RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-652
|
||||||||
*RMgm-652| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene mutation |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 21899698 |
| MR4 number | |
| 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 cl15cy1 |
| Other information parent line | A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255). |
| top of page | |
| The mutant parasite was generated by | |
| Name PI/Researcher | E. Laurentino, C.J. Janse, A.P. Waters |
| Name Group/Department | Leiden Malaria Research Group |
| Name Institute | Leiden University Medical Center |
| City | Leiden |
| Country | The Netherlands |
| top of page | |
| Name of the mutant parasite | |
| RMgm number | RMgm-652 |
| Principal name | 1045cl1,cl2 |
| Alternative name | Δfactl-gam 2a |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
| top of page | |
| Phenotype | |
| Asexual blood stage | Not different from wild type |
| Gametocyte/Gamete | Normal numbers of female and male gametocytes are produced. Fertility of female gametes is comparable to wild type female gametes as shown by crossing with wild type male gametes. Male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility is strongly reduced |
| Fertilization and ookinete | Normal numbers of female and male gametocytes are produced. Fertility of female gametes is comparable to wild type female gametes as shown by crossing with wild type male gametes. Male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility is strongly reduced, resulting in strongly reduced ookinete formation. |
| Oocyst | Male gamete fertility is strongly reduced, resulting in strongly reduced ookinete formation. Residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony. |
| Sporozoite | Not tested |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation BLAST-based homology searches with PBANKA_123220 identified clear homology to the eukaryotic SPT16 protein (e.g. 30% identity, 41% similarity to SPT16 of Saccharomyces cerevisiae) with an identical arrangement of the Rttp106-like, SPT16 and M24 peptidase domains Attempts to disrupt the fact-l gene in P. berghei were unsuccessful (see RMgm-255), indicating an essential role of FACT-L for blood stage development/multiplication In the 'promoter-swap' mutant the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060). Transcription of fact-l in mutant gametocytes was down-regulated compared with wild-type gametocytes. Phenotype analyses of the promoter-swap mutant indicate that FACT-L plays an important role in the production of fertile male gametes. The promoter-swap mutant produced normal numbers of female and male gametocytes. Fertility of female gametes is comparable to wild type female gametes as shown by crossing with wild type male gametes. Male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility is strongly reduced, resulting in strongly reduced ookinete formation. Residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony. In the same paper successful 'promoter-swap' mutants are described in which the promoter of fact-l is replaced by the 'asexual blood stage specific’ promoter of PBANKA_091420 that is silent in gametocytes. These mutants are: Δfact-lgam1a, 1044 in cl15cy1; Δfactlgam 1b, 1076cl1, cl3 in 507cl2; Δfact-lgam 1c, 1134cl1,cl2 in 820cl1m1cl1. Phenotype analyses of a mutant expressing c-myc-tagged FACT-L (RMgm-655) showed that FACT-L is located predominantly in the parasite nucleus with evidence that a small proportion of FACT-L is located in the cytoplasm. Expression was detected in all blood stages with high levels of FACT-L in the nuclei of male gametocytes. |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1232200 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0517400 | ||||||||||||||||||||||||||
| Gene product | FACT complex subunit SPT16, putative | ||||||||||||||||||||||||||
| Gene product: Alternative name | FACT-L; FACT | ||||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||||
| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | 'promoter-swap' mutant; the promoter of fact-l replaced by the promoter of PBANKA_140060 | ||||||||||||||||||||||||||
| 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 | tgdhfr | ||||||||||||||||||||||||||
| Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||||
| Additional remarks genetic modification | DNA constructs were generated to exchange or ‘swap’ the endogenous fact-l promoter with a promoter of a gene that was expected not to be transcribed in gametocytes (PBANKA_140060). Vector pL0001 (http://www.mr4.org) for integration via double-cross-over homologous recombination, was used as the backbone for four different promoter-swap constructs. This vector contains the dhfr-ts gene of Toxoplasma gondii under the control of the P. berghei dhfr-ts promoter as selectable marker cassette. Three subcloning steps were performed to generate the promoter-swap constructs: first, a 539 bp fragment located 2 kb upstream of fact-l, was PCR-amplified from genomic DNA using primer pair 3190/3191 (ggggtaccTACTATAACATGGTTTTGCC (F); ccaagcttAGGATAAAAACATAGGTGTG (R)). This fragment represents the left flanking target region and was cloned into the KpnI/HindIII restriction sites of pL0001. As the right flanking target region, a 623 bp fragment of the open reading frame of fact-l was PCR amplified using primer pair 3192/3193 (gctctagaTGAACTTTGTAAAAACAAGC (F); tccccgcggGAATTTTCAATGGTAGTTATCA (R)). This fragment was cloned into the SacII/XbaI restriction sites of the vector. Finally, a 2 kb fragment of the 5'UTR promoter region of PBANKA_140060 was amplified from genomic DNA using the primer pair 3196/3197 (CGgaattcATTCGATGTTTACATAATGAC (F); CGggatccCGAATTGACACCATTTTTCC (R) and cloned into the EcoRI/BamHI sites of the vector. The promoter-swap construct (pL1313) was linearized with KpnI/SacII before transfection. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
| |||||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||||
