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11.004 Pregnancy of transgenic-clone goat using mammary gland epithelial cells as donor cells
Abstracts - PGDIS: 8th International Symposium on PGD
11.004 Pregnancy of transgenic-clone goat using mammary gland epithelial cells as donor cells Liu LJ1, Ling Zhang Y1, Zhang Y2 1College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, Henan, 471003, China; 2Institute of Biotechnology, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi, 712100, China Objective: Mammary gland epithelial cells (MGEC) have many merits compared with other cell types on making mammary gland bioreactors by nuclear transfer (NT). However, their properties of short cell lifespan and high differentiation level greatly limit their application in cloning and producing transgenic animals. This study was designed to investigate the feasibility of production of transgenic animals using MGEC as donor cells. Materials/Methods: The isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. Next, 96-well plates were used to isolate the cell lineages from a single goat’s MGEC transfected with the pBLM-C1 plasmid. The conditioned medium was evaluated to make a single MGEC grow better. The neo gene was detected in isolated colonies. PCR assays were performed to detect the transfected fragment in positive cells. The isolated cell lineages were induced by hormones (5 Mg/ml each of prolactin, insulin, and hydrocortisone) to express human lactoferrin gene. The supernatant was collected, then enzyme-linked immunosorbent assay (ELISA) was carried out to test the expression of the target gene in the isolated cell lineages. The cell lineages of high expression were chosen to use for NT. Reconstructed karyoplast–cytoplast couplets were fused, activated and cultured in vitro. Partially cloned embryos were used for the detection of foreign genes and the remaining 401 cloned embryos were transferred into 43 synchronized recipients. All percentage data were tested by chi-squared analysis, and differences were considered significant at P < 0.05. Results: When single transfected cells were cultured in 100%, 50% and 0% conditioned medium, respectively, the colony rate in 100% group was significantly increased (13.6% versus 4.4%, 0%; P < 0.05). The co-culture of transgenic cells with non-transgenic cells markedly improved the colony rate after multiplication (33.3% versus 6.7%; P < 0.05). ELISA analysis showed that the exogenous gene expression level was variable from 0 to 60 mg/l in different cell lineages. Eleven recipients (pregnancy rate 25.6%) were confirmed to be pregnant on day 30–45 by ultrasonography. Regretfully, all of gravid goats aborted within the first 3 months of gestation. Conclusion: This study provided a reliable method for isolating transfected MGEC lineages for NT. The time and the cost of transgenic livestock production were greatly reduced by choosing the cell lineage of high expression in the cell level. The embryos derived from the transgenic MGEC could establish early pregnancies but did not go to term and further investigation is warranted. 14.002 Oligohydramnios diagnosis, treatment and consequences in term and post-term pregnancies Brandi A, Pomini, F Catholic University of S. Cuore of Rome, Rome, Italy Introduction: Oligohydramnios in term and post-term pregnancy is associated with an increasing risk of fetal distress in labour and Caesarean section. Maternal hydration with oral
water or intravenous hypo-osmotic solution has been shown to raise low AFI values. It is not known if a raised AFI can be maintained by daily hydration in order to obtain an effective labour induction. Aims of this study in term and post-term pregnancies with oligohydramnios (<5oPc) was to ascertain if combined daily oral and intravenous hyposmotic hydration raise AFI values at 24, 48 and 72 h compared with basal values and to non-hydrated similar controls. Materials/Methods: We excluded from the study patients with prelabour rupture of membranes, contraindications to vaginal birth, fetal anomalies and threatened labour. There were 72 women included in the study: 34 hydrated at term; 23 hydrated post-term; nine term controls; and six post-term controls. Hydrated pregnant women drank 3 l of water daily and were treated daily with intravenous infusion of 2 l of 5% glucose (278 mOsm/l). Results: In term pregnancies, AFI raised significantly from 57.9 ± 14.6 to 81.8 ± 30.4 at 24 h, 69.1± 27.4 at 48 h and 86.7 ± 39.3 at 72 h; all time values are significantly different from non-hydrated controls. In post-term pregnancies, AFI raised non significantly from 55.3 ± 19.4 to 61.9 ± 24.7 at 24 h, 72.5 ± 31.5 at 48 h and 51.5 ± 15.9 at 72 h. Maternal daily hydration raise low AFI values in term pregnancies. Post-term pregnancies show high variability. Conclusion: Further prospective study is necessary to assess the value of maternal hydration to improve efficacy of labour induction protocols. 