中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2013年
5期
371-376
,共6页
李哲海%张霄雁%刘国栋%呼和%武宇赤
李哲海%張霄雁%劉國棟%呼和%武宇赤
리철해%장소안%류국동%호화%무우적
基因表达%构建%质粒%DKK1蛋白
基因錶達%構建%質粒%DKK1蛋白
기인표체%구건%질립%DKK1단백
Gene expression%Construction%Plasmid%DKK1 proteins
目的 筛选构建针对骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSC)的小干扰RNA(shRNA)腺病毒载体.方法 根据shRNA靶位点的选择原则,从TRCshRNA数据库及Ambion shRNA数据库中择优选取3条DKK1 shRNA序列,通过DKK1构建过表达载体,筛选出最佳干扰序列,体外同源重组构建DKK1-shRNA腺病毒表达载体,经酶切、PCR鉴定、测序.结果 成功构建pYr-1.1-DKK1-shRNA-153、pYr-1.1-DKK 1-shRNA-155、pYr-1.1-DKK1-shRNA-Am表达载体,分别通过RT-PCR与Western印迹检测DKK1的表达,结果显示pYr-1.1-DKK 1-shRNA-153为最佳干扰序列.结论 成功构建了针对BMSC的小干扰RNA(shRNA)腺病毒载体,为后续靶向目的基因的实验研究提供了依据.
目的 篩選構建針對骨髓間充質榦細胞(bone marrow-derived mesenchymal stem cells,BMSC)的小榦擾RNA(shRNA)腺病毒載體.方法 根據shRNA靶位點的選擇原則,從TRCshRNA數據庫及Ambion shRNA數據庫中擇優選取3條DKK1 shRNA序列,通過DKK1構建過錶達載體,篩選齣最佳榦擾序列,體外同源重組構建DKK1-shRNA腺病毒錶達載體,經酶切、PCR鑒定、測序.結果 成功構建pYr-1.1-DKK1-shRNA-153、pYr-1.1-DKK 1-shRNA-155、pYr-1.1-DKK1-shRNA-Am錶達載體,分彆通過RT-PCR與Western印跡檢測DKK1的錶達,結果顯示pYr-1.1-DKK 1-shRNA-153為最佳榦擾序列.結論 成功構建瞭針對BMSC的小榦擾RNA(shRNA)腺病毒載體,為後續靶嚮目的基因的實驗研究提供瞭依據.
목적 사선구건침대골수간충질간세포(bone marrow-derived mesenchymal stem cells,BMSC)적소간우RNA(shRNA)선병독재체.방법 근거shRNA파위점적선택원칙,종TRCshRNA수거고급Ambion shRNA수거고중택우선취3조DKK1 shRNA서렬,통과DKK1구건과표체재체,사선출최가간우서렬,체외동원중조구건DKK1-shRNA선병독표체재체,경매절、PCR감정、측서.결과 성공구건pYr-1.1-DKK1-shRNA-153、pYr-1.1-DKK 1-shRNA-155、pYr-1.1-DKK1-shRNA-Am표체재체,분별통과RT-PCR여Western인적검측DKK1적표체,결과현시pYr-1.1-DKK 1-shRNA-153위최가간우서렬.결론 성공구건료침대BMSC적소간우RNA(shRNA)선병독재체,위후속파향목적기인적실험연구제공료의거.
Objective To screen and construct the small interfering RNA (shRNA) adenovirus vector of bone marrow-derived mesenchymal stem cells (BMSC).Methods Based on the principle of shRNA target sites selection,three DKK1 shRNA sequences were chosen from the shRNA database and Ambion shRNA database.The optimal interfering sequence was screened by construction of DKK1 overexpression vector and identified by restriction analysis,PCR and sequenced via construction of homologus recombination DKK1-shRNA adenovirus vector in vitro.Results Expression vectors,including pYr-1.1-DKK 1-shRNA-153,pYr-1.1-DKK1-shRNA-155 and pYr-1.1-DKK 1-shRNA-Am,were successfully constructed.DKK1 could be detected via RT-PCR and Western blotting,indicating that pYr-1.1-DKK1-shRNA-153 was the optimal interfering sequence.Conclusion The successful construction of shRNA (siRNA) adenovirus vector of BMSC provides useful information for further researches on target genes.
