RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4479

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1245300; Gene model (P.falciparum): PF3D7_0530900; Gene product: formin 1 Name tag: EGFP
Phenotype	Sporozoite;

Last modified: 16 August 2018, 16:59

*RMgm-4479

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene tagging
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 30011270
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	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Douglas RG, Frischknecht F
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-4479
Principal name	Formin 1::eGFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	No
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Phenotype
Asexual blood stage	Not tested
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	The mutant has not been analysed in detail. Formin 1::eGFP has a distinct localisation to the apical tip of the sporozoite
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses a C-terminal GFP-tagged version of formin-1 Protein (function) Formin 1 is an actin binding protein (ABP) (and regulates actin polymerization) Phenotype The mutant has not been analysed in detail. Formin 1::eGFP has a distinct localisation to the apical tip of the sporozoite Additional information Other mutants

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1245300

Gene Model P. falciparum ortholog

PF3D7_0530900

Gene product

formin 1

Gene product: Alternative name

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Details of the genetic modification

Name of the tag

EGFP

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

Type of plasmid/construct

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

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

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

In order to generate the endogenous reversibly switchable enhanced GFP (rseGFP) Formin 1 (PBANKA_1245300) tag, the PlasmoGEM system was used. The intermediate stage tagging vector PbGEM-int-309594 was ordered from PlasmoGEM. Also, the recombination vector GW R6K Emerald was used. The coding region of rseGFP (vector pQE31, a kind gift of Mhlanga lab) was amplified with primers 17 and 18 and cloned with SacII and ApaI into the GW R6K Emerald vector, replacing the coding region of Emerald with rseGFP. The ligated vector was transformed into pir1 cells and the rseGFP sequenced. Using the Gateway reaction, the GW R6K vector and the intermediate vector PbGEM-int-309594 were combined and transformed into TSA cells.

Additional remarks selection procedure

Primer information: Primers used for amplification of the target sequences Click to view information

Primer information: Primers used for amplification of the target sequences Click to hide information

Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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