RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-828

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_0208900; Gene model (P.falciparum): PF3D7_0104200; Gene product: StAR-related lipid transfer protein
Phenotype	No phenotype has been described

Last modified: 16 August 2013, 18:58

*RMgm-828

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_0208900

Gene Model P. falciparum ortholog PF3D7_0104200

Gene product StAR-related lipid transfer protein

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

GAACTCGTACTCCTTGGTGACGGGTACCAATTGGTGCCATAACCAGAACTAATATGTCTA

AAAATTAAATTTGTGTTTAAAACAATTTTTTCATACTAAAAAAAATAAATGAAAATAGAA

AATTACTAATATAGAAAAAATATAACTTTTTTCTGTTTCATTAATTTGAAGATTTAATAG

CACATTTTTTTGAATTATTTTATTTATTCTATTATAATCATATTTTTATTTTTCCAATGT

ACTTTTAAATAAAAATTTTTATTTTTGTTGTCATAATTTACAAAAAAACTTTTAGCGTTT

TTTCGATTTTCATTTCATAAATTAAATCGTTTATTTCTTTTTTTTCTATTCTACTTATTT

TATGTGCATTTTTTTACTACGATTTTATTGTTAATTGTTTGTTTTATTTAGTGCTTAATG

CAGTTACTATAAACTATTTATGGATCCTTTTTTTTTCATAATTTCTGTGGATATTATATA

GTTGTATAATGTTAACTCTCTTATATATGTGCATGTAATAATTAAACTTTATTTTCTATA

ATAATAGAAAAAAAAGTATATATTTTTTGTAGCTAAATCGGTTGATCGAGAGGTCGACGA

TGCTTGTAGATGAGTTAAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTT

AATATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACC

CCTCAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATAC

TATGCATATAATGTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAA

AATATGTATAAAAATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTG

CCCTTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAA

TAATATAATTAATATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATG

TAAATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTT

GCCTTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAAT

ATATAAAAATATATATTTGCTTATAAATAAATAAAAATTTTATAAAAATGGTTGGTTCGC

TAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCT

GGCCACCGCTCAGGAATGAATTCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAG

AAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGA

ATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCAC

AAGGTGCACATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAG

AATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAG

CCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAA

GTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAG

GTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGA

AGAATGATTAATGTTCGTTTTTCTTATTTATATATTTATACCAATTGATTGTATTTATAA

CTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATA

GCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAAT

GGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTA

AAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTAT

TATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGAC

TTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATT

AAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGATCCACTAGCACT

TGCATGCTATTGTCTTTATAATATTTTGTTTCAATATTTAATTTTTGTACATTACTTTCT

TGAGTTGTTTTCTTGTTTTATTTTTTTTAAGTATTTCAACGTTTTAATACAATCAACTTT

ATGTTTCAATTTTAAAATATAATTTAATTAACTCAAAGGTACAGTTATTAAAAAATGTCG

AAAAATATATGAACAATTCATAAAATAAAAGGTTAGCTAGCTAAAATGAAAAAAAAATAT

AGAAAGTTCACGCATTTACACATAAACATATAGATTGAACTGTTTTCACTTTTGATTTTT

TGCAGACGTCATTCCTATACTTACAGACTATTACTACATTATTTTATTTTTATTGGTTCT

TTATTATAAACATGGATGGTTTAGACTCAAACGAAAAAACACGAAAAAAATTATTAATTA

AAACTAAAAATAGTAAAGTATTATATTTATATATAGCTTAAGTTTTATTAGAGTTTTCAA

AAAAAGAAGGAAAATAATAAATTTCAAAAAAAAAAAATAAATGAATAAAAATACAAATCG

GTATCACTTCACATATGAAGGCATCATGGGTACCGCTGAGTGTCAATGACCAACCT

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 (1779; 1893).

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 (PCR1774) used the same targeting regions but differed by having the hdhfr::yfcu selectable marker cassette. The attempt was made in the 1037m1f1cl1 (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	GAACTCGTACTCCTTGGTGACGGGTACCAATTGGTGCCATAACCAG
Additional information primer 1	L5868; 5’-PBANKA_020890 targeting region F (Asp718)
Sequence Primer 2	CATCTACAAGCATCGTCGACCTCTCGATCAACCGATTTAGC
Additional information primer 2	L5867; 5’-PBANKA_020890 targeting region R
Sequence Primer 3	CCTTCAATTTCGGATCCACTAGCACTTGCATGCTATTGTC
Additional information primer 3	L5870; 3’-PBANKA_020890 targeting region F
Sequence Primer 4	AGGTTGGTCATTGACACTCAGCGGTACCCATGATGCCTTCATATGTG
Additional information primer 4	L5871; 3’-PBANKA_020890 targeting region R (Asp718)
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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