Andrews, Peter W., Beighton, Gemma, Bello, Paul A., Benvenisty, Nissim, Berry, Lorraine S., Bevan, Simon, Blum, Barak, Brooking, Justin, Chen, Kevin G., Choo, Andre B. H., Churchill, Gary A., Corbel, Marie, Damjanov, Ivan, Draper, Jon S., Dvorak, Petr, Emanuelsson, Katarina, Fleck, Roland A., Ford, Angela, Gertow, Karin, Gertsenstein, Marina, Gokhale, Paul J., Hamilton, Rebecca S., Hampl, Ales, Healy, Lyn E., Hovatta, Outi, Hyllner, Johan, Imreh, Marta P., Itskovitz-Eldor, Joseph, Jackson, Jamie, Johnson, Jacqueline L., Jones, Mark, Kee, Kehkooi, King, Benjamin L., Knowles, Barbara B., Lako, Majlinda, Lebrin, Franck, Mallon, Barbara S., Manning, Daisy, Mayshar, Yoav, Mckay, Ronald D. G., Michalska, Anna E., Mikkola, Milla, Mileikovsky, Masha, Minger, Stephen L., Moore, Harry D., Mummery, Christine L., Nagy, Andras, Nakatsuji, Norio, O'Brien, Carmel M., Oh, Steve K. W., Olsson, Cia, Otonkoski, Timo, Park, Kye-Yoon, Passier, Robert, Patel, Hema, Patel, Minal, Pedersen, Roger, Pera, Martin F., Piekarczyk, Marian S., Pera, Renee A. Reijo, Reubinoff, Benjamin E., Robins, Allan J., Rossant, Janet, Rugg-Gunn, Peter, Schulz, Thomas C., Semb, Henrik, Sherrer, Eric S., Siemen, Henrike, Stacey, Glyn N., Stojkovic, Miodrag, Suemori, Hirofumi, Szatkiewicz, Jin, Turetsky, Tikva, Tuuri, Timo, van den Brink, Steineke, Vintersten, Kristina, Vuoristo, Sanna, Ward, Dorien, Weaver, Thomas A., Young, Lesley A., Zhang, Weidong, Adewumi, Oluseun, Aflatoonian, Behrouz, Ahrlund-Richter, Lars, Amit, Michal, Andrews, Peter W., Beighton, Gemma, Bello, Paul A., Benvenisty, Nissim, Berry, Lorraine S., Bevan, Simon, Blum, Barak, Brooking, Justin, Chen, Kevin G., Choo, Andre B. H., Churchill, Gary A., Corbel, Marie, Damjanov, Ivan, Draper, Jon S., Dvorak, Petr, Emanuelsson, Katarina, Fleck, Roland A., Ford, Angela, Gertow, Karin, Gertsenstein, Marina, Gokhale, Paul J., Hamilton, Rebecca S., Hampl, Ales, Healy, Lyn E., Hovatta, Outi, Hyllner, Johan, Imreh, Marta P., Itskovitz-Eldor, Joseph, Jackson, Jamie, Johnson, Jacqueline L., Jones, Mark, Kee, Kehkooi, King, Benjamin L., Knowles, Barbara B., Lako, Majlinda, Lebrin, Franck, Mallon, Barbara S., Manning, Daisy, Mayshar, Yoav, Mckay, Ronald D. G., Michalska, Anna E., Mikkola, Milla, Mileikovsky, Masha, Minger, Stephen L., Moore, Harry D., Mummery, Christine L., Nagy, Andras, Nakatsuji, Norio, O'Brien, Carmel M., Oh, Steve K. W., Olsson, Cia, Otonkoski, Timo, Park, Kye-Yoon, Passier, Robert, Patel, Hema, Patel, Minal, Pedersen, Roger, Pera, Martin F., Piekarczyk, Marian S., Pera, Renee A. Reijo, Reubinoff, Benjamin E., Robins, Allan J., Rossant, Janet, Rugg-Gunn, Peter, Schulz, Thomas C., Semb, Henrik, Sherrer, Eric S., Siemen, Henrike, Stacey, Glyn N., Stojkovic, Miodrag, Suemori, Hirofumi, Szatkiewicz, Jin, Turetsky, Tikva, Tuuri, Timo, van den Brink, Steineke, Vintersten, Kristina, Vuoristo, Sanna, Ward, Dorien, Weaver, Thomas A., Young, Lesley A., and Zhang, Weidong

Abstract

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue- nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Published
2007
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17. A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

