熒光素標(biāo)記的siRNA（小干擾 RNA）每個基因設(shè)計有三個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，并選擇沉默效率高的一種，在整個轉(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
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
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。
