TY - JOUR
T1 - Stable transfection of E-cadherin gene into mouse embryonic stem cells and its effects on adhesive capacity of differentiated cells
AU - Hu, An Bin
AU - He, Xiaoshun
AU - Huang, Bing
AU - Cai, Ji Ye
PY - 2009/12/3
Y1 - 2009/12/3
N2 - 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 Lü 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.
AB - 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 Lü 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.
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U2 - 10.3969/j.issn.1673-8225.2009.49.040
DO - 10.3969/j.issn.1673-8225.2009.49.040
M3 - Article
AN - SCOPUS:77953767107
VL - 13
SP - 9787
EP - 9791
JO - Journal of Clinical Rehabilitative Tissue Engineering Research
JF - Journal of Clinical Rehabilitative Tissue Engineering Research
SN - 1673-8225
IS - 49
ER -