Author

Schulz, Thomas C., primary, Young, Holly Y., additional, Agulnick, Alan D., additional, Babin, M. Josephine, additional, Baetge, Emmanuel E., additional, Bang, Anne G., additional, Bhoumik, Anindita, additional, Cepa, Igor, additional, Cesario, Rosemary M., additional, Haakmeester, Carl, additional, Kadoya, Kuniko, additional, Kelly, Jonathan R., additional, Kerr, Justin, additional, Martinson, Laura A., additional, McLean, Amanda B., additional, Moorman, Mark A., additional, Payne, Janice K., additional, Richardson, Mike, additional, Ross, Kelly G., additional, Sherrer, Eric S., additional, Song, Xuehong, additional, Wilson, Alistair Z., additional, Brandon, Eugene P., additional, Green, Chad E., additional, Kroon, Evert J., additional, Kelly, Olivia G., additional, D’Amour, Kevin A., additional, and Robins, Allan J., additional

Published
2012
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21. A large-scale proteomic analysis of human embryonic stem cells

Author

Schulz, Thomas C, primary, Swistowska, Anna Maria, additional, Liu, Ying, additional, Swistowski, Andrzej, additional, Palmarini, Gail, additional, Brimble, Sandii N, additional, Sherrer, Eric, additional, Robins, Allan J, additional, Rao, Mahendra S, additional, and Zeng, Xianmin, additional

Published
2007
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23. An Efficient Method for Producing α(1,3)-Galactosyltransferase Gene Knockout Pigs

Author

Harrison, Sharon, primary, Boquest, Andrew, additional, Grupen, Christopher, additional, Faast, Renate, additional, Guildolin, Angelo, additional, Giannakis, Christopher, additional, Crocker, Lesley, additional, McIlfatrick, Stephen, additional, Ashman, Rodney, additional, Wengle, James, additional, Lyons, Ian, additional, Tolstoshev, Paul, additional, Cowan, Peter, additional, Robins, Allan, additional, O'Connell, Philip, additional, D'Apice, Anthony J.F., additional, and Nottle, Mark, additional

Published
2004
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24. Karyotypic Stability, Genotyping, Differentiation, Feeder-Free Maintenance, and Gene Expression Sampling in Three Human Embryonic Stem Cell Lines Derived Prior to August 9, 2001

Author

Brimble, Sandii N., primary, Zeng, Xianmin, additional, Weiler, Deborah A., additional, Luo, Yongquan, additional, Liu, Ying, additional, Lyons, Ian G., additional, Freed, William J., additional, Robins, Allan J., additional, Rao, Mahendra S., additional, and Schulz, Thomas C., additional

Published
2004
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25. Human Embryonic Stem Cell Lines Derived from Discarded Embryos

Author

Mitalipova, Maisam, primary, Calhoun, John, additional, Shin, Soojung, additional, Wininger, David, additional, Schulz, Thomas, additional, Noggle, Scott, additional, Venable, Alison, additional, Lyons, Ian, additional, Robins, Allan, additional, and Stice, Steven, additional

Published
2003
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27. THE ??-1,3-GALACTOSYLTRANSFERASE KNOCKOUT MOUSE

Author

Tearle, Rick G., primary, Tange, Margaret J., additional, Zannettino, Zara L., additional, Katerelos, Marina, additional, Shinkel, Trixie A., additional, Van Denderen, Bryce J.W., additional, Lonie, Andrew J., additional, Lyons, Ian, additional, Nottle, Mark B., additional, Cox, Timothy, additional, Becker, Christiane, additional, Peura, Anita M., additional, Wigley, Peter L., additional, Crawford, Robert J., additional, Robins, Allan J., additional, Pearse, Martin J., additional, and d'Apice, Anthony J.F., additional

Published
1996
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32. The Cell Surface Glycosphingolipids SSEA-3 and SSEA-4 Are Not Essential for Human ESC Pluripotency.

Author

BRIMBLE, SANDII N., SHERRER, ERIC S., UHL, ELIZABETH W., WANG, ELAINE, KELLY, SAMUEL, MERRILL JR., ALFRED H., ROBINS, ALLAN J., and SCHULZ, THOMAS C.

Subjects
GLYCOSPHINGOLIPIDS, EMBRYONIC stem cells, ANTIGENS, CELL differentiation, STEM cells
Abstract

The article examines the role of cell surface glycosphingolipids stage-specific embryonic antigen 3 (SSEA-3) and SSEA-4 in human embryonic stem cell (hESC) pluripotency. The results demonstrated that glycosphingolipids do not play a critical functional role in maintaining the pluripotency of hESC but suggested roles for the molecules during cellular differentiation.

