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O▪95 Transfection of embryonic stem cells with EGFP and BDNF genes by electroporation method
Abstracts - 6th International Symposium on Preimplantation Genetics 2005
O 95 Transfection of embryonic stem cells with EGFP and BDNF genes by electroporation method Fathi F1, Altiraihi T1, Mowahedin M1, Javad Mowla SJ2 1Department of Anatomy; 2Department of Genetics, Faculty of Basic Sciences, Tarbiat Modarres University, Ale Ahmad Highway, Tehran, Iran Introduction: Labelling of embryonic stem (ES) cells in culture and using them in clinical applications requires some genetic manipulation of the cells. The aim of the present study was to transfect choriocapillary endothelial (CCE) ES cells by EGFP and BDNF genes. Materials and methods: For this purpose, pIRES2-EGFP and pcDNA3-hBDNF-v5 plasmids were used. The plasmids were propagated, purified and transfected into ES cells by means of electroporation. To confirm expression of the EGFP and BDNF genes, inverse fluorescence microscopy and reverse transcriptionpolymerase chain reaction were used, respectively. Expression of EGFP was confirmed by examining the transfected cells with fluorescence microscopy. For BDNF, total RNA extracted from stably transfected cells was evaluated quantitatively by spectrophotometry and qualitatively by agarose gel electrophoresis. mRNAs were reverse transcripted and BDNF cDNA amplified by specific primers. The products of PCR were separated and visualized on agarose gel electrophoresis. Results: Both techniques confirmed successful transfection of CCE ES cells by both plasmids. Conclusions: The data obtained indicate that electroporation is an efficient method for transfection of CCE ES cells. The data also show that the CCE cell line is an appropriate donor cell for cell-mediated gene transfer.
O 96 Factors affecting PGD patients consent to donate embryos to stem cell research Franklin S1, Roberts C2, Throsby K1, Braude P3, Shaw J3, Lashwood A3, Pickering S3 1The BIOS Centre, London School of Economics and Political Science, London; 2Department of Sociology, Lancaster University; 3Centre for PGD, Guy’s and St Thomas’ Hospitals, London, UK Introduction: Although an increasing amount of research has been focused on patient perceptions of embryo research, and couples’ attitudes toward donating embryos to research, significant gaps remain in the ability to characterize factors affecting consent. The distinctive ethical issues raised by embryo donation to stem cell research reinforce the importance of more detailed study of patient perceptions of consent and consent procedures. Materials and methods: Eighty-five questionnaires were completed by couples undergoing PGD at Guy’s and St Thomas’ before being asked to consider donating embryos to stem cell research. Results: Seventy per cent of couples were willing to donate, 22% were uncertain, and 8% were unwilling. Among those willing to consent, knowledge of stem cells had no predictive influence. Conclusions: SPSS analysis of correlations between factors
affecting consent identified specific attitudes toward the embryo to be more prominent predictors than religious identification, educational achievement, professional training, age, or ethnicity.
O 97 Manipulation of stem cell proliferation and lineage commitment: visualization of label-retaining cells in whole mounts of mouse epidermis Braun K Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK Mammalian epidermis is maintained by stem cells that have the ability to self-renew and generate daughter cells that undergo terminal differentiation along the lineages of the hair follicles, interfollicular epidermis and sebaceous gland. Since stem cells divide infrequently in adult mouse epidermis, it is believed they can be visualized as DNA label-retaining cells (LRC). LRC are few in number and thus hard to visualize in conventional histological sections. We have overcome this problem by developing a whole mount labelling method that can be used to examine LRC in mouse tail epidermis. We demonstrate that LRC are not confined to the hair follicle, but also lie in sebaceous glands and interfollicular epidermis. Hair follicle LRC are not restricted to the bulge, but reside throughout the permanent portion of the follicle in a region of high α6β4 integrin expression. A few LRC divide, but they are not significantly depleted by successive hair growth cycles. Finally, we demonstrate the effects on LRC and lineage commitment of activating c-Myc or of expressing N-terminally truncated Lef1 to block β-catenin signalling. c-Myc stimulates proliferation and sebocyte differentiation without depleting LRC, while ΔNLef1-induces transdifferentiation of hair follicles and causes loss of LRC without stem cell depletion. We conclude that LRC and stem cells are not synonymous.
