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Updated: May 23, 2026
Mouse Genome Engineering Using Designer Nucleases
Published on: April 2, 2014
[Construction and identification of mouse wildtype-syndecan-1 and unshedding-syndecan-1 expressing vector]
Jun Liu1, Ji-de Wang, Jie-Si Kuang
1Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Aim:
To construct the expressing vector pcDNA3.0- wildtype-syndecan-1(WT-sdc1) and pcDNA3.0-unshedding-syndecan-1(uS-sdc1), and to explore the expression in vitro.
Methods:
(1)The mouse WT-sdc1 DNA was successfully amplified by PCR and then cloned into pcDNA3.0. uS-sdc1 was construct by Gene splicing by overlap extension- PCR on the basis of WT-sdc1. The two vectors confirmed by DNA sequencing. (2)there are 4 groups in our research: control group, pcDNA3.0 transfected group, WT-sdc1 transfected group and uS-sdc1 transfected group. each vecter was transfected into IEC-6 cells by Lipofectamine(TM);2000. RT-PCR, Western blot and Dot blot were performed to detect the expression of syndecan-1 before and after stimulation of phorbol 12-myristate 13-acetate (PMA) for 15 min.
Results:
The vector WT-sdc1 and uS-sdc1 were successfully constructed although an non-sense mutation was in uS-sdc1. Compared to control and pcDNA3.0 transfected groups, WT-sdc1 and uS-sdc1 groups showed a significant increase in the expression of syndecan-1 in both mRNA and protein levels. In response to the stimulation of PMA, the expression of syndecan-1 was down-regulated at the protein levels but not mRNA levels.
Conclusion:
The WT-sdc1 and uS-sdc1 are successfully constructed, which lays the foundation for further studying of syndecan-1 in gastrointestinal inflammation.
