| 1. |
- Gomes, Ana Rita, et al.
(author)
-
A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite
- 2015
-
In: Cell Host and Microbe. - : Cell Press. - 1931-3128 .- 1934-6069. ; 17:3, s. 404-413
-
Journal article (peer-reviewed)abstract
- The genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq). To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison. We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants. Thus, parallel phenotyping of barcoded mutants unlocks the power of reverse genetic screening for a malaria parasite and will enable the systematic identification of genes essential for in vivo parasite growth and transmission.
|
|
| 2. |
- Pfander, Claudia, et al.
(author)
-
A scalable pipeline for highly effective genetic modification of a malaria parasite
- 2011
-
In: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 8:12, s. 1078-1082
-
Journal article (peer-reviewed)abstract
- In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.
|
|
