Simple and efficient transfection methods for genetic manipulation of Plasmodium falciparum are desirable to identify, characterize and validate the genes with therapeutic potential and better understand parasite biology. Among the available transfection techniques for P. falciparum, electroporation-based methods, particularly electroporation of ring-infected RBCs is routinely used. Nonetheless, transfection of P. falciparum remains a resource-intensive procedure. Here, we report a simple and economic transfection method for P. falciparum, which is termed as the lyse-reseal erythrocytes for transfection (LyRET). It involved lysis of erythrocytes with a hypotonic RBC lysis buffer containing the desired plasmid DNA, followed by resealing by adding a high salt buffer. These DNA-encapsulated lyse-reseal erythrocytes were mixed with P. falciparum trophozoite/schizont stages and subjected to selection for the plasmid-encoded drug resistance. In parallel, transfections were also done by the methods utilizing electroporation of DNA into uninfected RBCs and parasite-infected RBCs. The LyRET method successfully transfected 3D7 and D10 strains with different plasmids in 63 of the 65 attempts, with success rate similar to transfection by electroporation of DNA into infected RBCs. The cost effectiveness and comparable efficiency of LyRET method makes it an alternative to the existing transfection methods for P. falciparum, particularly in resource-limited settings.

译文

:对于恶性疟原虫的基因操作,简单有效的转染方法对于鉴定,表征和验证具有治疗潜力的基因以及更好地了解寄生虫生物学是理想的。在恶性疟原虫的可用转染技术中,常规使用基于电穿孔的方法,特别是环感染的红细胞的电穿孔。尽管如此,恶性疟原虫的转染仍然是资源密集的过程。在这里,我们报告了一种简单而经济的恶性疟原虫转染方法,被称为用于转染的裂解-重新密封红细胞(LyRET)。它涉及用含有所需质粒DNA的低渗RBC裂解缓冲液裂解红细胞,然后通过添加高盐缓冲液进行重新密封。将这些DNA包被的裂解-重新密封的红细胞与恶性疟原虫滋养体/裂殖体阶段混合,并进行质粒编码的耐药性的选择。同时,转染也通过利用将DNA电穿孔到未感染的RBC和寄生虫感染的RBC中的方法进行。 LyRET方法在65次尝试中有63次成功用不同的质粒成功转染了3D7和D10菌株,成功率与通过将DNA电穿孔到感染的RBC中的转染相似。 LyRET方法的成本效益和可比的效率使其成为恶性疟原虫现有转染方法的替代方法,尤其是在资源有限的环境中。

+1
+2
100研值 100研值 ¥99课程
检索文献一次
下载文献一次

去下载>

成功解锁2个技能,为你点赞

《SCI写作十大必备语法》
解决你的SCI语法难题!

技能熟练度+1

视频课《玩转文献检索》
让你成为检索达人!

恭喜完成新手挑战

手机微信扫一扫,添加好友领取

免费领《Endnote文献管理工具+教程》

微信扫码, 免费领取

手机登录

获取验证码
登录