Pdsred2 1

The regulatory region of the human ubiquitin C promoter (1204 bp) was amplified by PCR, the vector pFUGW (Addgene) was used as the template as well as specific primers (Invitrogen). The fragment was then inserted into the promoterless vector pDsRed2-1 (Clontech). PCR of the YFP-I152L gene (720 bp) was performed using vector pcDNA3.1-YFPI152L (Invitrogen) as a template and specific primers (Invitrogen). The YFP-I152L-coding construct was kindly provided from Prof. Joe Lynch (Queensland Brain Institute, The University of Queensland, Brisbane, Australia). After removing the sequence of the red fluorescent protein from the vector phuUbC-DsRed2, YFP-I152L was sub-cloned into the vector. The expression of YFP-I152L is under the control of the constitutive active human ubiquitin C promoter, the plasmid carries the neomycin resistance gene for the selection with the antibiotic geneticin.

To investigate the effect of exogenous genes on gMDSC- and gMDSC-derived cloned embryo development, we used pDsRed2-1 (632405, Clontech, Santa Clara, CA) as an exogenous vector. gADSCs, gMDSCs and gFFCs were cultured in 24-well plates (1 × 10⁵ cells/well) for 24 h before transfection with pDsRed2-1 using Lipofectamine LTX and Plus Reagent (15338-100, Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s protocol. The transient transfection efficiency was evaluated by flow cytometry. Similarly, gADSCs, gMDSCs and gFFCs were cultured in 100 mm plates for G418 and fluorescence screening, resulting in gADSCs–pDsRed2-1, gMDSCs–pDsRed2-1 and gFFCs–pDsRed2-1. After 10 days, colony forming efficiency was calculated. Neurogenic, myogenic and insulin-producing cell differentiation was induced in gADSCs–pDsRed2-1, gMDSCs–pDsRed2-1 and gFFCs–pDsRed2-1, respectively, as described above.