RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4600

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1441400; Gene model (P.falciparum): PF3D7_1226600; Gene product: proliferating cell nuclear antigen 2 (PCNA2) Name tag: GFP
Phenotype	Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;

Last modified: 18 March 2019, 15:45

*RMgm-4600

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) : 30865364
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	Not applicable
Other information parent line
top of page
The mutant parasite was generated by
Name PI/Researcher	Pradhan S, Dhar SK
Name Group/Department	Special Centre for Molecular Medicine
Name Institute	Jawaharlal Nehru University
City	New Delhi
Country	India
top of page
Name of the mutant parasite
RMgm number	RMgm-4600
Principal name	PbPCNA2GFP::hDHFR
Alternative name	PbPCNA2‐GFP
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
top of page
Phenotype
Asexual blood stage	PbPCNA2‐GFP expression in all blood stages (diffused expression in whole parasite)
Gametocyte/Gamete	PbPCNA2‐GFP expression in all blood stages (diffused expression in whole parasite)
Fertilization and ookinete	PbPCNA2‐GFP expression in gametocytes and ookinetes. In ookinetes the signal was higher in nucleus than cytoplasm
Oocyst	PbPCNA2‐GFP expression in oocysts (diffused expression in whole parasite)
Sporozoite	PbPCNA2‐GFP expression in sporozoites (mainly in cytoplasm)
Liver stage	PbPCNA2‐GFP expression in liver stages (diffused expression in whole parasite)
Additional remarks phenotype	Mutant/mutation The mutant expresses a C-terminal GFP-tagged version of PCNA2 Protein (function) Proliferating cell nuclear antigen (PCNA) proteins play a role in DNA replication and DNA repair pathways. The genome of Plasmodium parasites encodes for two PCNA proteins. P. falciparum PCNA1 (PfPCNA1) has been previously implicated in DNA replication as well as DNA repair, whereas role of P. falciparum PCNA2 (PfPCNA2) has been assigned to DNA repair, during the asexual stage of the parasite. Phenotype PbPCNA2‐GFP expression in all stages. Additional information Other mutants

Tagged: Mutant parasite with a tagged gene

top of page

Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1441400

Gene Model P. falciparum ortholog

PF3D7_1226600

Gene product

proliferating cell nuclear antigen 2

Gene product: Alternative name

PCNA2

top of page

Details of the genetic modification

Name of the tag

GFP

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

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

In order to generate the PCNA2 knockout‐targeting construct, a920 bp fragment was amplified from the 5′UTR region of PbPCNA2 and a 609 bp from the 3′UTR of the gene. Both UTRs were cloned in knockout vector flaking the GFP and DHFR cassette.

For generating the PbPCNA2‐GFP tagging construct, 1898 bp fragment, containing 999 bp of 5′UTR and full length PbPCNA2 coding sequence excluding the stop codon, was amplified from the parasite genomic DNA.

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

top of page