成功轉染脂肪間充質(zhì)干細胞(ADMSCs)(IF6.684)文章分享

zeta life  Advanced DNA RNA 轉染試劑轉染脂肪間充質(zhì)干細胞(ADMSCs)(IF6.684)文章分享
zeta life轉染脂肪間充質(zhì)干細胞(ADMSCs)(IF6.684)文章分享編輯

一、轉染犬/狗脂肪間充質(zhì)干細胞(ADMSCs）siRNA基因沉默 簡(jiǎn)述

 熒光素標記的siRNA（小干擾 RNA）每個(gè)基因設計有三個(gè)siRNA，使用美國Zeta Life公司Advanced DNA RNA轉染試劑（Zeta Life，Menlo Park，CA，United States）用siRNA轉染犬/狗脂肪間充質(zhì)干細胞(ADMSCs),RT-qPCR用于檢測siRNA 的沉默效率，并選擇沉默效率高的一種，在整個(gè)轉染過(guò)程中，該基因每7天轉染一次以保持沉默。

二、轉染犬/狗脂肪間充質(zhì)干細胞(ADMSCs）基因過(guò)表達 簡(jiǎn)述

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

三、培養條件 簡(jiǎn)述

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

四、Adenovirus-Mediated Gene Overexpression原文分享

zeta life轉染脂肪間充質(zhì)干細胞(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轉染脂肪間充質(zhì)干細胞(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)舊金山校區聯(lián)合開(kāi)發(fā)用于哺乳動(dòng)物細胞、活體動(dòng)物轉染的 Advanced DNA RNA 第三代多肽小分子轉染試劑，此技術(shù)成為新的全球蛋白功能、免疫細胞及干細胞治療、研發(fā)及生產(chǎn)的主要關(guān)鍵技術(shù)之一。

七、文獻中所用轉染試劑產(chǎn)品信息

產(chǎn)品名稱(chēng)：Advanced DNA RNA 轉染試劑

貨號：AD600025、AD600050、AD600075、AD600150

規格：0.25ML、0.5ML、0.75ML、1.5ML