RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-5223
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Last modified: 9 August 2022, 17:11
*RMgm-5223| 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) : 23173711 |
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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 | Pornthanakasem W, Leartsakulpanich U |
| Name Group/Department | National Center for Genetic Engineering and Biotechnology |
| Name Institute | National Center for Genetic Engineering and Biotechnology |
| City | Khlong Luang, Pathum Thani |
| Country | Thailand |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1450200 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1235600 | ||||||||||||||||||||||||
| Gene product | Serine hydroxymethyltransferase, putative | ||||||||||||||||||||||||
| Gene product: Alternative name | SHMT | ||||||||||||||||||||||||
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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 |
The unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth/multiplication. Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites. Two genes have been identified in Plasmodium spp. encoding a cytosolic SHMT (cSHMT) and putative mitochondria SHMT (mSHMT). Plasmodium falciparum contains PF3D7_1235600, a previously characterized cSHMT gene (Pfcshmt), and PF3D7_1456100, a putative gene of mSHMT (Pfmshmt). Plasmids for the study of gene knockout in Plasmodium berghei ANKA strain were constructed based on the sequence of pL0017 vector (The Malaria Research and Reference Reagent Resource Center; MR4), which contains Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (Tgdhfr/ts) and green fluorescent protein gene (gfp) expression cassettes for pyrimethamine (PYR) selection and fluorescence detection of transfected parasites. The 553 and 1,018 bp of PCR amplicons, corresponding to 5′- and 3′UTR of Pbcshmt (PBANKA_145020) respectively, were produced initially from P. berghei genomic DNA (gDNA). The 5′UTR fragment was inserted into pL0017 at Hind III site, while the 3′UTR fragment was inserted at Kpn I and Sac II sites respectively. This construct, pL0017_Δshmt, was used in the knockout study. For allelic replacement construct, gfp in pL0017_Δshmt was replaced with Plasmodium vivax cshmt (Pvcshmt; PVX_100730) and named pL0017_(Pv)Δshmt. | ||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | |||||||||||||||||||||||||
| 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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