一、发表文章转染简述：

文章使用美国Zeta Life公司,#Advanced DNA RNA转染试剂（#AD600150，Zeta Life, USA) 将构建的pCMV-GSDMD-N-HA 载体质粒DNA（图 6A）转染到原代奶牛子宫内膜上皮细胞(BEECs)， 48小时后用Western blotting检测GSDMD，并检测到蛋白表达确定转染成功（图 6B）。

重建细胞炎症模型，提取总蛋白，GSDMD表达及其裂解的N使用蛋白质印迹法检测末端蛋白。结果表明 BEEC 暴露于 LPS（10 和 30 µg/ml）24 小时可以裂解 GSDMD 并导致焦亡（图 6C）

文章标题LPS Mediates Bovine Endometrial Epithelial Cell Pyroptosis Directly Through Both NLRP3 Classical and Non-Classical Inflflammasome Pathways。

发表文章单位：中国农业科学院畜牧与药学研究所，农业农村部兽药开发重点实验室，兰州研究所。

二、转染原代上皮细胞(BEECs)部分论文欣赏

1、LPS Exposure for 24 h Leads to BEEC Inflflammation and Cleaved GSDMD to Pyroptosis

We constructed a pCMV-GSDMD-N-HA vector (Figure 6A) and transfected it to BEECs to overexpress GSDMD, and detected protein expression withWestern blotting after 48 h to ensure the transfection was successful (Figure 6B). The cell inflflammation model was rebuilt,

total proteinwas extracted, and GSDMD expression and its cleaved N terminal protein were detected using Western blotting. The resultsshowed that BEEC exposed to LPS (10 and 30 µg/ml) for 24 h could cleave GSDMD and lead to pyroptosis (Figure 6C).

2、Genetic Recombination

Genetic Recombination Technology was adopted to ligate bovine GSDMD DNA fragments into the ampicillin-resistant pCMV-HA vector and transform it into E. coli. Brieflfly, the bacteria were grown on an LB agar medium containing ampicillin sodium for 24 h at 37°C. A single colony was picked and added to the LB liquid medium containing ampicillin sodium and culturedfor 16 h at 37°C with shaking at 300×g. Plasmid DNA was extracted with an Endo[1]free Plasmid Mini Kit I and quantifified using a Polluton100+. The Zeta Life Transfection Kit was used to transfect the plasmid into BEECs, and LPSwas used to construct the cell inflflammation model. The cell total protein was extracted and detected by an HA tag and Western blotting to evaluate the GSDMD protein expression and its cleavage during cell pyroptosis. The vector construction, restriction endonuclease cleavage verifification, and sequencing verifification were done by Genecreate Biological Company。

三、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。