RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-4599
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Last modified: 18 March 2019, 15:32
*RMgm-4599| Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
| The following genetic modifications were attempted | Gene disruption |
| Number of attempts to introduce the genetic modification | 3 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30865364 |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei ANKA |
| Name parent line/clone | Not applicable |
| Other information parent line | |
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| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | Pradhan S, Dhar SK |
| Name Group/Department | Special Centre for Molecular Medicine |
| Name Institute | Jawaharlal Nehru University |
| City | New Delhi |
| Country | India |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1441400 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1226600 | ||||||||||||||||||||||||
| Gene product | proliferating cell nuclear antigen 2 | ||||||||||||||||||||||||
| Gene product: Alternative name | PCNA2 | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | (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 | |||||||||||||||||||||||||
| Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | To pinpoint the significance of PCNA2 in the parasite lifecycle, three consecutive attempts were made to knock out the gene by double homologous recombination at the wild‐type pcna2 locus. However, after resistant parasites were detected, post selection using pyrimethamine drug pressure and insertion was confirmed by diagnostic PCR, attempts to obtain the clonal population (clones) of pcna2 KO parasites were unsuccessful. Proliferating cell nuclear antigen (PCNA) proteins play a role in DNA replication and DNA repair pathways. The genome of Plasmodium parasites encodes for two PCNA proteins. P. falciparum PCNA1 (PfPCNA1) has been previously implicated in DNA replication as well as DNA repair, whereas role of P. falciparum PCNA2 (PfPCNA2) has been assigned to DNA repair, during the asexual stage of the parasite. See also RMgm-3947 for evidence of knock-out of the PCNA2 gene (and with normal growth of blood stages) In order to generate the PCNA2 knockout‐targeting construct, a920 bp fragment was amplified from the 5′UTR region of PbPCNA2 and a 609 bp from the 3′UTR of the gene. Both UTRs were cloned in knockout vector flaking the GFP and DHFR cassette. For generating the PbPCNA2‐GFP tagging construct, 1898 bp fragment, containing 999 bp of 5′UTR and full length PbPCNA2 coding sequence excluding the stop codon, was amplified from the parasite genomic DNA. | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
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