RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-819

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_1127000; Gene model (P.falciparum): PF3D7_0628300.1; Gene product: choline/ethanolaminephosphotransferase, putative (CEPT)
Phenotype	No phenotype has been described

Last modified: 16 August 2013, 18:40

*RMgm-819

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	4
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_1127000

Gene Model P. falciparum ortholog PF3D7_0628300.1

Gene product choline/ethanolaminephosphotransferase, putative

Gene product: Alternative name CEPT

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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

AGGTTGGTCATTGACACTCAGCAGTACTGATCATTAGCATTATGGTGTGGTGGTAATGCT

GTTTATAAACGAAGAAAAGCATATACCTTTATTAAAAATGTTGACATTTATAATAATGTT

AATAATTAAAATATTTAATTACCTTTAATAGTTCGTTTTAGTTGCAATGTACATATATGT

ATTGTAGCACATTTTGTTTTTATTTCGAATTTTTAAATGTTACAAACATGCATAGCTTAT

TACAACGCCATTTCTACATTTCGTTTATTTATTATATTTTGTTATTTATTAACTTATTTT

ATTATAATACTATTTTAATAATTTTTTTACCCTTTGCACTTTTTTTTCTGAATTTTACAT

TCCCTTTAAATTTCTTTACTCATCTATTTATTATTTTTTTTTTAATTTATTTATTAGGAT

ACCATAATTTATTTTACTTTGCATATATTTATTTACCTACCTTTGTGCATATTATTATTT

ACAATTTGAATATTCATTTTCACATTACATAAAAATACCATATATGCTTGTTTTTATTTT

TTCTCATTTAATTTATATGATTAAGCTATTACATAGCGATGTATATTTTACCCGTCCTAG

TGGATCCGAAATTGAAGGAAAAAACATCATTTGTGTTTATATGTTTTTTTTTAATTTTTC

AACTTTTTTTATTTAACTATGCTAATCATATTATAAACATTTTAAAAATCAAGTCCAACT

ATTTATGAATCATTGAAGAGACAACATAATTATAACAGTAAAAATAATAATAATAATAAT

AATTTTGTCTTATTGTTTCAAACATATTATGTTCCATAAATATCTAAATTTTTAATATGG

TCATAAATTCATAGGATTTAATTTATATAAAATATGGCTATTCATACTAGCCATTTTATG

TGTGTAAAGTTTTTTTTTTCTGAACAAAAAATAAAATGTTCATAATTTTAGCTATTTACA

TGCATGTGCATGCACAAAAAAAAATATGCACACAACATACACATTTTTACAGTTATAAAT

ACAATCAATTGGTATAAATATATAAATAAGAAAAACGAACATTAATCATTCTTCTCATAT

ACTTCAAATTTGTACTTAATGCCTTTCTCCTCCTGGACATCAGAGAGAACACCTGGGTAT

TCTGGCAGAAGTTTATATTTCTCCAAATCAATTTCTGGAAAAAACGTGTCACTTTCAAAG

TCTTGCATGATCCTTGTCACAAATAGTTTAAGATGGCCTGGGTGATTCATGGCTTCCTTA

TAAACAGAACTGCCACCAACTATCCAGACCATGTCTACTTTATTTGCTAATTCTGGTTGT

TCAGTAAGTTTTAAGGCATCATCTAGACTTCTGGAAAGAAAATGTGCACCTTGTGGAGGT

TCCTTGAGTTCTCTGCTGAGAACTAAATTAATTCTACCCTTTAAAGGTCGATTCTTCTCA

GGAATGGAGAACCAGGTCTTCTTACCCATAATCACCAGATTCTGTTTACCTTCTACTGAA

GAGGTTGTGGTCATTCTCTGGAAATATCTGAATTCATTCCTGAGCGGTGGCCAGGGCAGG

TCCCCGTTCTTGCCGATGCCCATGTTCTGGGACACAGCGACGATGCAGTTTAGCGAACCA

ACCATTTTTATAAAATTTTTATTTATTTATAAGCAAATATATATTTTTATATATTTATAC

ACAAAGTATATATATTTTAGGAAATATTAAAATAAATTTAAATTTTGAAGGCAAATGAAA

AAATTACGTTTTTTGTTTCACAACAATTATATTTATTTTTTGAATTAATTTACATATTAT

TTTATATTAAAATTATAAAATAAAAAAAACATTACATATATTAATTATATTATTATATGA

GGATTTTTTTATAACATAGAAATATAAATAATTACGACTAGTTAATAAAGGGCACTTTAA

TTTTTATGCATAATTAGCTTATATTTAGTGCATATTTATTTTTATACATATTTTTATATA

TTTTTTGATTTACATGGCGTTTTATGTTTATATGTATAACATTATATGCATAGTATTTTT

TTGAAAATTTAATGGTATTTCAGTGGATGAAAATATTACTGGTGCTTTGAGGGGTGAGCA

TTTAAAGCACAATATCTAGGATACTACAAAAAATACTTAAAGTATGAATATTAAATTGTA

AACTTAAGCATAAAGAGCTCGAAAAGAATTAAGCTTAACTCATCTACAAGCATCGTCGAC

CTCTCTTCATAACTTGCATTTCTCCATTAACATGAATTTTATTGATATGGAACTAATGCT

ATAAAAATATAACTTAATTATTTATGAAAATTATATCATCATATATTTTCTCTTCAATTT

TTTTTATATTAATAAAAAATGAAAATGGGTATTGATCTAGAAATTTAGTTCTTAAAACTA

ATAAATATGTCAAATATTAAAAAAAATAGTAATATAAATAAAATTATTATTAAATATTTA

ACAAAATATATTATATATATATATATATTATAATAATATAAAATAAATAATTGGGATACA

AAATAAAAAATAAGCATTAAGAACTTTTTTTTTATATATTGCTATAATTAAAAAAAAATC

TCTTTGGAGTATTGCATTATTTATATAAATACGTATAAATGAACATTTTTCTCACATGAT

AATAAACTATAGCTATTATATAATTATATAATATGTTTTTGTGCTTACAATACAAATTAT

ATATGCATATATTATCATGCATTTTATAATAAATATATAATTAAAAAAAAAATTGATTTA

TTAATGTTCATTATTTAAAATTTTTTAATAACCCAGTTATGCATTTATGAAAATGGGTAC

CCGTCACCAAGGAGTACGAGTTC

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 several unsuccessful attempts to disrupt the gene (1528; 1610; 1650; 1683).

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

Attempts to disrupt the gene were carried out in both the 676m1cl1 and 1037m1f1mocl1 background (RMgm-32)

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	GAACTCGTACTCCTTGGTGACGGGTACCCATTTTCATAAATGCATAACTG
Additional information primer 1	L5205; 5’- cept targeting region F (Asp718)
Sequence Primer 2	CATCTACAAGCATCGTCGACCTCTCTTCATAACTTGCATTTCTC
Additional information primer 2	L5206; 5’- cept targeting region R
Sequence Primer 3	CCTTCAATTTCGGATCCACTAGGACGGGTAAAATATACATCG
Additional information primer 3	L5207; 3’- cept targeting region F
Sequence Primer 4	AGGTTGGTCATTGACACTCAGCAGTACTGATCATTAGCATTATGGTGTG
Additional information primer 4	L5208; 3’- cept 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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