SummaryRMgm-5230
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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) : 35998188 |
MR4 number | |
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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 | RMgm-4478 |
Other information parent line | The mutant contains a modified/mutated actin-1 gene locus. It contains the wild-type actin 1 gene under control of the actin 1 5'-UTR whereas the 3'-UTR is from the dhfs gene. In addition, the yfcu::hdfr selection cassette is located in the 3'-UTR region. This mutant is used as an 'actin-1' recipient line for the introduction of mutated forms of actin-1. Constructs to integrate mutated forms of actin 1 integrate by double homologous integration at the 5'- and 3'-UTR regions of actin-1 thereby replacing the wild-type copy of actin 1 and the yfcu::hdfr selection cassette. These mutants are selected by negative selection using 5-FC |
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The mutant parasite was generated by | |
Name PI/Researcher | Yee M, Frischknecht F, Douglas RG |
Name Group/Department | Integrative Parasitology, Center for Infectious Diseases |
Name Institute | Heidelberg University Medical School |
City | Heidelberg |
Country | Germany |
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Name of the mutant parasite | |
RMgm number | RMgm-5230 |
Principal name | see below |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | ATET/SEPQ developed into mature ookinetes in a manner suggesting no defects with this mutant in early mosquito infection. While development remained unaffected, ATET/SEPQ had highly reduced numbers of moving ookinetes. Other mutants showed normal oocyst production, except for A272S(see below). |
Oocyst | With the exception of A272S, which showed slightly reduced oocyst levels, individual mutants displayed similar oocyst loads to the wild-type control experiment and thus proceeded normally through the initial stages of mosquito infection. The ATET/SEPQ mutant showed highly reduced total oocyst numbers. |
Sporozoite | Single actin mutant sporozoites were impaired in salivary gland invasion, yet only the subdomain 2 single mutant exhibited aberrant motility. Actin mutants are less transmissible due to deficiency in salivary gland invasion. |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation The mutants are generated using the an 'actin-1' recipient line (RMgm-4478) for the introduction of mutated forms of actin-1. Constructs to integrate mutated forms of actin 1 integrate by double homologous integration at the 5'- and 3'-UTR regions of actin-1 thereby replacing the wild-type copy of actin 1 and the yfcu::hdfr selection cassette. These mutants are selected by negative selection using 5-FC Protein (function) With the exception of A272S, which showed slightly reduced oocyst levels, individual mutants displayed similar oocyst loads to the wild-type control experiment and thus proceeded normally through the initial stages of mosquito infection. The ATET/SEPQ mutant showed highly reduced total oocyst numbers. Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1459300 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1246200 | ||||||||||||||||||||||||||
Gene product | actin I | ||||||||||||||||||||||||||
Gene product: Alternative name | ACT1; actin1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | Different mutants are described with different point mutations in (subdomains of) actin 1 | ||||||||||||||||||||||||||
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/yfcu | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | No | ||||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||||
Additional remarks genetic modification | Actin 1 ORF replacement constructs were generated as described before. Briefly, a pair of complementary primers of approximately 25 to 30 base pairs containing the mutation(s) of interest were designed. These mutagenesis primers (primers 1–10) were utilized to amplify the sequencing plasmid containing the codon-modified actin 1 ORF. After confirming that these amplicons were of the correct sizes via agarose gel electrophoresis, 1.5 μL of DpnI restriction endonuclease was directly added (New England Biolabs, #R0176S (20 U/μL)) into the remaining 15 μL PCR product. This reaction mix was incubated at 37°C/ 2 h to digest the parental methylated template. Next, 5 μL of this DpnI-digested PCR product was transformed into competent E. coli and the mutation verified via both restriction enzyme digestion and sequencing. The mutated ORFs were subsequently cloned into transfection vector Pb238 via BamHI and XbaI restriction sites. The transfection construct was linearised with SalI and PmlI, transfected into the actin recipient line (RMgm-4478) and integration selected by negative selection using 5-fluorocytosine (1 mg/ml in drinking water) [36,79]. This rendered a line free of the selection cassette and contained the desired change in actin sequence. Isogenic parasites from the transfection mixture of integrants and resistant recipient line was obtained by limiting dilution. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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