一、转染犬/狗脂肪间充质干细胞(ADMSCs）siRNA基因沉默 简述

荧光素标记的siRNA（小干扰 RNA）每个基因设计有三个siRNA，使用美国Zeta Life公司Advanced DNA RNA转染试剂（Zeta Life，Menlo Park，CA，United States）用siRNA转染犬/狗脂肪间充质干细胞(ADMSCs),RT-qPCR用于检测siRNA 的沉默效率，并选择沉默效率高的一种，在整个转染过程中，该基因每7天转染一次以保持沉默。

二、转染犬/狗脂肪间充质干细胞(ADMSCs）基因过表达 简述

使用美国Zeta Life公司Advanced DNA RNA转染试剂（Zeta Life，Menlo Park，CA,USA）转染AAV-293细胞收集重组腺病毒颗粒并感染犬/狗脂肪间充质干细胞(ADMSCs)，用 RT-qPCR和蛋白质印迹检测目的基因的表达。

三、培养条件 简述

脂肪间充质干细胞(ADMSCs),使用美国Zeta Life公司z7010FBS-500胎牛血清培养。

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、美国Zeta Life 公司与美国加利福尼亚大学旧金山校区联合开发用于哺乳动物细胞、活体动物转染的 Advanced DNA RNA 第三代多肽小分子转染试剂，此技术成为新的蛋白功能、免疫细胞及干细胞治疗、研发及生产的主要关键技术之一。