RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-749

Malaria parasite	P. berghei
Genotype
Transgene	Transgene not Plasmodium: GFP-Luciferase Promoter: Gene model: PBANKA_1400800; Gene model (P.falciparum): PF3D7_1302200; Gene product: early transcribed membrane protein 13 (UIS3; ETRAMP13; upregulated in infective sporozoites) 3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts) Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype	Sporozoite;

Last modified: 2 May 2019, 11:00

*RMgm-749

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) : 22574152
MR4 number
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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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The mutant parasite was generated by
Name PI/Researcher	C. Ramakrishnan; E.A. Levashina
Name Group/Department	Institut de Biologie Moléculaire et Cellulaire
Name Institute	Université de Strasbourg
City	Strasbourg
Country	France
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Name of the mutant parasite
RMgm number	RMgm-749
Principal name	uis3::GFP-LUC clone 4.4
Alternative name	1096, 1097
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	Not different from wild type
Oocyst	Not different from wild type
Sporozoite	No GFP was detected in non-sporulating or sporulating oocysts at 11 and 21 days post infection. Salivary gland sporozoites expressed GFP (and luciferase).
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses the fusion protein GFP-Luciferase under the control of the uis3 promoter. The gfp-luciferase transgene has been integrated into the silent 230p locus Phenotype In blood stage ring-forms, trophozoites, gametocytes and ookinetes no GFP signal above background was observed for uis3::GFP-LUC. A faint (and unexpected) but distinct GFP signal was observed in developing schizonts. No GFP was detected in non-sporulating or sporulating oocysts at 11 and 21 days post infection. Salivary gland sporozoites expressed GFP (and luciferase). Additional information The luciferase activity of this reporter line and reporter line RMgm-750 has been exploited to establish a simple and fast biochemical assay to evaluate salivary gland sporozoite loads in whole mosquitoes Other mutants RMgm-750: A mutant expresses the fusion protein GFP-Luciferase under the control of the promoter of glyceraldehyde-3-phospho-dehydrogenase (glyc, PBANKA_132640). The gfp-luciferase transgene has been integrated into the silent 230p locus. Also in this mutant no GFP was detected in non-sporulating or sporulating oocysts at 11 and 21 days post infection. Salivary gland sporozoites expressed GFP (and luciferase).

