Stable transfection of E-cadherin gene into mouse embryonic stem cells and its effects on adhesive capacity of differentiated cells
10.3969/j.issn.1673-8225.2009.49.040
- VernacularTitle:上皮型钙粘蛋白基因稳定转染小鼠胚胎干细胞及对分化细胞黏附能力的影响
- Author:
Anbin HU
;
Xiaoshun HE
;
Bing HUANG
;
Jiye CAI
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(49):9787-9791
- CountryChina
- Language:Chinese
-
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.