Published
2007
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33. BG01V: A variant human embryonic stem cell line which exhibits rapid growth after passaging and reliable dopaminergic differentiation.

Author

Zeng, Xianmin, Chen, Jia, Liu, Ying, Luo, Yongquan, Schulz, Thomas C., Robins, Allan J., Rao, Mahendra S., and Freed, William J.

Subjects
EMBRYONIC stem cells, CELL lines, IMMUNOCYTOCHEMISTRY, CELL culture, DOPAMINERGIC mechanisms, STEM cells
Abstract

Purpose: To explore a karyotypically abnormal variant human embryonic stem cell (hESC) line, BG01V, as a potential model for studies of dopaminergic neuronal differentiation. Methods: The properties of BG01V cells were compared to those of normal BG01 cells using immunocytochemistry, RT-PCR, focused microarrays and in vitro differentiation, including dopaminergic differentiation, by culturing with the stromal cell line PA6. Results: Despite the karyotypic abnormality (49, +12, +17 and XXY), undifferentiated BG01V cells expressed pluripotent ESC markers similar to BG01 cells, and retained the ability to differentiate into cell types characteristic of all three germ layers. When co-cultured with the stromal cell line PA6, BG01V cells differentiated into dopaminergic cells which exhibited properties similar to those of mature dopaminergic neurons. Conclusions: BG01V cells were easier to maintain in culture than karyotypically normal BG01 cells and can be used as an alternative pluripotent hESC type for in vitro developmental studies. [ABSTRACT FROM AUTHOR]

Published
2004
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34. Bicyclic C21Terpenoids from the Marine Sponge Clathria compressa

Author

Gupta, Prasoon, Sharma, Upasana, Schulz, Thomas C., McLean, Amanda B., Robins, Allan J., and West, Lyndon M.

Abstract

Three new bicyclic C21terpenoids, clathric acid (1) and two N-acyl taurine derivatives, clathrimides A (2) and B (3), were isolated from the marine sponge Clathria compressa. The structures of these compounds were elucidated by interpretation of spectroscopic data. Clathric acid showed mild antibacterial activity against several Gram-positive bacteria.

Published
2012
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37. THE α-1,3-GALACTOSYLTRANSFERASE KNOCKOUT MOUSE.

Author

Tearle, Rick G., Tange, Margaret J., Zannettino, Zara L., Katerelos, Marina, Shinkel, Trixie A., Van Denderen, Bryce J.w., Lonie, Andrew J., Lyons, Ian, Nottle, Mark B., Cox, Timothy, Becker, Christiane, Peura, Anita M., Wigley, Peter L., Crawford, Robert J., Robins, Allan J., Pearse, Martin J., and D'apice, Anthony J.f.

Published
1996
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38. Non–Radioactive Photobiotin–Labeled Probes Detect Single Copy Genes and Low Abundance mRNA

Author

McInnes, James L., Dalton, Stephen, Vize, Peter D., and Robins, Allan J.

Abstract

We show that the non–radioactive reagent, photobiotin, can replace the use of radioactivity in preparing gene probes for Southern, Northern and DNA dot–blot analyses. Using a photobiotin–labeled probe, the single copy human metallothionein IIA (MT IIA) gene is readily detected in genomic DNA by Southern transfer. Similarly, low abundance chicken histone H5 mRNA is easily detected in total cytoplasmic RNA isolated from an erythroleukemic cell line by Northern analysis. Finally, a photobiotin–labeled fragment of the pig growth hormone gene is used to detect the presence of this gene in transgenic mice by dot–blot analysis.

Published
1987
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41. A spiral bridge

Author

Robins, Allan

Subjects
fictocriticism, allegory, literary and cultural theory, creative artefact and exegesis, teaching literature and writing
Abstract

Commencing tertiary students in writing and communications programs often struggle to absorb literary and cultural theory as it is presented in existing texts. Finding alternate strategies for presenting such material might prove productive. One possible alternate strategy evolved from the preparation of the theoretical component of my PhD thesis, during which I realised that I was telling a story not only about the object of analysis (the creative artefact), but also about the very selection and synthesis of theory for my epistemological apparatus. Forming the view that the discursive and experiential composition of a writing subject is central not only to literary practice but also to critical and theoretical practice, I recognised this as one of the defining attributes of a fictocritical approach, which validates exploration, construction and application of literary theory by using the textual strategies, traditions and conventions of literature itself, so that theory might ‘don the clothing’ of literature and ‘walk about in it’, much as an actor does to understand and interpret a character for an audience. Thus writing literary and cultural theory into a narrative might prove useful for commencing tertiary students,who are likely to be familiar with literary strategies and conventions. Refereed/Peer-reviewed