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

TCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCT

CCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCG

CAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAA

AGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACC

TTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGTTG

GTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTC

TCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCG

GCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGA

CACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGG

GCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACAGACTACA

CAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAG

ATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGC

CTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGT

ATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTT

TGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGG

GGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAAC

CGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGATT

TCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGA

TGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTT

ATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTT

GTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAA

AAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAAT

CCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAA

CAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTT

CAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGT

TAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCT

TCAATTTCGATATCGCGGCCGCGGACATATTTTGGGACTATCCAGGTATAGTGTGTTAAG

AATTTATATTTGTTAAACTATATACATACGAATAATTAATTAATTGGTTGCTTTTTTCTC

TAAATTTTATAGATTAATAAATCAAATGCTTGCCAAAAAATTGCACCACATTTGTTTTGG

ACAGTGTTCACCTTAGTAACAATGATTTGGTAAAAATATAAATAAAATGTGTATTTATAT

TATTTTGTTTGCACTGTTTTTATATGGTTCGATATATATATATATATATATATATATATA

TATAACTGTAAAACATTAATTTTTTCCGATTTAGGTGTTCTATTGTAGGATTGATTTTAA

GTTTAATGATTATAACAATAGTAGGAAAAAAAACAAAATACTTTCAAAAAATACGAGAAA

ATCATTTATTTAAAAAGAAATATTTAAATAAAAATATAGGATATATGTATACATATATAA

TGAAAGAAAGATATGTCATGGTTAAATTGTGCAAATAAAACACAATTTCATTTCGTTAGG

GATCGTTTTTTTTTACAAAACCAAAGAAAGTGAAATTATAATTGGATTTAAATTTTGGGG

ACTTTATGATTTATTGTTAATTTTTTTAGTAAAAAATTAATTGATCAACATAATGTTATA

TTACTAAAATGTATAACATTATTTTATAAAAATATATAAATTATACAATTATGACTGTTT

TCATGATATGCATTATTTAAATAGTATAAAAACGCATGGCTATTTTATATATGTCTTTCT

CGCTAACCCATAAAATATTTTATATATAATTTAACATATTAATAAAAAAATTATAAATTA

AAGGGATATGAAATTTAATTTTTTCATAAATATAATTGGGAAAATGAATCATTATATTGT

GCTATAAAGCGTATTATGAATTAATATAAAAGGATTATATTTTATAATGTTTCACATAAA

AAAATGTATTTGGCTAACTATGCATGTACATATTTAGTGGTTATATCTATAATTACAACC

ATTTCAGTGATAAAATATATTGAACCATATGGCCAATATTTAAAAACATATAATAATATG

GTATAGTAACGATTTACTAAAGTTAAAAGCTAGCATTAGCATAACATCTCATTGGCATGT

TCATATTCATTAGAATATATATATTATTATCTTTAGTTCTAAGTACTATTCTTCTTGTAT

TTTATTTGTTTTTATGTTTTTTTTTCAATGGTTGTTTTATTTAACATAAATATATTTTGA

ATTAAAAATTAAACATAATAAATTATTTTTACCAGAATTACGTATTTGATATAATATTTT

TCCACACAAAAATGACCCATTTAAATGTTTAAGTGACTATGTTGCAAACAAATGGTAAAT

TATAAAAAAAAGAGGTCATGCATTTATTTAATTTGTGTAATTAACATAGATGAAATAATT

TAATCTATAATATTTAATATTTGTAAATATAAATTTATTGGAAATAATAAATTATATATT

TGTTAATTTAATATTATAAACCATTTTTTGTAATATACAATCTTAAATATATATTTAAAC

ATTATCCTTTGAATTTAATAAACATTAATTTTGTTTATGTTGTGGTTGCATGCAATTATT

CGTATTTGGTAAGCCTACAAATAAAAATGTCTCAGTTTTTTACATGCATAATTTATATTA

ATAAATATTTTAAAGAGCATGATTGACTGAAAATATTATTTATAATAAATAGTATCACAT

TTATCATTGTGATTCAAAAATTATCACTTTATTTAATAATTTTTTGTTGATACATAAAAA

GAAAATATAATATTAACAATAATAATGATAAAAAATTGTACATAATTATTTGATCTTTAT

AGTAATTTAATTTTTTTGCATTATTTAGACAAATAACAAATATCCATGGGGATCCATGAG

TAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGT

TAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACT

TACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCTTGGCCAACACTTGTCAC

TACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGA

CTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGA

TGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAG

AATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGA

ATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAA

AGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTA

TCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTC

CACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGA

GTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAGGGATCCTGGC

TAGCCAGTCGACCTGCAGGCATGCAAGCTTGCGGCCGATCCAAATGGAAGACGCCAAAAA

CATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACT

GCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACA

TATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGC

TATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCT

TCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAA

CGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGT

GTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAAT

CCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACAC

GTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTT

TGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAA

GGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTAT

TTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGG

TTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTA

TAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTT

GCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTT

ATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGC

GGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTAC

ATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGT

TCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCA

GAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGA

AGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTG

GGACGAAGACGAACACTTCTTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGG

ATATCAGGTGGCCCCCGCTGAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGA

CGCGGGCGTGGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGT

TTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGT

AACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCT

TACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG

AAAGATCGCCGTGTAATTCTAGAAGATCCCGTTTTTCTTACTTATATATTTATACCAATT

GATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCA

CATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTT

TACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAAT

TTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGAC

AAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTC

ATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAA

AAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCG

GGTACCGAGCTCGAATTCTCTTGAGCCCGTTAATGAAATAGATACAATTCATTCATGTTA

TATACATCTAGAACATAATCTGAATATGGTTCAAGTTAAATGTCCAAAAATTATAAAAAG

TGATGATATTTTTGATGGTAATACCATAATAGACACCAAGGTAACATCACGAAGTAGTCA

ACAAAATAATTTTTATTTAGAAAATACAGATGTTGAACCAGAAGAAATAGAGAAATATAA

AAATATAGAATACATACCAGAAAACGATGAAGTAATGCATCTAGACAAAAAAGAAAAGCT

AGATGATATATTACCAGGTGTTATCATATTAGATAAAAATAAAATGTTCAAAGAAAAAGG

ACATTTCACTTTTGTTACTCCATTAATTGTAGAAAAGGTATTAATATTAAAAATATATTG

TGATAATACTAAAACAATAATTAATAATATGAAAGGGAAAAAAGGTATTACAGTAATAAG

GATTTCTCAAAATACAACAAAAAATAAATTTTATGGATGTGACTTTTCAGGTAATTCTAA

AAAAACATTTTACTATTCCAATGTTTATGATTTAGAAAAAAAAAATGAGTTTTGTGAAAT

AGAATTAAAAGAAAATATAGTAGTTAGCTTAAATTGTCCAACTGGTAAAATTAATCCAAA

AAATTGTTTTAGAAATGTATATATAAAAAGTAATATGAATGAACAAACAACCGAAAATAT

AGAAAATATATTTAACGAAATAAAAGTTATAGATGCAGATTATTTTATAAATAATTCATC

AACCTTTTTGATGATTTCCAAAATTACAAAAAAAGAGTTTGATTTTTATTGTACATGTGA

AGATTATAAAACCAAAAATATAGGAACAATATATATTAAAAATTATGAATATCTAGATTC

