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Fig. 2 | Parasites & Vectors

Fig. 2

From: Plasmodium berghei Brca2 is required for normal development and differentiation in mice and mosquitoes

Fig. 2

A–D Generation of PbBrca2-knockout (KO) parasites. A Schematic representation of targeted PbBrca2 disruption. Targeting vector (bottom) containing puromycin resistance gene (PuromycinR) upstream (5′) of PbEf1α and downstream (3′) of PbDhfr-ts was integrated into the PbBrca2 locus (top) by double crossover. Arrows indicate genomic DNA (B) and reverse transcription (RT)-polymerase chain reaction (PCR) (C) primers. Black bar indicates southern blot probe. B PbBrca2 locus amplified in wild type (WT) and PbBrca2-KO1 (KO1) and PbBrca2-KO2 (KO2) clones. In the WT parasite, PCR products were longer than those detected for KO1 and KO2 parasites. C PbBrca2 KO was detected by southern blot. EcoRV-digested genomic DNA from WT and KO parasites demonstrated hybridization with upstream PbBrca2. KO1 and KO2 clones detected relatively short bands. D To analyze mRNA-level PbBrca2 expression, RT-PCR was performed using primers amplifying the region between PbBrca2 exon 3 and exon 4 in blood-stage parasites. PbBrca2 PCR products were detected for WT but not for KO1 or KO2 parasites. Amplified 18S ribosomal RNA was the positive control. M DNA size marker

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