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The Constrution and Identification of Mice CX3CR1Gene Lentivial Overexpression Vector

Author: LiuFang
Tutor: LiFaZuo
School: Chongqing Medical University
Course: Geriatrics
Keywords: CX3CR1 Chemokines Lentiviral vectors
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Type: Master's thesis
Year: 2013
Downloads: 24
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Abstract


Objective：To construct recombinant lentivirus over-expressing vectorof mice CX3CR1gene.Methods:The CX3CR1gene sequence of mice publicated onGendbank was analyzed by Oligo software,and designed a couple of primercontaining both Age I and BamH I.The full length CX3CR1cDNA wasobtained by PCR and cloned directedly into multiple cloning sites ofpUbi-MCS-EGFP，then the recombinant plasmid was selected by Ampresistance.Purified pUbi-MCS-CX3CR1-EGFP plasmids from the positiveclones was confirmed by PCR and sequencing.The production of lentiviruswere obtained by using pHelper1.0,pHelper2.0and Lipofectamine2000to contransfect293T cells.The titer of lentivirus and the expression level ofenhanced green fluorescent protein was tested by RT-PCR.Result：The PCR and sequencing confirmed that the recombinantlentivirus plasmid of pUbi-MCS-CX3CR1-EGFP was correctlyconstructed.And the titer of lentivirus was2×108TU/ml.Conclusion：Lentivirus vector of CX3CR1gene over-expresseion were successfully constructed,It will lay the foundation for our further study ofmesenchymal stem cells homing capacity.

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