Comparasite Full-length cDNA Database

Comparasite Full-length cDNA Database
Home > Experimental Procedures

Experimental Procedures

Experimental Procedures

I) Preparation of parasites

Plasmodium faliciparum

    P. falciparum 3D7 parasites were cultured either in a candle jar (XPFn, XPF2n) or in the mixed gas consisting of 3% oxygen, 5% carbon dioxide and 92% nitrogen using blood type A erythrocytes and frozen human plasma.

Plasmodium vivax

    Blood samples were collected from the Indonesian patients with P.vavax who were diagnosed by Giemsa staining of the blood smear and gave the written informed consent. Heparinized whole blood was filtrated through Plasmodipur filter (Eurodiagnostica) to remove white blood cells. Parasites were homogenized using the Polytron Homogenizer, frozen in liquid nitrogen and sent to Japan with dry ice.

Plasmodium yoelii and Plasmodium berghei

    P. yoelii 17XL (XPYw), P. yoelii 17XNL (XPYs) and P. berghei ANKA were propagated in vivo in five-week old female ICR mice. Blood was filtrated through Plamsodipur filter to remove white blood cells.

Toxoplasma gondii

    The RH strain of Toxoplasma gondii tachyzoites (1x107 parasites) were infected to Vero (T25) cells and incubated in EMEM medium supplemented with 8% fetal bovine serum for 72 hours in vitro. Infected cells were collected with a scraper and passed through 27G needle for three times and purified by filtration through Millex SV filters (pore size 5 um, Millipore). Cells were collected by centrifugation at 2,000 rpm for 10 min and washed in EMEM medium without FBS. Parasites were suspended at 1x107/ml in EMEM medium without FBS. Five-week old ICR female mice were infected with 1 ml parasites intraperitoneally.. Three days after infection, mice were sacrificed by cervical dislocation. Parasites were collected by washing the peritoneal cavity with 5 ml EMEM, passed through 5 um filter, washed in PBS and suspended at 2-3x109 parasite/ml. 20 ml TRI REGENT (Sigma) was added to 1 ml parasite solution, mixed by pipetting, frozen in liquid nitrogen and kept at -80C until used.

Cryptosporidium parvum

    C. parvum HNJ-1 strain (genotype 2) was originally isolated from a Japanese immunocompetent patient with diarrhea in 1989 and has been maintained by subinoculation in SCID (severe combined immunodeficiency) mice.
    Five-week old female SCID mice were infected by oral inoculation of 105 oocysts. Feces were collected in water under the cage and oocysts were purified by sugar floatation followed by diethyl ether method. Oocysts were incubated in 0.1N HCl at 37oC for 1 hour. After three washes with MEM they were incubated in MEM supplemented with 0.1% deoxycholate at 37oC for 30 min. Excysted sporozoites were homogenized in Trizol (Invitrogen) using Polytron homogenizer and RNAs were isolated.

II) Isolation of RNA.
    Cytoplasmic RNA and Poly A+ RNA were isolated using standard methods. Oligo-dT cellulose was obtained from Collaborative Biomedical Products and Roche.

III) Oligo-capping.
    Oligo-capping was performed as described (Maruyama and Sugano, 1994), with some modifications. In brief, 10 to 50 ug of polyA+ RNA was treated with 1.2 units of bacterial alkaline phosphatase (BAP; TaKaRa) in 100ul of 100mM Tris-HCl (pH 8.0), 5mM 2-mercaptoethanol with 100 units of RNasin (Promega) at 37oC for 60 min. After extraction with phenol:chloroform (1:1) and ethanol precipitation, the polyA+ RNA was treated with 20 units of tobacco acid pyrophosphatase (TAP) in 100ul of 50mM sodium acetate (pH 5.5), 1mM EDTA, 5mM 2-mercaptoethanol with 100 units of RNasin at 37oC for 60 min. After phenol:chloroform extraction and ethanol precipitation, 2 to 4ug of the BAP-TAP treated polyA+ RNA were ligated with 0.4ug of 5'-oligo (KM-02; 5'-AGC AUC GAG UCG GCC UUG UUG GCC UAC UGG-3') using 250 units of RNA ligase (TaKaRa) in 100ul of 50mM Tris-HCl (pH7.5), 5mM MgCl2, 5mM 2-mercaptoethanol, 0.5mM ATP, 25% PEG8000 with 100 units of RNasin at 20oC for 3 hours.

IV) cDNA synthesis.
    After unligated 5'-oligo was removed, cDNA was synthesized with RNaseH free reverse-transcriptase (Superscript II, Gibco BRL). Ten picomoles of dT adapter-primer (5'-GCG GCT GAA GAC GGC CTA TGT GGC CTT TTT TTT TTT TTT TTT-3') was used in 50ul with 2 to 4ug of oligo-capped polyA+ RNA. The reaction conditions were as recommended by the supplier and reactions were incubated at 42oC for 3 hours.

V) cDNA amplification.
    After first-strand cDNA synthesis, RNA was degraded in 15mM NaOH by incubating at 65oC for 1 hour. The cDNA which is made from 1ug of "Oligo-capped" polyA+ RNA was amplified in a volume of 100ul using an XL PCR kit (Perkin-Elmer) with 16pmol of 5' (5'-AGC ATC GAG TCG GCC TTG TTG-3') and 3' (5'-GCG GCT GAA GAC GGC CTA TGT-3') PCR primers. For dT-adapter primer primed cDNA, amplification were carried out by 15 cycles of PCR reactions at 94oC for 1 min, 58oC for 1 min, and 72oC for 10 min. PCR products were extracted with phenol:chloroform (1:1) once, ethanol precipitated and digested with SfiI. SfiI-digested PCR products were separated by agarose gel electrophoresis and products longer than 1 kb were isolated and cloned into DraIII-digested pME18S-FL3. In this way, we could clone the cDNA into the vector in an orientation-defined manner.

VI) Sequencing.
    Plasmid DNA was isolated using PI-100 and PI-200 auto-plasmid-isolators (KURABO).

    Sequences were determined by the dideoxy termination method using an AutoCycle sequencing kit (Pharmacia) and a reaction robot R. O. B. DNA processor (Pharmacia) or a BigDye sequencing kit (ABI). The sequence was read using an ALF DNA (Pharmacia) and an ABI 377XL (ABI) auto-sequencer.

VII) Sequence similarity test.
    Sequence similarity of cDNA was tested against GenBank non-redundant nucleotide library (Release 98) using the BLASTN or FASTA program.

VIII) Database construction.

Help Sitemap Contact About Comparasite Search Database Related Sites