Figure 1.
Replacement of PfEMP1 Exon I with the Selectable Marker bsd
(A) Schematic diagram showing integration of the bsd cassette from the construct pVbBB/IDH into the NF54 var PFB1055c locus through double crossover recombination.
(B) Southern analysis was performed using HpaI digested DNA and probed for bsd and pUC18. The size of the DNA fragment is shown on the left. The absence of a 6.7-kb band when using a bsd probe and the absence of hybridization with plasmid backbone are indicative of a double crossover recombination event, resulting in replacement of exon I with the bsd cassette. This arrangement was confirmed by additional Southern blots and by sequencing across the sites of integration.
Figure 2.
Outline of Experimental Design
NF54 parasites were cloned by limiting dilution to create a clonal population (NF54/C3) that predominantly expressed PFD1005c. These parasites were transfected with the plasmid pVbBB/IDH and the clone B12E3 containing a double crossover integration was isolated. After blasticidin selection, B12E3 exclusively expressed bsd, and all other var genes were transcriptionally silent. Drug pressure was then removed for two months during which time the var gene expression pattern became heterogenous. This heterogeneous B12E3 population was then re-cloned and clone DC-J isolated. DC-J predominantly expressed the var gene PFD1015c and had silenced the bsd gene. Growth of DC-J back under blasticidin pressure however results in reactivation of bsd expression and silencing of the rest of the var gene family, thus demonstrating the reversibility of the phenomenon.
Figure 3.
Analysis of Levels of Transcription of the Entire var Family
All values are presented as relative copy number to the housekeeping gene seryl-tRNA synthetase (PF07_0073).
Top panel: NF54 parasites were cloned by limiting dilution, and var gene expression was measured by Q-RT-PCR as soon as the culture reached the required parasitemia, approximately 6 wk after plating. The clone NF54/C3, which was used to generate all transgenic lines, was predominantly expressing var PFD1005c (located on Chromosome 4 internal cluster) while expression of the rest of the var family was virtually undetectable.
Second panel: The recombinant line B12E3 growing under blasticidin pressure only transcribed bsd (red), while transcription levels of the rest of the var family was close to zero (blue).
Central panel: Expression of additional var genes was easily observed after the parasites were grown for 10 wk without drug pressure. At this point the culture is transcriptionally heterogeneous and bsd is no longer the dominant gene. Five genes were expressed at levels equal to or greater than the control (copy number = 1).
Fourth panel: The transcription pattern of clone DC-J that was re-cloned from this culture represents a population that had switched away from bsd and now predominantly expresses var PFD1015c. Applying blasticidin to DC-J resulted in the selection of parasites that had switched back to exclusively expressing bsd (bottom panel).
Figure 4.
Integration of the bsd Expression Cassette into a Telomeric var Locus
(A) Schematic diagram showing integration of the construct pVbBB/IDH into the var PFL0020w locus through a single homologous recombination event within the var upstream region.
(B) Southern analysis was performed using BamHI-digested gDNA and hybridized with probes specific to bsd and pUC18. The linearized plasmid within the multiple copy concatameric insertion appears as a high intensity 6.7 kb band with both probes. The 6.1-kb bsd band and the ~13-kb pUC18 band correspond to fragments flanking the site of integration. This arrangement was confirmed by additional Southern blots and by sequencing across the sites of integration.
(C) Analysis of the level of transcription from each var gene in the genome shows that selection for bsd expression results in silencing of the entire gene family. The only “on” gene in parasites growing under drug pressure (top panel) is the locus expressing the bsd cassette. Analysis performed 10 wk after drug removal demonstrated that the culture has become transcriptionally heterogenous (bottom panel) with the majority of the genes upregulated, of which nine are expressed at levels greater than the control (relative copy number > 1).
Figure 5.
Integration of a bsd Expression Cassette into an Internal Chromosomal var Locus
(A) Schematic diagram showing integration of the construct pVcBB/IDH into the var PFL1960w locus through a single homologous recombination event within the var upstream region.
(B) Southern analysis was performed using BamHI and StyI digested gDNA hybridized with probes to bsd and pUC18. The 8.3-kb bsd band and the 13.2-kb pUC18 band correspond to the fragments flanking the integrated concatamer. The doublet at ~7.1 kb corresponds to fragments from within the integrated concatamer.
(C) Analysis of the level of transcription of each var gene within the parasite genome shows that selection with blasticidin results in silencing of all members of the var gene family. The only var locus expressed by parasites grown under drug pressure (top panel) was the gene that contained the integrated bsd expressing plasmid. 10 wk after drug removal, the culture became transcriptionally heterogenous (bottom panel).
Figure 6.
PfEMP1 Is Not Expressed in the Knock-Out Transgenic Lines
Western blot analysis of whole cell extracts isolated from NF54 and the three transgenic lines B12E3, B15C2, and C7G12 growing under blasticidin pressure. Extract were probed with antibodies to either the conserved C terminus of PfEMP1 (α-ATS) or to the ER protein Pf39 (α Pf39). α-ATS signal appears only in NF45 parasites and not in any of the transgenic lines.
Figure 7.
An Active var Promoter on an Episome Is Exclusively Expressed
NF54/C3 parasites stably carrying the episome pVBB/IDH were grown under blasticidin pressure.
(A) Plasmid map of pVBB/IDH.
(B) Transcription levels were then measured by Q-RT-PCR and indicated that the parasites exclusively express bsd while all endogenous var genes are silent.