AAAACCTAAATATAAAAATAAACAAATTTCCTATATAGATGTAGTTCCATACCCGCGGGG

AAAGGGCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTA

CCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGG

CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGC

GGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTA

CAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACG

CGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCG

GGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCC

TCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAG

GTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT

CAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA

GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTT

GCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGT

TGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT

TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGG

TATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGA

ATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAA

GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGA

CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA

CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACA

CCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA

CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCAC

TTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGC

GTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAG

TTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGA

TAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTT

AGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATA

ATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG

AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAA

CAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTT

TTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGC

CGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAA

TCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA

GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC

CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAA

GCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA

CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCG

GGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC

TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTG

CTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTG

AGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG

AAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAAT

GCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATG

TGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGT

TGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACG

CCAAGCTTCCGCGGGTATATGGTAAAGAACCTACTAACACAATAAAATATTTAAATAATG

TATTTCCTATAAATAAATTTACAGATTTATTTTTTAATACAAAAGATATAGATATACCAG

AAATAAATGATCAGTTTAAAGGTTTTAAATTCTTTATGACATCATTTATAAATCATGGAT

CATATCCACTAACAATAGAATGTGGTGTAACAAATGGTGGAACTAGTTATAAAAGAGCAA

TTATTTTATTGCATGTTCGAACTGATTTAAAAGATAGACCAGTTTCATTTTGTGATTTTC

GAAAAGGAGAATTATATAATTATTTGAATGCTTATACTGAAGGGGATGTATGCATAATAA

TTTCCAAATCAAATACAAGTTTTGGTTTTAGATGCCCAGTAAATACAAAAAAAATGCCAA

AAAATTGTTTTACGCAAGTATATGAAAAAGGGTATCTAAATGACGCCAATAAAATTAATA

CTAAAAATGTTATTAACTATTCATTTGAAAATCCAGAATATGCGCTAGCTGGTTYTAATT

ATACATTAACAAAATCGTATCAATTTGAATGTCATTGTGTAGATAAAGAAACAGAACAAA

TTGTAAAAACGGTTTTAGTCAAATATGTAAATGAAGATGAAATATATGATTATAATGATT

TTCCAATGGTGAATCACAAACCTATTATTGCACATCCAAATAAAACACATCAAGCTTGCA

TGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATT

ATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATA

ATAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTT

TGTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAAT

AAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTT

TTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTC

ATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTA

ACATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACAT

TTTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATATATATATAT

ATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATAT

GAACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCA

ATAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATAT

ATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATAT

GGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTT

ATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCG

GATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTA

TGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTAT

AATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCA

AAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAA

AAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAA

ATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATAC

ATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTAT

ACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTT

ATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAA

AATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAG

GCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACA

CTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATC

AATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATATTTATAAT

TTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGG

TAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTG

AAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAG

CGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAA

TAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATA

ATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATA

AATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTT

TATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAA

ATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTA

TATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGT

GTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTG

GCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGA

AGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACT

TCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGA

AAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTC

CCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCG

AGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGT

CGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGT

TGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCC

TGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCC

TGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGC

GACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTT

TGTGGTTC

Restriction sites to linearize plasmid SpeI, BglI

Selectable marker used to select the mutant parasite tgdhfr

Promoter of the selectable marker pbdhfr

Selection (positive) procedure pyrimethamine

Selection (negative) procedure No

Additional remarks genetic modification Primers 2601 (5′-CAAGATAGAAGAAGCCGTTCACAAGCC-3′) and 2602 (5′-GTACCGATATCCCCGGGCTGCA-3′) were used to amplify GFP from plasmid pL0031 and were cloned into an intermediate vector between the promoter of P. berghei elongation factor (pbef) 1α and the 3′ untranslated region (UTR) of pbdhfr using BamHI and XbaI. The GFP and the P. berghei dihydrofolate reductase (pbdhfr) 3′ UTR were excised together using BamHI and KpnI and inserted into another intermediate vector where the insert was flanked by the AMA1 promoter and by a fragment of p230p located in the 3′ region of p230p. The plasmid contained also an ampicillin resistance gene, a 5′ fragment of p230p and the tgdhfr/ts gene flanked by 5′ and 3′ UTRs of pbdhfr. The UIS10 promoter was amplified by primers 2603.