Published
2010

42. Representing Indigeneity: a reflection on motivation and issues

Author

Robins, Allan John

Subjects
representation, postcolonialism, Indigeneity, children's literature
Abstract

Non-Indigenous Australian authors representing Indigeneity in their work need to address a number of issues, including the fundamental question of whether to do so. Having incorporated representations of Indigeneity in a children's novel written as part of my PhD thesis, I traced the origins and evolution of my representational practices in the novel through the other component of the thesis, a multi-genre exegesis. The processes of writing both components raised a range of issues, including questions of motivation; my right, responsibility and competence to represent; and the strategies employed in both components to address some of these questions. In this paper I reflect on these issues, and conclude that my efforts have been unavoidably far from perfect, yet worthwhile as a stage in ongoing negotiations of meaning and power between non-Indigenous and Indigenous cultures in Australia, and that non-Indigenous writers like myself need to be conscious that our efforts are never, and can never be, purely benevolent and/or selfless. Refereed/Peer-reviewed

Published
2008

43. The spiral travelled: an exegesis with accompanying novel, The diary of Jeremy Prior

Author

Robins, Allan, University of South Australia Centre for Professional and Public Communication, University of South Australia School of Communication, Woods, Claire, and Page, Sue

Subjects
fictocriticism, representation, indigeneity, 420299 Literature Studies not elsewhere classified, children's literature
Abstract

PhD Doctorate The thesis focuses on representations of Indigeneity by non-Indigenous writers and in particular the author's practice in the writing of a children's novel, in which the relationship between non-Indigenous and Indigenous characters is represented as part of a colonial and post-colonial, contemporary world. The exegesis takes an experimental approach to representing theory in a fictocritical, multi-genre form.

Published
2007

44. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.

Author

Adewumi O, Aflatoonian B, Ahrlund-Richter L, Amit M, Andrews PW, Beighton G, Bello PA, Benvenisty N, Berry LS, Bevan S, Blum B, Brooking J, Chen KG, Choo AB, Churchill GA, Corbel M, Damjanov I, Draper JS, Dvorak P, Emanuelsson K, Fleck RA, Ford A, Gertow K, Gertsenstein M, Gokhale PJ, Hamilton RS, Hampl A, Healy LE, Hovatta O, Hyllner J, Imreh MP, Itskovitz-Eldor J, Jackson J, Johnson JL, Jones M, Kee K, King BL, Knowles BB, Lako M, Lebrin F, Mallon BS, Manning D, Mayshar Y, McKay RD, Michalska AE, Mikkola M, Mileikovsky M, Minger SL, Moore HD, Mummery CL, Nagy A, Nakatsuji N, O'Brien CM, Oh SK, Olsson C, Otonkoski T, Park KY, Passier R, Patel H, Patel M, Pedersen R, Pera MF, Piekarczyk MS, Pera RA, Reubinoff BE, Robins AJ, Rossant J, Rugg-Gunn P, Schulz TC, Semb H, Sherrer ES, Siemen H, Stacey GN, Stojkovic M, Suemori H, Szatkiewicz J, Turetsky T, Tuuri T, van den Brink S, Vintersten K, Vuoristo S, Ward D, Weaver TA, Young LA, and Zhang W

Subjects
Alkaline Phosphatase metabolism, Antigens, CD biosynthesis, Biotechnology methods, Cell Differentiation, Cell Lineage, Cell Membrane metabolism, Cells, Cultured, Cluster Analysis, Female, Gene Expression Profiling, Genotype, Glycolipids chemistry, Humans, Membrane Glycoproteins biosynthesis, Tetraspanin 29, Embryonic Stem Cells cytology, Gene Expression Regulation, Developmental
Abstract

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Published
2007
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45. An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs.

Author

Harrison S, Boquest A, Grupen C, Faast R, Guildolin A, Giannakis C, Crocker L, McIlfatrick S, Ashman R, Wengle J, Lyons I, Tolstoshev P, Cowan P, Robins A, O'Connell P, D'Apice AJ, and Nottle M

Subjects
Animals, Animals, Genetically Modified genetics, Female, Pregnancy, Fibroblasts, Galactosyltransferases genetics, Gene Deletion, Nuclear Transfer Techniques, Oocytes, Swine genetics
Abstract

We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.

Published
2004
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