RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-827

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_1230700; Gene model (P.falciparum): PF3D7_0615900; Gene product: protein phosphatase, putative
Phenotype	No phenotype has been described

Last modified: 16 August 2013, 18:55

*RMgm-827

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	2
Reference (PubMed-PMID number)	Not published (yet)
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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	P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line	676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
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Attempts to generate the mutant parasite were performed by
Name PI/Researcher	J. Lin; C.J. Janse; S.M. Khan
Name Group/Department	Leiden Malaria Research Group
Name Institute	Leiden University Medical Center
City	Leiden
Country	The Netherlands

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_1230700

Gene Model P. falciparum ortholog PF3D7_0615900

Gene product protein phosphatase, putative

Gene product: Alternative name

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct used PCR construct double cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

GAACTCGTACTCCTTGGTGACGGGTACCATATATCATTCTACGCATATCCTAAATTTTGC

TTCCAAGTATTCATATTTGTATTTTAAGCTCTGTTTCCTATTTTATTTTTTAAACGGTTT

ATTTCTTAAATTTATATTTTTTTTTATTACACACACATTTCCATACAATCATGTGTGGTT

CTAAAAATGTATGAACATATTTATATAAAAAAAAAATATAATAAAAAATACCAAAGTATA

TTGCTTAATTCCTAATCATACTATTATTCCTTACATTTTAAAATGAAATTTTATAGTTAT

TAATAAAGTAAAAACATTTATTAAATATTTAAATGCACACCTATTTAATATCAAGATATA

TGTAATATTATGCATTCTAATAAAGCTTCTTTATAGTCTTTTTAATTATTATATTGCAGT

AGTAACAACAATTTGTATTATTTTTATTTTTTATGATTATTATTCTTACTTTTGTTGTTA

TTATTATTCAATGATTATATATTATAGATATAATAAAAAAAAATGATAAAAGCTAGTTTC

ACAGTAATTAACAATAACAACTTTGAATATTTTTATTTTATTATAAGCTAAGGAAAAATG

TGTGTCATAAAAAAGGACTATATACATGATTGTAAAATAATCCCACAAATGCATAAATGT

GCGATTATTTATATAACTCCAAACTAATACATGCTAAGAGGTCGACGATGCTTGTAGATG

AGTTAAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACT

TTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACC

AGTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAAT

GTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAA

AATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAAC

TAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAA

TATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCA

AAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATT

TAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATAT

ATATTTGCTTATAAATAAATAAAAATTTTATAAAAATGGTTGGTTCGCTAAACTGCATCG

TCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGGCCACCGCTCA

GGAATGAATTCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGA

ATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAA

AGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGTGCACATT

TTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATA

AAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACC

CAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTT

TTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTG

ATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAAT

GTTCGTTTTTCTTATTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGT

GTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATG

AACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAA

TAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATA

TTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTT

TACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAA

ATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACAT

ATAAACACAAATGATGTTTTTTCCTTCAATTTCGGATCCACTAGCAAGAGAAATGAAGGA

ACAGATTTCGATAAGCAATGATTTAAATGATTGGATCATAAAAATAAATAAATATATTTT

AAAAGAAGATGGAAAACAAATAAATATAGGCAATGTTCCATTTAATTTAATTAAATTTAA

CAGATTATATGAATGTTTACACCCATGCAGAGTAATAGGTGATTATGATTTAAAAGGGAA