(5′-CGGATATCGCGGCCGCGAGTATAGGATAGATAATTTTTTTTGTGG-3′) and 2604 (5′CGGGATCCCCATGGTCTTTCACATTTACGCCAATAATTTTTTTAATG-3′). The EcoRV/BamHI digested PCR product was then inserted into the digested vector. After an HpaI/KpnI digest, the luciferase gene was inserted into the vector to yield pL1163. The UIS10 promoter could be exchanged with the UIS3 or Glyc promoters, which were amplified with primers 2605 (5′-ATAAGAATGCGGCCGCGGACATATTTTGGGACTATCCAGGTATAGTGTG -3′) and 2606 (5′-CATGCCATGGATATTTGTTATTTGTCTAAATAATGC-3′) or 2607 (5′-ATAAGAATGCGGCCGCGTATAAACTGAGTTAAGGGAAGTGG-3′) and 2608 (5′-CATGCCATGGTTTTATGTTTTTTTAAAATATTATATTGCTTG-3′), respectively. NotI/NcoI-digested fragments were used for replacement of the promoter yielding pL1171 for the Glyc promoter. The linker between GFP and luciferase was exchanged in the vector carrying the UIS3 promoter by using AG465 (5′-CGGGATCCGGAGGACCATCAGGAATGGAAGACGCCAAAAACa-3′) which annealed to the 3′ end of GFP and AG466 (5′- ACCCAGTAGATCCAGAGGAATTCATTATCAGT-3′) annealing downstream of the start codon to amplify the linker region. The PCR product was excised, ligated upstream to the UIS3 promoter fragment and downstream to the luciferase gene and cloned into the UIS3 intermediate construct lacking gfp described above. The GFP gene was then re-inserted using the BamHI restriction site to yield pL1327.

Gfp-luc (gfp mutant 3 and firefly luciferase IAV) fusion genes under the control of the salivary gland-specific promoters uis3 (PBANKA_140080), uis10 (PBANKA_112810 ) or glyceraldehyde-3-phospho-dehydrogenase (glyc, PBANKA_132640 ) were cloned into a plasmid containing the Toxoplasma gondii dihydrofolate reductase/thymidylate synthase (tgdhfr/ts) gene (selectable marker for pyrimethamine resistance) flanked by the 5′ and 3′ UTRs of the pbdhfr gene. For the uis3, uis10 and glyc promoters, fragments of 1895 bp, 1824 bp and 1914 bp, respectively, were used to generate the constructs. It has been previously observed that transgenic parasites producing the fusion protein GFP-LUC were considerably less fluorescent compared to a parasite lines expressing GFP alone, possibly due to steric hindrance of the fluorophore. Therefore in the uis3::GFP-LUC line, the linker between GFP and luciferase was changed from Gly-Ile-Leu-Ala-Ser to Gly-Gly-Pro-Ser-Gly, allowing more flexibility between GFP and luciferase. The DNA vector (derived from pAMA1RFP230p [pL1157]) also contained two flanking arms homologous to the p230p gene which served to introduce the transgene into the genome and stably disrupt the non-essential p230p locus by homologous recombination. The resulting plasmids pL1327 (uis3::gfp-luc), pL1163 (uis10::gfp-luc) and pL1171 (glyc::gfp-luc) were then linearised and individually transfected into P. berghei ANKA cl15cy1 parasites as described

pL1163 and pL1171 were linearised with SacII and pL1327 with SpeI and BglI before transfection.

Additional remarks selection procedure

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Other details transgene

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Promoter

Gene Model of Parasite PBANKA_1400800

Gene Model P. falciparum ortholog PF3D7_1302200

Gene product early transcribed membrane protein 13

Gene product: Alternative name UIS3; ETRAMP13; upregulated in infective sporozoites

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	ATAAGAATGCGGCCGCGGACATATTTTGGGACTATCCAGGTATAGTGTG
Additional information primer 1	2605 (NotI); UIS3 sense
Sequence Primer 2	CATGCCATGGATATTTGTTATTTGTCTAAATAATGC
Additional information primer 2	2606 (NcoI); UIS3 antisense

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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 Replacement locus

Gene Model of Parasite PBANKA_0306000

Gene product 6-cysteine protein

Gene product: Alternative name 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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