11.004 Pregnancy of transgenic-clone goat using mammary gland epithelial cells as donor cells

Abstract

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 g/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.