轉(zhuǎn)染脂肪間充質(zhì)干細(xì)胞(ADMSCs)(IF6.684)文章分享

zeta life轉(zhuǎn)染脂肪間充質(zhì)干細(xì)胞(ADMSCs)(IF6.684)文章分享

zeta life轉(zhuǎn)染脂肪間充質(zhì)干細(xì)胞(ADMSCs)(IF6.684)文章分享編輯

一、轉(zhuǎn)染犬/狗脂肪間充質(zhì)干細(xì)胞(ADMSCs）siRNA基因沉默 簡述

 熒光素標(biāo)記的siRNA（小干擾 RNA）每個(gè)基因設(shè)計(jì)有三個(gè)siRNA，使用美國Zeta Life公司Advanced DNA RNA轉(zhuǎn)染試劑（Zeta Life，Menlo Park，CA，United States）用siRNA轉(zhuǎn)染犬/狗脂肪間充質(zhì)干細(xì)胞(ADMSCs),RT-qPCR用于檢測siRNA 的沉默效率，并選擇沉默效率為最高的一種，在整個(gè)轉(zhuǎn)染過程中，該基因每7天轉(zhuǎn)染一次以保持沉默。

二、轉(zhuǎn)染犬/狗脂肪間充質(zhì)干細(xì)胞(ADMSCs）基因過表達(dá) 簡述

使用美國Zeta Life公司Advanced DNA RNA轉(zhuǎn)染試劑（Zeta Life，Menlo Park，CA,USA）轉(zhuǎn)染AAV-293細(xì)胞收集重組腺病毒顆粒并感染犬/狗脂肪間充質(zhì)干細(xì)胞(ADMSCs)，用 RT-qPCR和蛋白質(zhì)印跡檢測目的基因的表達(dá)。

三、培養(yǎng)條件 簡述

 脂肪間充質(zhì)干細(xì)胞(ADMSCs),使用美國Zeta Life公司z7010FBS-500胎牛血清培養(yǎng)。

四、Adenovirus-Mediated Gene Overexpression原文分享

zeta life轉(zhuǎn)染脂肪間充質(zhì)干細(xì)胞(ADMSCs)(IF6.684)文章分享編輯

The cDNA sequences for genes were synthesized by Wuhan Gene Create Biological Engineering Co., Ltd. (Wuhan, China). The cDNA for each gene was PCR-amplifified and inserted into pAdTrack-CMV previously digested with BglII/HindIII using the In-Fusion HD cloning kit (Takara, Japan) (Sleight et al., 2010). The resulting plasmid was linearized with PmeI, and then homologous recombination was performed with pAdeasy-1 in E. coli BJ5183. The recombinant plasmid with PacI linearization was used to transfect AAV-293 cells to

produce recombinant adenovirus particles using the Advanced DNA RNA transfection reagent (Zeta Life, Menlo Park, CA, United States). RT-qPCR (Nolan et al., 2006) and Western-blot (following the Elabscience Western blot detection kit operation manual) were used to detect the expression of target gene. The canine ADMSCs were transfected with the recombinant adenovirus particles.

五、siRNA-Mediated Gene Silencing原文分享

zeta life轉(zhuǎn)染脂肪間充質(zhì)干細(xì)胞(ADMSCs)(IF6.684)文章分享編輯

Fluorescein-labeled siRNA . Each gene was designed with three siRNAs. In the process of transdifffferentiation, the canine ADMSCs were transfected with siRNAs using the Advanced DNA RNA transfection reagent (Zeta Life, Menlo Park, CA, United States). RT-qPCR (Nolan et al., 2006) was used to detect the silencing effiffifficiency of siRNAs and the one with the highest silencing effiffifficiency was selected. The gene was silenced every 7 days throughout the transdifffferentiation to keep it silent.

六、美國Zeta Life 公司與美國加利福尼亞大學(xué)舊金山校區(qū)聯(lián)合開發(fā)用于哺乳動物細(xì)胞、活體動物轉(zhuǎn)染的 Advanced DNA RNA 第三代多肽小分子轉(zhuǎn)染試劑，此技術(shù)成為新的蛋白功能、免疫細(xì)胞及干細(xì)胞治療、研發(fā)及生產(chǎn)的主要關(guān)鍵技術(shù)之一。

七、文獻(xiàn)中所用轉(zhuǎn)染試劑產(chǎn)品信息

產(chǎn)品名稱：Advanced DNA RNA 轉(zhuǎn)染試劑

貨號：AD600025、AD600050、AD600075、AD600150

規(guī)格：0.25ML、0.5ML、0.75ML、1.5ML

安培生物科技有限公司是美國 Zeta Life 公司的中國代理商。Advanced DNA RNA轉(zhuǎn)染試劑，均經(jīng)過反復(fù)充分驗(yàn)證，轉(zhuǎn)染效果好且重復(fù)性佳，提供完整售后服務(wù)和技術(shù)支持。

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安培生物科技有限公司介紹：

安培生物堅(jiān)持為生命科學(xué)研究、活體動物轉(zhuǎn)染、大規(guī)模生物生產(chǎn)、基因和細(xì)胞治療領(lǐng)域客戶，提供*細(xì)胞體內(nèi)可代謝的核酸轉(zhuǎn)染試劑；inviCELL™Platelet lysate無動物血清產(chǎn)品旨在支持廣泛的細(xì)胞擴(kuò)增和生產(chǎn)，包括培養(yǎng)間充質(zhì)干細(xì)胞和多種免疫細(xì)胞系等，為制藥公司或者生物技術(shù)公司提供無血清細(xì)胞培養(yǎng)規(guī)模生產(chǎn)服務(wù)；提供以植物生物為平臺基因序列全人源化、*的細(xì)胞培養(yǎng)可分解3D基質(zhì)膠產(chǎn)品；提供內(nèi)毒素≦ 1.0 EU/mL、對細(xì)胞無毒性影響、高純度的一型膠原蛋白產(chǎn)品。安培生物專注為生物醫(yī)藥領(lǐng)域提供優(yōu)質(zhì)的產(chǎn)品和技術(shù)服務(wù)，努力發(fā)展為基因治療、細(xì)胞治療的生物科技企業(yè)。