Stable transfection of E-cadherin gene into mouse embryonic stem cells and its effects on adhesive capacity of differentiated cells

Anbin HU; Xiaoshun HE; Bing HUANG; Jiye CAI

ABSTRACT

BACKGROUND:

During the embryonic stage, E-cadherin expression plays a critical role in the formation of hepatic tissue.

OBJECTIVE:

E-cadherin gene was transfected into mouse embryonic stem cells (ESCs) to observe its effects on adhesive capacity of differentiated cells.DESIGN, TIME AND

SETTING:

A cytological in vitro observation was performed at the Laboratory of Surgery, First Hospital Affiliated to Sun Yat-sen University between December 2007 and December 2008.MATERIALS BALB/c mice at gestational 13 days, of clean grade, were provided by Laboratory Animal Center, Sun Yat-sen University. BALB/c mouse ESCs were preserved by professor Huang Bing from the Department of Ophthalmology, Sun Yat-sen University. CMV promoter-containing eukaryotic expression plasmid pEGFP-N1 was gifted by doctor Lu Zhi-yue from Medical College, Sun Yat-sen University.

METHODS:

Total RNA was extracted from BALB/c mouse fresh hepatic tissue and synthesized into cDNA by reverse transcription (RT). The synthesized cDNA was used as a template to perform a polymerase chain reaction (PCR) that amplifies a targeted fragment. Following double enzyme digestion, pEGFP-E-cadherin plasmids were reconstructed and transfected into mouse ESCs. In vitro differentiation of transfected mouse ESCs was performed.MAIN OUTCOME

MEASURES:

Detection of E-cadherin expression in the differentiation system using RT-PCR and immunocytochemistry and observation of adhesive capacity of differentiated cells.

RESULTS:

E-cadherin gene-transfected ESCs could stably express E-cadherin during differentiational 1-17 days, while non-transfected ESCs expressed a decreasing amount of E-cadherin. The adhesive capacity of differentiated cells that stably expressed E-cadherin was markedly enhanced. Compact cell connection and multi-layer growth state remained at 19 days. While non-transfected ESCs gradually changed from embryoid bodies into noncohesive cell populations.

CONCLUSION:

Differentiating E-cadherin ESCs exhibit markedly enhanced adhesive capacity and maintain multi-layer growth state.