基因君官网 
2015年7月17日/生物谷BIOON/–近期的研究表明，隐孢子虫（Cryptosporidium）是在全球范围内引起婴幼儿严重腹泻的第二大病原体。腹泻据估计是占到婴幼儿总死亡案例的10.5%。引起严重的腹泻的首要病原体是轮状病毒，其次是隐孢子虫。这种隐孢子虫还是艾滋病患者，以及器官移植患者体内的机会病原体。这种致病毒对于营养不良的儿童和免疫缺陷患者是巨大的威胁，然而至今还没有针对隐孢子虫的疫苗，唯一的一种药物效果也很差。针对隐孢子虫的连续培养系统、动物模型以及分子遗传学工具都很缺乏。
美国佐治亚大学的一个课题组近期在《Nature》发表了一篇文章介绍了一种实验体系，可以通过基因改造这种重要病原体，使得今后针对隐孢子虫的研究更容易进行。他们建立并优化了小球隐孢子虫（Cryptosporidium parvum）转染组织培养的细胞方法。为了获得稳定的转基因隐孢子虫，他们建立了小鼠模型能够被这种隐孢子虫稳定感染。
这种隐孢子虫的基因组可以被Cas9系统编辑，他们引入一些报告基因（荧光素基因），使得其对于氨基糖苷类抗生素有耐药性。通过手术注射的方法，研究人员试图将这种基因改造过的隐孢子虫注入小鼠的小肠。这种小鼠是一种基因敲除的小鼠，对隐孢子虫敏感。感染基因改造的隐孢子虫三十天后，这些小鼠肠道和粪便中能检测到隐孢子虫的卵囊。基因编辑后的隐孢子虫也能够感染组织培养的细胞。
增加了荧光素标记基因的隐孢子虫系统，能够让科学家定量研究很多分子遗传学特性，这些对于以后的药物研究以及疫苗开发都提供了分子基础。由于这种寄生虫对于药物不敏感，耐药性很强。利用这种基因改造后的隐孢子虫系统用于体内或体外的药物筛选。通过基因改造使得隐孢子虫弱化，或许能够成为一种有希望的疫苗。他们还测试基因敲除之后的隐孢子虫对药物的敏感性。他们希望通过转基因改造这种寄生虫为未来的研究提供基础，并为针对隐孢子虫的药物研发打开了大门。（基因宝jiyinbao.com）
本文系生物谷原创编译整理。欢迎转载！转载请注明来源并附原文链接。更多资讯请下载生物谷资讯APP。
PMC:
PMID:
Genetic modification of the diarrhoeal pathogenCryptosporidium parvum
Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus1, 2, 3. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality4. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation5,6. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients7, 8. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools3, 9. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivoselection for aminoglycoside resistance. We derived reporter parasites suitable for in vitroand in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.