O 98 Epigenetic mechanisms of mouse germ cell specification and reprogramming Surani A Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK Investigations on the mouse germ cell lineage could provide fundamental insights into the epigenetic mechanisms of cell fate determination and epigenetic reprogramming of the genome. Concerning specification of primordial germ cells (PGC), both germ cells and somatic cells originate from common pluripotent epiblast precursor cells in response to signalling molecules. In particular, the proximal epiblast cells respond to signalling molecules, including BMP4, to acquire germ cell competence. However, these germ cell competent cells are initially destined for a somatic mesoderm fate as judged by expression of genes such as Brachyury as well as Hox genes, including Evx1, Lim1 and Hoxb1. A critical event for PGC specification implicates an epigenetic mechanism that is necessary for wide-ranging repression of genes, including Hox genes, which ensures that PGC escape the fate of
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O 95 Transfection of embryonic stem cells with EGFP and BDNF genes by electroporation method Fathi F1, Altiraihi T1, Mowahedin M1, Javad Mowla SJ2 1Department of Anatomy; 2Department of Genetics, Faculty of Basic Sciences, Tarbiat Modarres University, Ale Ahmad Highway, Tehran, Iran Introduction: Labelling of embryonic stem (ES) cells in culture and using them in clinical applications requires some genetic manipulation of the cells. The aim of the present study was to transfect choriocapillary endothelial (CCE) ES cells by EGFP and BDNF genes. Materials and methods: For this purpose, pIRES2-EGFP and pcDNA3-hBDNF-v5 plasmids were used. The plasmids were propagated, purified and transfected into ES cells by means of electroporation. To confirm expression of the EGFP and BDNF genes, inverse fluorescence microscopy and reverse transcriptionpolymerase chain reaction were used, respectively. Expression of EGFP was confirmed by examining the transfected cells with fluorescence microscopy. For BDNF, total RNA extracted from stably transfected cells was evaluated quantitatively by spectrophotometry and qualitatively by agarose gel electrophoresis. mRNAs were reverse transcripted and BDNF cDNA amplified by specific primers. The products of PCR were separated and visualized on agarose gel electrophoresis. Results: Both techniques confirmed successful transfection of CCE ES cells by both plasmids. Conclusions: The data obtained indicate that electroporation is an efficient method for transfection of CCE ES cells. The data also show that the CCE cell line is an appropriate donor cell for cell-mediated gene transfer.
O 96 Factors affecting PGD patients consent to donate embryos to stem cell research Franklin S1, Roberts C2, Throsby K1, Braude P3, Shaw J3, Lashwood A3, Pickering S3 1The BIOS Centre, London School of Economics and Political Science, London; 2Department of Sociology, Lancaster University; 3Centre for PGD, Guy’s and St Thomas’ Hospitals, London, UK Introduction: Although an increasing amount of research has been focused on patient perceptions of embryo research, and couples’ attitudes toward donating embryos to research, significant gaps remain in the ability to characterize factors affecting consent. The distinctive ethical issues raised by embryo donation to stem cell research reinforce the importance of more detailed study of patient perceptions of consent and consent procedures. Materials and methods: Eighty-five questionnaires were completed by couples undergoing PGD at Guy’s and St Thomas’ before being asked to consider donating embryos to stem cell research. Results: Seventy per cent of couples were willing to donate, 22% were uncertain, and 8% were unwilling. Among those willing to consent, knowledge of stem cells had no predictive influence. Conclusions: SPSS analysis of correlations between factors
affecting consent identified specific attitudes toward the embryo to be more prominent predictors than religious identification, educational achievement, professional training, age, or ethnicity.
O 97 Manipulation of stem cell proliferation and lineage commitment: visualization of label-retaining cells in whole mounts of mouse epidermis Braun K Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK Mammalian epidermis is maintained by stem cells that have the ability to self-renew and generate daughter cells that undergo terminal differentiation along the lineages of the hair follicles, interfollicular epidermis and sebaceous gland. Since stem cells divide infrequently in adult mouse epidermis, it is believed they can be visualized as DNA label-retaining cells (LRC). LRC are few in number and thus hard to visualize in conventional histological sections. We have overcome this problem by developing a whole mount labelling method that can be used to examine LRC in mouse tail epidermis. We demonstrate that LRC are not confined to the hair follicle, but also lie in sebaceous glands and interfollicular epidermis. Hair follicle LRC are not restricted to the bulge, but reside throughout the permanent portion of the follicle in a region of high α6β4 integrin expression. A few LRC divide, but they are not significantly depleted by successive hair growth cycles. Finally, we demonstrate the effects on LRC and lineage commitment of activating c-Myc or of expressing N-terminally truncated Lef1 to block β-catenin signalling. c-Myc stimulates proliferation and sebocyte differentiation without depleting LRC, while ΔNLef1-induces transdifferentiation of hair follicles and causes loss of LRC without stem cell depletion. We conclude that LRC and stem cells are not synonymous.
O 98 Epigenetic mechanisms of mouse germ cell specification and reprogramming Surani A Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK Investigations on the mouse germ cell lineage could provide fundamental insights into the epigenetic mechanisms of cell fate determination and epigenetic reprogramming of the genome. Concerning specification of primordial germ cells (PGC), both germ cells and somatic cells originate from common pluripotent epiblast precursor cells in response to signalling molecules. In particular, the proximal epiblast cells respond to signalling molecules, including BMP4, to acquire germ cell competence. However, these germ cell competent cells are initially destined for a somatic mesoderm fate as judged by expression of genes such as Brachyury as well as Hox genes, including Evx1, Lim1 and Hoxb1. A critical event for PGC specification implicates an epigenetic mechanism that is necessary for wide-ranging repression of genes, including Hox genes, which ensures that PGC escape the fate of
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