top of page |
Details of the target gene |
Gene Model of Rodent Parasite |
PBANKA_1315700
|
Gene Model P. falciparum ortholog |
PF3D7_1452000
|
Gene product | rhoptry neck protein 2 |
Gene product: Alternative name | RON2 |
top of page |
Details of the genetic modification |
Short description of the mutation | The mutant contains a mutated ron2 gene with two LoxN sites (up- and downstream of the gene) |
Inducable system used | No |
Short description of the conditional mutagenesis | Not available |
Additional remarks inducable system |
|
Type of plasmid/construct | (Linear) plasmid double cross-over |
PlasmoGEM (Sanger) construct/vector used | No |
Modified PlasmoGEM construct/vector used | No
|
Plasmid/construct map |
|
Plasmid/construct sequence |
|
Restriction sites to linearize plasmid |
|
Selectable marker used to select the mutant parasite | hdhfr |
Promoter of the selectable marker | unknown |
Selection (positive) procedure | pyrimethamine |
Selection (negative) procedure | No |
Additional remarks genetic modification | In order to target different genes of interest, we first generated a generic plasmid, pDownstream1Lox (Addgene #164574), containing a GFP-2A-hDHFR cassette under the control of a P. yoelii hsp70 promoter and followed by the 3’UTR of P. berghei calmodulin (cam) gene and a single LoxN site. The plasmid also contains a yFCU cassette to enable the elimination of parasites carrying episomes by negative selection with 5-fluorocytosine. The ama1Con plasmid was designed to excise only ~30 bp downstream of P. berghei ama1 3’UTR. Two fragments were inserted on each side of the GFP-2A-hDHFR cassette of the pDownstream1Lox plasmid: a 5’ homology region (HR) homologous to the terminal portion of ama1 (ORF and 3’ UTR) followed by a single LoxN site, and a 3’ HR homologous to a sequence downstream of the 3’ UTR of ama1 gene. The ama1Dutr plasmid was assembled similarly to the ama1Con construct except that the 5’ HR consisted in the terminal portion of ama1 ORF followed by a LoxN site and the 3’ UTR of P. yoelii ama1, to allow excision of the 3’UTR upon rapamycin activation of DiCre. The ama1cKO plasmid was designed to introduce a single LoxN site upstream of ama1 in the ama1Conrapa parasites, which already contained a residual LoxN site downstream of the gene. To generate the ama1cKO plasmid, the pDownstream1Lox vector was first modified to remove the downstream LoxN site. Then, a 5’ HR and a 3’ HR, both homologous to sequences located upstream of ama1 gene, were cloned into the modified plasmid on each side of the GFP-2A-hDHFR, with a single LoxN site introduced upstream of the GFP-2A-hDHFR cassette. To generate ron2cKO and ron4cKO constructs, two separate plasmids, P1 and P2, were generated to insert a LoxN site upstream of the promoter and downstream of the gene of interest, respectively, in two consecutive transfections. P1 plasmids were constructed by insertion of 5’ and 3’ HR on each side of the GFP-2A-hDHFR cassette in the pDownstream1Lox plasmid, with a second LoxN site introduced upstream of the GFP cassette. The 5’ HR and 3’ HR correspond to consecutive fragments located in the promoter region of the GOI. Because the intergenic sequence between ron4 gene and its upstream gene is short, and in order to maintain expression of the upstream gene and exclude any unwanted duplication and spontaneous recombination events, we introduced the 5’ HR of ron4 in two fragments, with fragment 1 corresponding to the region just upstream of the ORF while fragment 2 corresponded to the 3’ UTR from the P. yoelii ortholog of the upstream gene. P2 plasmids were constructed in a similar manner by insertion of a 5’ HR and a 3’HR on each side of the GFP-2A-hDHFR cassette in the pDownstream1Lox plasmid. The 3’ HR regions corresponded to the 3’ UTR sequences of RON2 or RON4, respectively. For both target genes, the 5’ HR was divided into two fragments, where fragment 1 corresponded to the end of the ORF followed by a triple Flag tag, and fragment 2 corresponded to the 3’ UTR from the P. yoelii ortholog gene, in order to avoid duplication of the 3’ UTR region and spontaneous recombination |
Additional remarks selection procedure | |
Primer information: Primers used for amplification of the target sequences 
Primer information: Primers used for amplification of the target sequences
Sequence Primer 1 | |
Additional information primer 1 | |
Sequence Primer 2 | |
Additional information primer 2 | |
Sequence Primer 3 | |
Additional information primer 3 | |
Sequence Primer 4 | |
Additional information primer 4 | |
Sequence Primer 5 | |
Additional information primer 5 | |
Sequence Primer 6 | |
Additional information primer 6 | |
|
|
top of page |