RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-689

Malaria parasite	P. yoelii
Genotype
Transgene	Transgene not Plasmodium: GFP-Luciferase Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a) 3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts) Insertion locus: Gene model: PY17X_0306600; Gene product: 6-cysteine protein (P230p; 230p)
Phenotype	Asexual bloodstage; Gametocyte/Gamete; Oocyst; Sporozoite; Liver stage;

Last modified: 24 December 2016, 17:38

*RMgm-689

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Introduction of a transgene
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 22216235
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. yoelii
Parent strain/line	P. y. yoelii 17XNL
Name parent line/clone	RMgm-688
Other information parent line	GIMO-Py17X (RMgm-688)contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PY04774) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. yoelii 17XNL line is used for rapid introduction of transgenes free of drug-resistance genes (RMgm-688; PubMed: PMID: 22216235).
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The mutant parasite was generated 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
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Name of the mutant parasite
RMgm number	RMgm-689
Principal name	PyGFP-luc(con); PyGFP-luccon
Alternative name	1971cl1, cl2, cl3
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	GFP-Luciferase expression in all stages; increase of GFP-Luciferase expression during development from ring form into mature trophozoite/immature schizont; decrease of GFP-Luciferase expression in mature schizonts.
Gametocyte/Gamete	GFP-Luciferase expression in female and male gametocytes
Fertilization and ookinete	Not tested
Oocyst	Strong GFP-Luciferase expression in maturing oocysts
Sporozoite	(Weak) GFP-Luciferase expression in salivary gland sporozoites
Liver stage	Strong GFP-Luciferase expression in maturing liver stages
Additional remarks phenotype	Mutant/mutation The mutant expresses the fusion protein GFP-Luciferase under the control of the constitutive P. berghei eef1a promoter. The mutant does not contain a drug-selectable marker. The mutant has been generated and selected using the GIMO transfection method ('gene insertion/marker out') of transfection of GIMO mother lines that contain the hdhfr::yfcu selectable marker into the silent 230p locus. The mutant has been generated in the reference line GIMO_Py17x (RMgm-688). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice. Phenotype GFP-Luciferase is expressed in all life cycle stages and expression of the transgene has no influence on parasite viability and infectivity. Additional information To introduce the GFP-Luciferase into the 230p locus a PCR-amplified construct was used. See 'Additional remarks genetic modification' for details of the generation of the construct. Such 'GIMO-constructs' contain only the two genome targeting sequences and the transgene expression cassette. The simple structure of GIMO constructs permits the cloning of larger transgenes (the GIMO constructs are smaller as the selectable marker cassette is absent) and improves the retention of plasmids in bacteria as internally repetitive regions of AT-rich Plasmodium DNA are absent. Further, after transfection with the GIMO construct, the selection of integration mutants is improved as no episomal construct DNA is maintained in the parasites and negative selection kills parasites expressing yfcu. Other mutants RMgm-29: A P. berghei mutant expressing the fusion protein GFP-Luciferase under the control of the constitutive P. berghei eef1a promoter. The mutant does not contain a drug-selectable marker.

Transgene: Mutant parasite expressing a transgene

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Type and details of transgene

Is the transgene Plasmodium derived Transgene: not Plasmodium

Transgene name GFP-Luciferase

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct (Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC

ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGC

TCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAA

TTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTG

GTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGCCCTTGAACTC

GTACTCCTTGGTGACGGGTACCGTGATGGAATGGCAACATCTGATCACATTTTAAAAATA

GTTGATGATGATAGTAAAACGATTAAATATTTTAACGATATACCTTATCAGATGTGTAAT

TTTGATTATAATTTAAGCAAATTAAGTGAAATACAAATATGTGAAAAAACAATAAATGAA

TTTAGTTTATTCATGTATAATTGTGAGCAAATAACAGATAATAGAATCGTATATGGTAAA

GAACCTATTAATACCATAAAATATTTAAGTAATGTATTTCCTATAAATAAATTTACAGAT

TTATTTTTTAATACAAAAGATATAGATATACCAGAAATAAATGAACAATTTAAAGGTTTT

AAATTTTTTATGACCTCATTTATAAATCATGGATCATATCCACTAACCATAGAATGTGGT

GTAACAAATGGTGGGACTGATTATAGAAGAGCAATTATTTTATTACATGTTCGAACAGAT

TTAAAAGATAGACCAGTTTCATTTTGTGATTTTAGAAAAGGTGAATTATATAATTATTTA

AATGCTTATAGTGAAGGAGATCTATGTATAATAATTTCCAAATCAAATACAAGTTTTGGT

TTTAGATGCCCCGAAAATACAAAAAAAATGCCAGAAAAATGTTTTACTCAAGTATATGAA

AAAGGCTATCTACATGATTCCTATAAAATTAATACTAAAAATATTATTAACTATTCATTT

GAAAATCCAGAATATGCATTAGCTGGTTTTAATTATACATTAACAAAATCATATCAATTT

GAATGTCATTGTGTAGATAAAGAAACAGAACAAATTGTAAAAACTGTTTTAGTAAAATAT

GTAAATGAAGATGAAATATATGATTATAATGATTTACCATTGGTTAATCATAAATCTATT

GTTGCACATCCAAATAAAACACATCTATGTGACTTTATGACATCTGATAATATGTTATCA

CCTAAAAAAGAAGATTCAGTAAATTATGTTTGTAATGTATTTCCAAAACCATTAGAATAT

GTAGCATTACATTGTCCAACCGAGGTCGACGATGCTTGTAGATGCCCGGGGCCCAGCTTA

ATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTT

TTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTT

CATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATAT

AAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATATGC

ACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATT

ATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTAATG

TTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAAATA

TAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTT

TAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGCTTA

TAAATAAATAAAAAATTTTATAAAACATAGGGGGATCTATGAGTAAAGGAGAAGAACTTT

TCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTT

CTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTT

GCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTG

TTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCA

TGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA

CACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTA

TTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCAC

ACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTA

GACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAA

TTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTT

CGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTG

GGATTACACATGGCATGGATGAACTATACAAAGGGATCCTGGCTAGCCAGTCGACCTGCA

GGCATGCAAGCTTGCGGCCGATCCAAATGGAAGACGCCAAAAACATAAAGAAAGGCCCGG

CGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGA

GATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCA

CGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGC

TGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGG

TGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAAC

GTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGG

GGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCA

TGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATC

TACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAA

TTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGC

ATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCA

TTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTA

CACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGC

TGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTAT

TTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAA

TTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCC

ATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTA

CACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGA

AGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTG

TCAGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGA

TTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACT

TCTTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGTGGCCCCCG

CTGAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTC

TTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGA

CGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGT

TGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACG

CAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTAAT

TCTAGAAGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTG

TAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCT

AAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGC

TAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAA

ATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTAT

TATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTG

ATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAA

AAAAAACATATAAACACAAATGATGTTTTTTCCTTCAACCCGGGCCTTCAATTTCGGATC

CACTAGAAGTAAAAGGGGTAAGACAGCAATTGTTAAAATTAATGTTCATCCAAATTCTTT

AAAGATTATGGGATGTGATTTTGTCGGAGCATATTCTTCTTATTTTATATTTAGTAAAAA

ATGGGATCAAATAACTCCAAATTATGTTTGTGAAATTAATGTAGAAGATGATTCTATTAT

TGGATTGGCTTGTCCTTATAATACAAAAATATATCCTTCTGATTGTTTTGAAAGTGTTAT

AAAAAATAATAAAGTTTACAAAAGAGATACCTTAATTGAATATAAAAATACATTTTTTTA

TCAAAAAAATGGAAAACCTACATTATCTTTTATACAAATTAAAAAAGTTTATTCTGACCA

TCTTACTTGTAAGTGCTTTGAAAATAATAATGGAAATTACAAAGAAGTTACTATCAAATT

AATTTATAAATCATATATATTAGGAACACCAAAAATGACTTTAAATAAACCATTTATGAA

ATATAAAAGTGTTAATTTTTTGGATTATCTGATTGGGAAGAAATTCTAATTTTTATAGTA

TCCCCAGCCCCTTTTGATTCAATATAGTTTTAGCTTGCCGATTTCTTTGGGTTCGTGTTT

TGCGATTTCTTTGGGTTCGTGTTTTGCGATTTCTTTGGGTTCGTGTTTTGCGATTTGTTT

GGGTTCGTGTTTTGCGATTTGTTTGGGTTCGTGTTTTGTGATTTCTTTGCGATTTTTGTG

TGTTTAGTGATTTTTGTGTGTGTCGATTGTGCGTTTTTTATTATATTATTGTTATCATAT

TTGATTTACCTTTTGATATTAGGACATATATATATGCATATATTTTTTTGTATTTCCTGA

TTTGTTTGTGATCATAATTTTCGATAAAATGAGTTTTTATTATTGATGCTTTAAACATTC

CAGTTGGTTCCAGATCTCTTAGTACTGCTGAGTGTCAATGACCAACCTAAGGGCGAATTC

TGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGCCC

TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAAC

CCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAAT

AGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGG

ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG

CTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA

CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTA

GTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGC

CATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG

GACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTAT

AAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA

ACGCGAATTTTAACAAAATTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAA

AGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGAC

AAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATA

GCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTC

TGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTG

ATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGA

ACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGA

CTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGG

GCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGA

GGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGT

TGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCT

GTCATCCCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCT

GCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCG

AGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCA

GGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGA

TCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTT

TTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTT

GGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCT

TTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTT

CTTCTGAATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCC

TTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAA

GATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGT

AAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT

CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGC

ATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACG

GATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCG

GCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAAC

ATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCA

AACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTA

ACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGAT

AAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA

TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAG

CCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTT

TACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTG

AAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGA

GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTA

ATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAA

GAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACT

GTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA

TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTT

ACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGG

GGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAG

CGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTA

AGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT

CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCG

TCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC

TTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAAC

CGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGC

GAGTCAGTGAGCGAGGAAGCGGAAG

Restriction sites to linearize plasmid

Selectable marker used to select the mutant parasite hdhfr/yfcu

Promoter of the selectable marker eef1a

Selection (positive) procedure No

Selection (negative) procedure 5-fluorocytosine (5-FC)

Additional remarks genetic modification First a basic P. yoelii 230p-targeting construct (pL1849) was generated using a modified 2-step PCR method. In the first PCR reaction, 5’-and 3’- targeting sequences (both ~1kb) of 230p were amplified from P. yoelii 17XNL genomic DNA with the primer set 6523/6524 (py230p 5’- targeting sequence, F: GAACTCGTACTCCTTGGTGACGGGTACCGTGATGGAATGGCAACATCTG; py230p 5’- targeting sequence, R: CATCTACAAGCATCGTCGACCTCGGTTGGACAATGTAATGCTAC) and 6525/6526 (py230p 3’- targeting sequence, F: CCTTCAATTTCGGATCCACTAGAAGTAAAAGGGGTAAGACAGC; py230p 3’- targeting sequence, R: AGGTTGGTCATTGACACTCAGCAGTACTAAGAGATCTGGAACCAACTGG). These primers contain 5’- extensions homologues to the hdhfr::yfcu selectable marker cassette and 5’-terminal extensions with an anchor-tag suitable for the second PCR reaction. A 55nt oligo (oligo 6598; GAGGTCGACGATGCTTGTAGATGCCCGGGCCTTCAATTTCGGATCCACTAG) containing a XmaI restriction site flanked by 2 sequences homologues to the hdhfr::yfcu selectable marker cassette was used to join the two 230p targeting regions. In the second PCR reaction an fragment containing both 230p targeting sequences interrupted by the XmaI site was amplified, using the external anchor-tag primers 4661/4662 (anchor-tag primer, F: GAACTCGTACTCCTTGGTGACG; anchor-tag primer, R: AGGTTGGTCATTGACACTCAGC), resulting in the PCR product of ~2 kb. The PCR product was cloned into TOPO TA vector (TOPO TA Cloning® Kit, Invitrogen, Groningen, The Netherlands) resulting in construct pL1849.

Next a construct (pL1847) for GIMO-transfection in the GIMOPy17X mother line was generated by cloning an PCR-amplified GFP-luciferase expression cassette into the XmaI site of the basic P. yoelii 230p targeting construct pL1849 (see above). The GFP-luciferase expression cassette (5’ eef1α-gfp::luciferase-3’pbdhfr) was amplified from pL1603 (MRA-852, www.mr4.org) using primers 6599 and 6600 (5’pbeef1α, F: TCCCCCCGGGGCCCAGCTTAATTCTTTTCGAGCTC; 3’pbdfhr/ts, R: TCCCCCCGGGTTGAAGGAAAAAACATCATTTGTG).

Additional remarks selection procedure This reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPy17x (RMgm-688). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.

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Other details transgene

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Promoter

Gene Model of Parasite PBANKA_1133300

Gene Model P. falciparum ortholog PF3D7_1357100

Gene product elongation factor 1-alpha

Gene product: Alternative name eef1a

Primer information details of the primers used for amplification of the promoter sequence Click to view information

Primer information details of the primers used for amplification of the promoter sequence Click to hide information

Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2

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3'-UTR

Gene Model of Parasite PBANKA_0719300

Gene product bifunctional dihydrofolate reductase-thymidylate synthase, putative

Gene product: Alternative name dhfr/ts

Primer information details of the primers used for amplification the 3'-UTR sequences Click to view information

Primer information details of the primers used for amplification the 3'-UTR sequences Click to hide information

Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2

Insertion/Replacement locus

Replacement / Insertion Insertion locus

Gene Model of Parasite PY17X_0306600

Gene product 6-cysteine protein

Gene product: Alternative name P230p; 230p

Primer information details of the primers used for amplification of the target sequences Click to view information

Primer information details of the 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

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