Zeta Life轉(zhuǎn)染原代奶牛子宮內(nèi)膜上皮細(xì)胞(BEECs)發(fā)表文章分享IF6.684

一、發(fā)表文章轉(zhuǎn)染簡(jiǎn)述：

文章使用美國(guó)Zeta Life公司,#Advanced DNA RNA轉(zhuǎn)染試劑（#AD600150，Zeta Life, USA) 將構(gòu)建的pCMV-GSDMD-N-HA 載體質(zhì)粒DNA（圖 6A）轉(zhuǎn)染到原代奶牛子宮內(nèi)膜上皮細(xì)胞(BEECs)，  48小時(shí)后用Western blotting檢測(cè)GSDMD，并檢測(cè)到蛋白表達(dá)確定轉(zhuǎn)染成功（圖 6B）。

重建細(xì)胞炎癥模型，提取總蛋白，GSDMD表達(dá)及其裂解的N使用蛋白質(zhì)印跡法檢測(cè)末端蛋白。結(jié)果表明 BEEC 暴露于 LPS（10 和 30 µg/ml）24 小時(shí)可以裂解 GSDMD 并導(dǎo)致焦亡（圖 6C）
   文章標(biāo)題LPS Mediates Bovine Endometrial Epithelial Cell Pyroptosis Directly Through Both NLRP3 Classical and Non-Classical Inflflammasome Pathways。
  發(fā)表文章單位：中國(guó)農(nóng)業(yè)科學(xué)院畜牧與藥學(xué)研究所，農(nóng)業(yè)農(nóng)村部獸藥開發(fā)重點(diǎn)實(shí)驗(yàn)室，蘭州研究所。

二、轉(zhuǎn)染原代上皮細(xì)胞(BEECs)部分論文欣賞

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 Endofree 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 公司與美國(guó)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(dòng)物細(xì)胞、活體動(dòng)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。

四、文獻(xiàn)中所用產(chǎn)品信息

以上就是本期的內(nèi)容，更多資訊，歡迎點(diǎn)擊安培生物網(wǎng)站鏈接了解更多技術(shù)與產(chǎn)品資訊。

安培生物科技有限公司介紹：

安培生物堅(jiān)持為生命科學(xué)研究、活體動(dòng)物轉(zhuǎn)染、大規(guī)模生物生產(chǎn)、基因和細(xì)胞治療領(lǐng)域客戶，提供*細(xì)胞體內(nèi)可代謝的核酸轉(zhuǎn)染試劑；inviCELL™Platelet lysate無動(dòng)物血清產(chǎn)品旨在支持廣泛的細(xì)胞擴(kuò)增和生產(chǎn)，包括培養(yǎng)間充質(zhì)干細(xì)胞和多種免疫細(xì)胞系等，為制藥公司或者生物技術(shù)公司提供無血清細(xì)胞培養(yǎng)規(guī)模生產(chǎn)服務(wù)；提供以植物生物為平臺(tái)基因序列全人源化、*的細(xì)胞培養(yǎng)可分解3D基質(zhì)膠產(chǎn)品；提供內(nèi)毒素≦ 1.0 EU/mL、對(duì)細(xì)胞無毒性影響、高純度的一型膠原蛋白產(chǎn)品。安培生物專注為生物醫(yī)藥領(lǐng)域提供優(yōu)質(zhì)的產(chǎn)品和技術(shù)服務(wù)，努力發(fā)展為基因治療、細(xì)胞治療的生物科技企業(yè)。