15.001 Blastocyst biopsy in PGD practice: laboratory considerations Traversa M, Marshall JT, de Boer KA, Leigh DA, McArthur SJ Sydney IVF, Sydney, NSW, Australia Objective: Preimplantation genetic diagnosis (PGD) is typically done using blastomere biopsy where a single cell(s) is removed on day 3 post-fertilization from typically 6–8-cell embryos. An alternative method is blastocyst biopsy, which involves the removal of approximately 3–6 trophectoderm cells from a day-5 embryo containing approximately 100 cells. For PGD centres considering a move to blastocyst biopsy, we provide some insights based on our experiences since its routine implementation in our PGD laboratory in 2003. Materials/Methods: Progression of testing requirements for blastocyst biopsy was compared with current practice for blastomere biopsy. A contrast of the biopsy techniques, sample processing, types of testing and impact on laboratory resources is discussed. Results: IVF laboratories must account for extra time needed in patient management and extended incubation requirements. Blastocyst biopsy is performed at a later stage of embryo development (day 5) and results in a larger amount of genetic material available for analysis (average 3–6 trophectoderm cells). Techniques suitable for hatching as well as fully hatched embryos are available and work effectively. For the analysis laboratory, however, the biggest impact is on the amount of time available for genetic analysis (typically 4–6 h post biopsy for same day transfers but longer for day-6 transfer following biopsy on day 5). Test design can accommodate these time constraints and our own results suggest there is little loss of embryo viability from day 5 to day 6. Embryos do not all develop at the same rate and several rounds of biopsy may be required to produce all results. We feel this biopsy-when–ready approach is less harmful to embryos and typically the desired embryo is analysed before
S-55 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008
11.004 Pregnancy of transgenic-clone goat using mammary gland epithelial cells as donor cells Liu LJ1, Ling Zhang Y1, Zhang Y2 1College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, Henan, 471003, China; 2Institute of Biotechnology, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi, 712100, China Objective: Mammary gland epithelial cells (MGEC) have many merits compared with other cell types on making mammary gland bioreactors by nuclear transfer (NT). However, their properties of short cell lifespan and high differentiation level greatly limit their application in cloning and producing transgenic animals. This study was designed to investigate the feasibility of production of transgenic animals using MGEC as donor cells. Materials/Methods: The isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. Next, 96-well plates were used to isolate the cell lineages from a single goat’s MGEC transfected with the pBLM-C1 plasmid. The conditioned medium was evaluated to make a single MGEC grow better. The neo gene was detected in isolated colonies. PCR assays were performed to detect the transfected fragment in positive cells. The isolated cell lineages were induced by hormones (5 Mg/ml each of prolactin, insulin, and hydrocortisone) to express human lactoferrin gene. The supernatant was collected, then enzyme-linked immunosorbent assay (ELISA) was carried out to test the expression of the target gene in the isolated cell lineages. The cell lineages of high expression were chosen to use for NT. Reconstructed karyoplast–cytoplast couplets were fused, activated and cultured in vitro. Partially cloned embryos were used for the detection of foreign genes and the remaining 401 cloned embryos were transferred into 43 synchronized recipients. All percentage data were tested by chi-squared analysis, and differences were considered significant at P < 0.05. Results: When single transfected cells were cultured in 100%, 50% and 0% conditioned medium, respectively, the colony rate in 100% group was significantly increased (13.6% versus 4.4%, 0%; P < 0.05). The co-culture of transgenic cells with non-transgenic cells markedly improved the colony rate after multiplication (33.3% versus 6.7%; P < 0.05). ELISA analysis showed that the exogenous gene expression level was variable from 0 to 60 mg/l in different cell lineages. Eleven recipients (pregnancy rate 25.6%) were confirmed to be pregnant on day 30–45 by ultrasonography. Regretfully, all of gravid goats aborted within the first 3 months of gestation. Conclusion: This study provided a reliable method for isolating transfected MGEC lineages for NT. The time and the cost of transgenic livestock production were greatly reduced by choosing the cell lineage of high expression in the cell level. The embryos derived from the transgenic MGEC could establish early pregnancies but did not go to term and further investigation is warranted. 14.002 Oligohydramnios diagnosis, treatment and consequences in term and post-term pregnancies Brandi A, Pomini, F Catholic University of S. Cuore of Rome, Rome, Italy Introduction: Oligohydramnios in term and post-term pregnancy is associated with an increasing risk of fetal distress in labour and Caesarean section. Maternal hydration with oral
water or intravenous hypo-osmotic solution has been shown to raise low AFI values. It is not known if a raised AFI can be maintained by daily hydration in order to obtain an effective labour induction. Aims of this study in term and post-term pregnancies with oligohydramnios (<5oPc) was to ascertain if combined daily oral and intravenous hyposmotic hydration raise AFI values at 24, 48 and 72 h compared with basal values and to non-hydrated similar controls. Materials/Methods: We excluded from the study patients with prelabour rupture of membranes, contraindications to vaginal birth, fetal anomalies and threatened labour. There were 72 women included in the study: 34 hydrated at term; 23 hydrated post-term; nine term controls; and six post-term controls. Hydrated pregnant women drank 3 l of water daily and were treated daily with intravenous infusion of 2 l of 5% glucose (278 mOsm/l). Results: In term pregnancies, AFI raised significantly from 57.9 ± 14.6 to 81.8 ± 30.4 at 24 h, 69.1± 27.4 at 48 h and 86.7 ± 39.3 at 72 h; all time values are significantly different from non-hydrated controls. In post-term pregnancies, AFI raised non significantly from 55.3 ± 19.4 to 61.9 ± 24.7 at 24 h, 72.5 ± 31.5 at 48 h and 51.5 ± 15.9 at 72 h. Maternal daily hydration raise low AFI values in term pregnancies. Post-term pregnancies show high variability. Conclusion: Further prospective study is necessary to assess the value of maternal hydration to improve efficacy of labour induction protocols. 15.001 Blastocyst biopsy in PGD practice: laboratory considerations Traversa M, Marshall JT, de Boer KA, Leigh DA, McArthur SJ Sydney IVF, Sydney, NSW, Australia Objective: Preimplantation genetic diagnosis (PGD) is typically done using blastomere biopsy where a single cell(s) is removed on day 3 post-fertilization from typically 6–8-cell embryos. An alternative method is blastocyst biopsy, which involves the removal of approximately 3–6 trophectoderm cells from a day-5 embryo containing approximately 100 cells. For PGD centres considering a move to blastocyst biopsy, we provide some insights based on our experiences since its routine implementation in our PGD laboratory in 2003. Materials/Methods: Progression of testing requirements for blastocyst biopsy was compared with current practice for blastomere biopsy. A contrast of the biopsy techniques, sample processing, types of testing and impact on laboratory resources is discussed. Results: IVF laboratories must account for extra time needed in patient management and extended incubation requirements. Blastocyst biopsy is performed at a later stage of embryo development (day 5) and results in a larger amount of genetic material available for analysis (average 3–6 trophectoderm cells). Techniques suitable for hatching as well as fully hatched embryos are available and work effectively. For the analysis laboratory, however, the biggest impact is on the amount of time available for genetic analysis (typically 4–6 h post biopsy for same day transfers but longer for day-6 transfer following biopsy on day 5). Test design can accommodate these time constraints and our own results suggest there is little loss of embryo viability from day 5 to day 6. Embryos do not all develop at the same rate and several rounds of biopsy may be required to produce all results. We feel this biopsy-when–ready approach is less harmful to embryos and typically the desired embryo is analysed before
S-55 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008