RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-639

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_1426200; Gene model (P.falciparum): PF3D7_0811300; Gene product: CCR4-associated factor 1 \| poly(A) ribonuclease POP2 (carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1))
Phenotype	No phenotype has been described

Last modified: 28 August 2011, 17:59

*RMgm-639

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)	Reference 1 (PMID number) : 21803864
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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 1037m1f1mocl1 (RMgm-32)
Other information parent line	P. berghei ANKA 1037m1f1mocl1 (1037cl1; RMgm-32) is a reference ANKA mutant line which expresses GFP-luciferase under control of a schizont-specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 20019192).
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Attempts to generate the mutant parasite were performed by
Name PI/Researcher	B. Balu; J.H. Adams
Name Group/Department	Department of Global Health, College of Public Health
Name Institute	University of South Florida, College of Public Health
City	Tampa, Florida
Country	USA

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_1426200

Gene Model P. falciparum ortholog PF3D7_0811300

Gene product CCR4-associated factor 1 | poly(A) ribonuclease POP2

Gene product: Alternative name carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1)

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct used PCR construct

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

ATTGGGGAACTTGTTCACCATTTCTATTACATTATATATATTAGGACACAATATAATAAA

TTTATTAAAAATATTTTTAGGATATTACTTTTTTATATTAGTTAAAAAGAAAAAAGAGTA

CCCTAAATCGAAATGCTGTTTTTAAATAAAATATTGGATATTGAAAAAACAATTGCTAAG

TGAAAAGCATATACATAATACACATTCATGCATTCATAATATATATACATATATCGTGCT

AGTACTATTATTATTTTTTTGTGTGTTGTGTTGTCAATTTTTATGAAAATGCTGGATACA

TTTCATCAATACAATTTTAAAATATACGACTAGAAAGAGTAAAACATTAAAAACGCGCAT

ATATATACATATATGAGTGTACAATATGTATATCAGCAAATACGAATAAATGCCCTGAAT

TTTTTGTTTTTTAGTAAGTTGGAAAAAACACATTCAAGCGTTTTTCGGTATAAAGAAACA

ATTTTTAATAAGTTTATATTTTTTCTAAATTTGGTGCAAAAAGCGTATATGCATTTATAT

ATATATAAGCCGACGTAAAAGGAAATAAATATAGCATTGTTGAAGTAATGCCAATTACAG

GAATTATGATTTTTTTTGTGTGTTAGAAAATAAGATAGATGATTGATGGATTAAAAAAAA

AAATACAAAAGATAAAAGTGATAAAATACGAGGCTTGTTTATATACATATATGCACATGT

ACAATTTGATATTCCCGTTTTATATATGTGTAGTATGCAAAAAGTGAGAGGTCGACGATG

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

AAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGATCCACTAGTGTGGC

TTGTGTTAAAGACATTGCTAAAAGTAATAGCAATACTAGTAGGAGTAATAGCAAAAATAG

TTTGATGGATGGCTCTAAAGGTAGCAATTATAGTAGTAATAACAATAATAAAAATGGTAA

AAATAATAATAGTAATAAAAATGGAAATAAAAATAAGAGCAAAGGAAAAAATAAAAATAT

TAATGATAATAATCATATTAACGGTAATAAAAATAATGAAAAAAATAGTAATAAAGGCAA

TGATAATAATGGAAGTAACAATAAGAGTCGAAATAACAACAATAATAGTAACAATAATAA

TTCTATGAAAAATAAAGTTGGTGATAAAAATAATGAAGATAGCATTTTTGATAATAATGC

AACAGATAATAATAGTAACTATGTTTTAATTAATCAAAGAAAAAATGAAAACAAAAATTA

TGCAAATTTAAGTACCAAAGGCGATGATAATATTTCAAGAATCAATAATAATAATAATAG

TGCTGTAAATAATTCAAATAAAGAATTAATTGACGAGCAAGCCAAAGCACGAAACTCAGA

TAAAAATATATACAATAATTTTTACAACAATGTGAATTATTTTAAATCTGATAATAACAT

GAAACAGCATAGCAGAATAATGGGAAGAGGC

Restriction sites to linearize plasmid Asp718, ScaI

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 The carbon catabolite repressor protein 4 (CCR4)-Not complex is a well-conserved, eukaryotic gene regulatory complex, broadly divided into two modules: CCR4 and CCR4-associated factor 1 (CAF1), regulating mRNA decay, and the Not proteins, regulating transcription and protein degradation. In yeast and other eukaryotes, CAF1 has a major role in determining transcript levels through regulated deadenylation of mRNA. Members of the CCR4-Not complex are well conserved in all Plasmodium species, suggestive of their significance in parasite gene regulation.

