Pattaradilokrat Sittiporn, Li Jian, Su Xin-zhuan
National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA.
J Vis Exp. 2011 Jan 3(47):2365. doi: 10.3791/2365.
Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents and humans. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh. Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.
疟原虫对抗疟药物的反应以及致病性的差异具有生物学和医学重要性。连锁图谱分析已成功鉴定出啮齿动物和人类疟原虫各种性状的相关基因或基因座。约氏疟原虫是从野生非洲啮齿动物中分离出的众多疟原虫物种之一，已适应在实验室中生长。该物种重现了人类疟原虫的许多生物学特性；还为该寄生虫开发了微卫星和扩增片段长度多态性（AFLP）标记等遗传标记。因此，可以进行啮齿动物疟原虫的遗传研究以补充恶性疟原虫的研究。在此，我们展示了在约氏疟原虫中进行遗传杂交的技术，这些技术最初是由爱丁堡大学的大卫·沃利克博士、理查德·卡特博士及其同事开创的。约氏疟原虫和其他啮齿动物疟原虫的遗传杂交是通过用含有两种基因不同克隆的配子体的接种物感染小家鼠来进行的，这两种克隆在感兴趣的表型上有所不同，并在感染后4天让蚊子叮咬受感染的小鼠。在蚊子叮咬之前，通过显微镜确认小鼠血液中存在雌雄配子体。在叮咬后48小时内，在蚊子的中肠中，单倍体配子体分化为雄配子和雌配子，受精并形成二倍体合子（图1）。在合子发育成动合子的过程中，似乎会发生减数分裂。如果合子是通过两种基因不同的寄生虫的配子之间的杂交受精产生的，可能会发生遗传交换（染色体重排以及一对同源染色体的非姐妹染色单体之间的交叉；图2），从而导致同源基因座处的遗传物质重组。每个合子经历两次连续的核分裂，产生四个单倍体核。动合子进一步发育成卵囊。一旦卵囊成熟，就会形成数千个孢子体（杂交后代）并释放到蚊子的血腔中。从唾液腺中收集孢子体并注入新的小鼠宿主，在那里进行前红细胞期和红细胞期发育。在进行遗传连锁图谱分析之前，对红细胞形式进行克隆并根据区分亲本品系的特征进行分类。单个亲本品系的对照感染与遗传杂交的产生方式相同。