ATGTAATGATTCAAATTTTATTTTGTCTAACAATTCTAATATATATAAATATGACATGAA

TAATATAAATTTGATTAATAATATTCATTATATTTATAAAATACAAAAATGTATAAATTG

CAAAATGATATATTTATTGAATAGTTATGGAAAAATAAATTCAATCAAAAATATTGCTTT

ACTTAATACGAAAAGCGAATTATATCAAAATAGTAACATACTTGTAAATTACATTGAAAA

AGAACAAACAAACGATTGTTGTAATAATGTGATAGATAAATATGAAAGTTATAATTCCTT

TGAAGAAAAACTTGAAAGTATTATGACCATTTATAAAGACAATGAAAATATTAATGACTC

TAAAAAAATGAAAAAAAAAAATACTAACTTATGTTTGAATGAATCATTATCTCCATATTT

GCATATTAAAAATAATTCATTAAATAAAAATTATGTATATCGATCAGGAAAAAAAGAAAA

CAAACATTTTTTTCATTTGCTAATTATCGCATCGGAAGTACTGCTGAGTGTCAATGACCA

ACCT

Restriction sites to linearize plasmid

Partial or complete disruption of the gene Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite hdhfr

Promoter of the selectable marker eef1a

Selection (positive) procedure pyrimethamine

Selection (negative) procedure No

Additional remarks genetic modification The protein contains the following domain: IPR001932 - Protein phosphatase 2C-related (pp2c).

Several unsuccessful attempts to disrupt the gene (exp 1782 and 1957 with PCR constructs 1782 and 1957 respectively).

The unsuccessful attempts indicate an essential role during blood stage development/multiplication.

The locus was unsuccessfully targeted using a linear construct that was generated using a 2-step, anchor tagging PCR method (for primer details see below).

The 5’- and 3’ targeting regions of the gene were PCR amplified from genomic DNA using primer pairs 1&2 and 3&4. Primers 2 and 3 have 5’-terminal extensions homologues to the hDHFR selectable marker cassette. Primers 1 and 4 both have a 5’-terminal overhang with an anchor-tag which serves as a primer site in the 2nd PCR reaction.

The target fragments from the first PCR reaction were annealed to either side of the selectable marker cassette by PCR with anchor-tag primers 5 and 6, resulting in the 2nd PCR product. The hDHFR selectable marker cassette used in this reaction was digested from pL0040 using restriction enzymes XhoI and NotI. pL0040 is available from The Leiden Malaria Research Group.

To remove the anchor-tags from the final KO construct, and to eliminate contaminating pL0040, the 2nd PCR product was digested with Asp718/ScaI and DpnI respectively. DpnI only cuts methylated plasmid DNA but not the PCR product.

For a more detailed understanding of the 2-step anchor tagging PCR method, please see the following publications:

J.W. Lin, S.M. Khan et al
A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites
PLoS One. 2011;6(12)

T. Annoura et al
Assessing the adequacy of attenuation of genetically modified malaria parasite vaccine candidates
Vaccine. 2012 Mar 30;30(16):2662-70

One more unsuccessful attempt to disrupt the gene was carried out using a second linear construct that was generated using a 2-step, anchor tagging PCR method. This construct (PCR1827) used the same targeting regions but differed by having the hdhfr::yfcu selectable marker cassette. The attempt was made in the WT cl15cy1 background.

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	GAACTCGTACTCCTTGGTGACGGGTACCATATATCATTCTACGCATATCC
Additional information primer 1	L5844: 5’- pp2c targeting region F (Asp718)
Sequence Primer 2	CATCTACAAGCATCGTCGACCTCTTAGCATGTATTAGTTTGGAG
Additional information primer 2	L5845: 5’- pp2c targeting region R
Sequence Primer 3	CCTTCAATTTCGGATCCACTAGCAAGAGAAATGAAGGAACAG
Additional information primer 3	L4725: 3’- pp2c targeting region F
Sequence Primer 4	AGGTTGGTCATTGACACTCAGCAGTACTTCCGATGCGATAATTAGC
Additional information primer 4	L4673: 3’- pp2c targeting region R (ScaI)
Sequence Primer 5	GAACTCGTACTCCTTGGTGACG
Additional information primer 5	L4661; anchor tag primer
Sequence Primer 6	AGGTTGGTCATTGACACTCAGC
Additional information primer 6	L4662: anchor tag primer

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