RMgmDB - Rodent Malaria genetically modified Parasites
Back to search resultsSummaryRMgm-1119
|
||||||||||
Last modified: 6 September 2014, 14:04
*RMgm-1119| 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 | ≥ 5 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 25185663 |
| top of page | |
| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei ANKA |
| Name parent line/clone | P. berghei ANKA 2.34 |
| Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
| top of page | |
| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | Tremp AZ; Dessens JT |
| Name Group/Department | Pathogen Molecular Biology Department, Faculty of Infectious and Tropical Diseases |
| Name Institute | London School of Hygiene and Tropical Medicine |
| City | London |
| Country | UK |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_0402700 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0304100 | ||||||||||||||||||||||||
| Gene product | inner membrane complex protein 1e, putative | ||||||||||||||||||||||||
| Gene product: Alternative name | IMC1e; ALV2 | ||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||
| 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 | pbdhfr | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | The unsuccessful attempts to knock-out imc1e indicates an essential role of IMC1e in blood stage development/multiplication. The three motile and invasive stages (zoites) of Plasmodium species (i.e. ookinetes, sporozoites and merozoites), as well as zoites of other apicomplexan parasites, possess a similar cortical structure termed the pellicle. The pellicle is essentially made up of the plasma membrane and an underlying double membrane structure termed the inner membrane complex (IMC). Closely associated with the IMC on its cytoplasmic side is a network of intermediate filaments termed the subpellicular network (SPN), which supports the pellicular membranes and provides mechanical strength to the cell. Several members of an Apicomplexa-specific family of proteins termed IMC1 proteins have been identified as components of the SPN. Structurally related proteins from ciliates and dinoflagellate algae have since been added to this protein family renamed ‘alveolins’, which now defines the Alveolata infrakingdom. In the genus Plasmodium, the number of members of the alveolin family has risen to 12, which are encoded by conserved and syntenic genes. The alveolin family members display differential expression between the three zoite stages of the parasite, with the largest repertoires present in the ookinete and sporozoite according to proteomic studies. It has been shown in the rodent malaria species Plasmodium berghei that the disruption of individual alveolin family members expressed in sporozoites (PbIMC1a), in ookinetes (PbIMC1b) or in both these zoites (PbIMC1h) results in morphological abnormalities that are accompanied by reduced tensile strength of the zoite stages in which they are expressed. Besides roles in morphogenesis and mechanical strength, the Plasmodium alveolins are also involved in gliding motility in both ookinetes and sporozoites, most likely through interactions with components of the glideosome that are situated within the pellicular cytoplasm. PbIMC1e (PBANKA_040270) is composed of 512 amino acids encoded by a single exon. Sequence conservation is limited to an IMCp domain (Pfam12314) in their central portions. The Plasmodium imc1e locus is located directly downstream of its family member imc1a in the opposite orientation. Analysis of a mutant expressing fluorescent-tagged IMC1e (GFP; RMgm-1121) showed weak expression in blood stages whereas mature ookinetes displayed much stronger GFP fluorescence that was distributed predominantly in the cell cortex, consistent with a pellicular localization of the protein. PbIMC1e was also present in an unknown structure situated at one extremity (posterior) of the ookinete. Sporulated oocysts and sporozoites also displayed GFP florescence, which localized to the periphery of the sporozoites. Sporozoites possessed a small fluorescent spot at one extremity (posterior). Evidence is presented for temporal recruitment to the SPN of ookinetes. The entire pbimc1e coding sequence plus ca. 0.58 kb of upstream sequence was PCR amplified from genomic DNA with primers pDNR-IMC1e-F ACGAAGTTATCAGTCGACGGTACCGCATAAATTAACTTAGTTTCATTGAACTTC) and pDNR-IMC1e-R (ATGAGGGCCCCTAAGCTTTCGTTTAAGACGGGTGGTAC) and cloned into SalI/HindIIIdigested pDNR-EGFP by in-fusion cloning to give plasmid pDNR-IMC1e/GFP. The 3′UTR of pbimc1e was amplified with primers pLP-IMC1e-F (ATATGCTAGAGCGGCCTTTGGCTTCGATTTTTGTG) and pLP-IMC1e-R (CACCGCGGTGGCGGCCTAACAGCATTATGAAAGATTGGC) and the resulting ca. 0.87 kb fragment cloned into NotI-digested pLP-hDHFR by in-fusion cloning to give plasmid pLPhDHFR/IMC1e. The pbimc1e/gfp-specific sequence from pDNR-IMC1e/GFP was transferred to pLP-hDHFR/IMC1e by Cre/loxP recombination to give the final construct pLPIMC1e/GFP. This plasmid served as template in a PCR-based site-directed mutagenesis using primers MC1e-KO-F (AATATGTGATGAGTAAAGGAGAAGAACTTTTCAC) and IMC1e-KO-R (TTACTCATCACATATTTAGTGCCACAATTGC). The resulting PCR product was circularized using infusion to give plasmid pLP-IMC1e-KO. In this plasmid, the pbimc1e coding sequence except for the first amino acids has been removed. | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
|
Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
| |||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||