The unsuccessful attempts to disrupt CAF1 indicates an essential role during asexual blood stage development of P. berghei.

In P. falciparum a 'CAF1-knock out mutant' has been generated and selected by piggyBac-mediated mutagenesis. Asexaul blood stages of this mutant show a severely attenuated growth rate. Phenotype analyses of the P. falciparum mutant indicates that the average number of merozoites/schizont and the length of the cell cycle is similar to that of wild type parasites. However, merozoites show a defect in egress from the host erythrocyte.

In order to create a P. berghei caf1 deletion mutant, constructs were generated which would target the gene by double-crossover homologous recombination. These constructs were amplified using an adapted anchor-tagging PCR-based method for generation of gene deletion constructs. This method employs a two-step PCR. In the first PCR, two flanking fragments (5' and 3' target) were amplified using genomic DNA (cl15cy1) as the template with the primer pairs 4674/4726 (5' UTR in construct pL1518;
GAACTCGTACTCCTTGGTGACGGGTACCATTGGGGAACTTGTTCAC/CATCTACAAGCATCGTCGACCTCTCACTTTTTGCATACTACAC) or 5342/5343 (5'UTR in construct pL1585; GAACTCGTACTCCTTGGTGACGGGTACCTCCTTATGTAGCCATTGAC/CATCTACAAGCATCGTCGACCTCCCAAGCCATATATAATACCTG) and 4727/4675 (3'UTR both constructs; CCTTCAATTTCGGATCCACTAGTGTGGCTTGTGTTAAAGAC/AGGTTGGTCATTGACACTCAGCAGTACTGCCTCTTCCCATTATTCTG). The 5'UTR targeting region in pL1585 was moved approximately 800 bp downstream compared to construct pL1518, leaving 770 bp of the 5’ ORF intact. The primers 4726, 5343, and 4727 have 5'-terminal extensions homologous to the hdhfr selectable marker (SM) cassette. This cassette contains hdfr under the control of the ef1a promoter region and the 3' untranslated region (UTR) pbdhfr/ts and is obtained from plasmid pL0040 by digestion with restriction enzymes XhoI and NotI (pL0040 is available from The Leiden Malaria Research Group). Primers 4674, 5342, and 4675 have a 5'-terminal overhang with an anchor tag suitable for the second PCR. In the second PCR, the fragments were annealed to either side of the hdhfr selectable marker cassette with anchor tag primers 4661/4662 (GAACTCGTACTCCTTGGTGACG/AGGTTGGTCATTGACACTCAGC), resulting in the second PCR fragment with the expected size, i.e., 3.0 kb (1.6 kb of the selectable marker cassette plus two targeting fragments). To remove the anchor tag from the final DNA construct, the second PCR fragment was digested with Asp718 and ScaI.

Both constructs were used in two transfection experiments each, resulting in four independent attempts to disrupt P. berghei cafI. Transfections were carried out in 3 different parasite lines. Construct pL1518 was transfected into P. berghei ANKA 1037m1f1mocl1 (RMgm-32) and P. berghei ANKA 676m1cl1. P. berghei ANKA 1037m1f1mocl1 (1037cl1; RMgm-32) is a reference ANKA mutant line which expresses GFP-luciferase under control of a schizont-specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 20019192). 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). Construct pL1585 was transfected into P. berghei ANKA 1037m1f1mocl1 (RMgm-32) and P. berghei ANKA cl15cy1. P. berghei ANKA cl15cy1 is a reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).

See RMgm-318 for independent attempts to disrupt P. berghei caf1

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
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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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