title: [Expression of exogenous porcine transforming growth factor beta-1 gene in ES cells and its effect on their differentiation in vitro].
abstract: A TGF-beta 1 gene expression plasmid was constructed by inserting the porcine 1.7 Kb TGF-beta 1 cDNA into BamHI site of retrovirus vector Dol. The plasmid DNA was introduced into mouse embryonic stem cells (ES-5 line) by calcium phosphate mediated transfection, and transfected ES-5 cells were then selected by stepwise increase in G418 concentration. Finally, we obtained 21 clones that could be stably grown in culture medium with G418 at 500 micrograms/ml and were designated as ES-T cells. Dot blot and Northern analysis of total RNA and polyA+ RNA extracted from those ES-T cells were shown in FIg. 2 and 3, demonstrating that 6 clones could express exogenous porcine TGF-beta 1 mRNA. The stronger hybridized signal in two clones (ES-T6 and ES-T 16) of them were further proved by southern hybridization of genomic DNA from these ES-T cells with 1.7 Kb TGF-beta 1 cDNA probe (Fig. 4). The product of TGF-beta 1 gene overexpression in ES-T 6 cells was shown in Fig. 5 and 6 by SE-LISA for TGF-beta 1 immunoreactivity to TGF-beta 1 antibodies and biological assay for CCL/64 cell growth inhibition, respectively. With respect to some biological characteristics, ES-T 6 cells, like their parent ES-5 cells, retained their pluripotent properties and positive SSEA-1 antigen (Plate I, Fig. 1). ES-T6 cells were expanded and used for studies of in vitro differentiation. Both of ES-T 6 cells and control ES-5 cells could form a lot of simple aggregates and differentiate into embryoid bodies by hanging drop culture for 3 days in the presence of retinoic acid (RA) at 10(-9) mol/L. Then individual embryoid bodies were plated on gelatinized tissue culture wells. On the third day of further culture without RA, a large amounts of epithelial-like and round cells occurred around the embryoid bodies formed either from ES-T 6 cells or ES-5 cells (Plate I, Fig. 4). However, with further culture of embryoid bodies, only the cells differentiated from ES-T6 embryoid bodies could arrange themselves and differentiate into a lot of radially arranged tubular structures (Plate II, Fig. 5). The frequency of tubular structures present in ES-T6 embryoid bodies were about 95.5%, but in ES-5 group there was only about 17.8% cases giving less defined tubular structures (Plate III, Fig. 8).(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Shanghai Institute of Cell Biology, Academia Sinica.
authors.forename: H C
authors.initials: HC
authors.lastname: Tsung
authors.forename: S H
authors.initials: SH
authors.lastname: Xia
authors.forename: L X
authors.initials: LX
authors.lastname: Xu
authors.forename: X L
authors.initials: XL
authors.lastname: Li
authors.forename: W K
authors.initials: WK
authors.lastname: Shi
authors.forename: Z
authors.initials: Z
authors.lastname: Yao
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D015870:Gene Expression; D051379:Mice; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D013552:Swine; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D016212:Transforming Growth Factor beta