Mutant/mutation
The mutant expresses blasticidin S deaminase (bsd) under control of the 5'- and 3'-regulatory sequences of HSP70

Protein (function)
The blasticidin S deaminase (bsd) gene is isolated from Aspergillus terreus; it confers blasticidin S (blasticidin) resistance and is widely used for genetic manipulation of eukaryotic cells.
As blasticidin is toxic to rodents and thus cannot be used for selection of P. berghei in vivo, a pac-puromycin system was developed based on a short-term in vitro culture method

Phenotype
Mutant blood stages have decreased sensitivity to blasticidin compared to wild type blood stages (see additional information)

Additional information
We analyzed the IC50 values for blasticidin of wild type parasites (WT), pyrimethamine resistant pbdhfr-ts marker integrated parasites (PBDHFR), and pXL/hdhfr-bsd-egfp transfected parasites that were selected by pyrimethamine (BSD). The IC50 values were 29.87 ± 12.55 μg ml-1 for WT, 27.13 ± 8.47 μg ml-1 for PBDHFR, and 159.15 ± 103.54 μg ml-1 for BSD. The IC50 value of BSD tended to be increased, as compared with that of WT (p=0.0684) and PBDHFR (p=0.0637). BSD showed significantly decreased susceptibility to blasticidin at the concentrations above the IC70 value compared with WT and PBDHFR (p < 0.05).
Drug sensitivity test described above confirmed that the optimum concentration of blasticidin was 500 μg ml-1 (approximately IC70 value of BSD and 5-fold IC90 value of PBDHFR). The in vitro selection procedure for the pXL/hdhfr-bsd-egfp transfected parasite was performed three times with the 500 μg ml-1 concentration of blasticidin, and the egfp-expressing mutant ratio was monitored after each selection. The ratio was 3.29 ± 1.57, 92.33 ± 1.97, and 94.64 ± 1.98 (Mean ± S.D.) % after each selection, respectively. It took 1-2 days from transfection to 1st selection (parasitemia 1.89 ± 1.53%), 6-7 days from 1st to 2nd selection (parasitemia 1.46 ± 0.23%), and 5 days (parasitemia 0.98 ± 0.51%) from 2nd to 3rd selection.
Two clones were isolated from the mutant parasites population by in vivo limiting dilution method to confirm that bsd was integrated into the genome. Southern blot analysis confirmed that bsd was integrated into the genome.

We constructed a targeting vector that contained a bsd expression cassette which replaced the gfp expression cassette of the GFP-expressing mutant (PbDHFR-GFP) through double crossover homologous recombination. The targeting vector was transfected to PbDHFR-GFP, and the gfp negative mutant ratio was monitored after each in vitro selection. The ratio was 4.74 ± 0.99, 6.88 ± 0.69, and 98.57 ± 2.35 (Mean ± S.D.) % after each selection, respectively. It took 2-3 days from transfection to 1st selection (parasitemia 1.09 ± 0.92%), and 5-6 days from 1st to 2nd selection (parasitemia 1.80 ± 0.86%). Two clones were isolated and Southern blot analysis confirmed that bsd was integrated into the genome. A typical transfection line was also analyzed using flow cytometry. More than 99% of the parasites did not express GFP after the second selection. Therefore, bsd can be used for gene targeting.

In conclusion: Blasticidin S deaminase (bsd) can be used as  a (additional) selectable marker for genetic modification of P. berghei
However, to be able to use bsd as a (additional) selectable marker for genetic modification of P. berghei multiple rounds of in vitro selection and in vivo (in mice) propagation are required to select for the desired mutants

Other mutants