Construction of recombinant plasmid pEGFPN1-tailless-like protein and transfection into dermal multipotential stem cells

Tao WANG; Anchun QIU; Lusi LI; Yanping LIU; Jifu QU; Wenyue XU

ABSTRACT

BACKGROUND:

It is reported that tailless-like protein (TLX) plays critical roles in the regulation of early developmental processes in vertebrates, and it plays a key role in stem cells proliferation and differentiation into neurons.

OBJECTIVE:

To construct recombinant plasmid pEGFPN1-TLX and study the transfection into dermal multipotential stem cells. DESIGN, TIME AND

SETTING:

Cytogene experiment was performed at the Department of Pathogen Biology, School of Basic Medical Science, the Third Military Medical University of Chinese PLA from March to December 2007. MATERIALS An adult SD was obtained from the Experimental Animal Center of the Third Military Medical University of Chinese PLA; dermal moltipotential stem cells (DMSCs) were cultured by the Institute of Combined Injury of the Third Military Medical University of Chinese PLA; pEGFPN1 and DH5α was gifted by professor Xu.

METHODS:

Total RNA was extracted from rat brain tissue to amplify TLX-coded cDNA sequence using RT-PCR. T/A was cloned on pMD18-T vector and determined using BamHI and Hindlll. The products were positive recombinant plasmid pMD18-T-TLX segments, which were sub-cloned in pEGFPN1 to construct recombinant plasmid pEGFPN1-TLX. Finally, pEGFPN1-TLX was transfected into DMSCs.MAIN OUTCOME

MEASURES:

The fluorescence protein expression was observed under fluorescence microscope at 24 hours after transfection; TLX mRNA expression was detected using RT-PCR; neuronal differentiation was observed using immunohistochemical staining.

RESULTS:

TLX full length cDNA was successfully cloned into pEGFPN1, and pEGFPN1-TLX was successfully constructed by means of sequence analysis and enzyme cutting identification. As compared with non-transfected DMSCs, pEGFPN1-TLX transfected DMSCs were observed after 10 days, formed resistant clones after 15 days, and shown a green fluorescent protein expression. However, non-transfected DMSCs died at day 10. RT-PCR indicated that pEGFPN1-TLX transfected DMSCs could express TLX mRNA. At day 3 after induction, NF200 positive cells were increased, but glial fibrillary acidic protein positive cells were decreased after induction of pEGFPN1-TLX transfected DMSCs.

CONCLUSION:

TLX was successfully constructed and transfected into DMSCs. After transfection, neuronal differentiation of DMSCs was enhanced, and the differentiation to gliocytes was inhibited.