Zeta Life转染原代上皮细胞BEECs信息分享及相关文献欣赏_产品知识

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。