RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-824

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_1319100; Gene model (P.falciparum): PF3D7_1455400; Gene product: hemolysin III
Phenotype	No phenotype has been described

Last modified: 23 December 2016, 17:47

*RMgm-824

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)	Reference 1 (PMID number) : 25941254
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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 cl15cy1
Other information parent line	A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255)
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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_1319100

Gene Model P. falciparum ortholog PF3D7_1455400

Gene product hemolysin III

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 (Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

GAACTCGTACTCCTTGGTGACGGGATCCTTCTAAAATCCCCATATACACTCCATAAAATA

AAGGCTTGTCAATTATGTGTAGAAACAAATATATAATTAATTTTAAGCTAATTTCCATAA

ATATCTATTTCTATTGTATAAAGGCATATTAGTATATATCACCAGATATATATAAAGAAA

AAACATGTAATAATAATATTTATTGATTTTTATTAATATTCGGATTTAATAATATGTGTA

TCTATAAATTTAAAAATATAAAATTATGCTTTTTATTAAAATAAAAAAGAATGGTATTTT

CTATAACATTTATGTGCAATTGATTTAACATTGTATGTGCATTTCATTTTAATGAAATGT

AATATGCTATATAAATCAAATTTATATTTTTAACAAATCATAAAGAAAATTAAAAAATAT

GTAAGAATTTTTATAAATAAAGCATATATTCATAAAATAATATCATTAAAATAAAGTGGA

TAAAAAATAAATAAAAAATAAAATAACATAACATATTAAATTTCACAATTAATAATAATA

GCAAAACAACCATATTATCATTCCAATTCTCACATAAAACCCCAAAGGAGGTCGACGATG

CTTGTAGATGAGTTAAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAA

TATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCC

TCAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTA

TGCATATAATGTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAA

TATGTATAAAAATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCC

CTTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATA

ATATAATTAATATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTA

AATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGC

CTTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATAT

ATAAAAATATATATTTGCTTATAAATAAATAAAAATTTTATAAAAATGGTTGGTTCGCTA

AACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGG

CCACCGCTCAGGAATGAATTCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAA

GGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAAT

CGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAA

GGTGCACATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAA

TTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCC

ATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGT

GACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGT

GTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAG

AATGATTAATGTTCGTTTTTCTTATTTATATATTTATACCAATTGATTGTATTTATAACT

GTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGC

TAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGG

CTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAA

AATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTA

TTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTT

GATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAA

AAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGATCCACTAGATATGT

CCCAATCAAATACACAATCAATTATATGTACATGTGTATATTTTTTTTTTAATCCTTTTT

AGGAATTTTTGGATTTCACGAGCTTTTTCATGCTTGTTGCTTAGGAAGCCTTTTATTCAC

ATTTTCTTTGAACCGTAGTGTTCTAATGAGAAAATCTTAGAATATTTATCCTTTGTACGT

TTATTGCATATATTTTTAAATATGCATATATTGTTTTTTTTAGTTTTCAAAAATTTAATA

CCACTAGTATTGAATTTTTTATAACCATAGATTATAGTTAAAATTCCTTTATTTATTTTT

TATTTAATGTATAGTACTATAAAAATATATATAGCTAATTGAAGGATGCATGGTATAAAT

ATATGCGTAAATTTGTTAGTGAGTATTCGTTTTAATATTAAATTTTAGAATGCTTATAGT

TAATATTTGTATATACAATTTTTACTAAATTTATTATATATTTTATTCCATTATATAATG

CCAATATTGATTTAGACAATTTTTTTAATATTTTTTTTAAATAACATTTGTAACTATATA

TTATCTTATATTTATTGTATTATATTTTTGGTGTAACTACGTGTGCGTATGTTTCAAGTA

ATAGTACTGCTGAGTGTCAATGACCAACCT

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 (1594; 1618).

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

The locus was 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

A second attempt to disrupt the gene was carried out in the 1037m1f1mocl1 (RMgm-32) 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	GAACTCGTACTCCTTGGTGACGGGATCCTTCTAAAATCCCCATATACAC
Additional information primer 1	L5386; 5’- hemolysin targeting region F (BamHI)
Sequence Primer 2	CATCTACAAGCATCGTCGACCTCCTTTGGGGTTTTATGTGAG
Additional information primer 2	L5387; 5’- hemolysin targeting region R
Sequence Primer 3	CCTTCAATTTCGGATCCACTAGATATGTCCCAATCAAATACAC
Additional information primer 3	L5388; 3’- hemolysin targeting region F
Sequence Primer 4	AGGTTGGTCATTGACACTCAGCAGTACTATTACTTGAAACATACGCAC
Additional information primer 4	L5389; 3’- hemolysin 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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