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- 11 July 2014 - Last week 56 new ‘phosphatase’ mutants were added to the RMgm database.
The RMgm database now contains information on the genotype and phenotype of more than 1000 different genetically modified malaria parasites.
These 56 new mutants (gene-deletion mutants and tagging-mutants) have been generated in a systematic functional analysis of the entire P. berghei ‘protein phosphatome’, which encompasses all 30 predicted protein phosphatases (PPs). In this study, published in Cell Host & Microbe, 30 and 29 PPs were identified in the genomes of P. berghei and P. falciparum respectively, 28 of these are direct orthologs within 5 PP families. Gene disruption analysis of all P. berghei PPs revealed that half of the genes are likely to be essential for asexual blood stage development; whereas six are essential for sexual development/sporogony in the mosquito.
In addition, the first reported gene-deletion mutant in a rodent parasite that has been generated using the 150 mg viagra for sale CRISPR/Cas9 genome editing system has been added to the database (published in Mbio by J. Yuan and collaborators)
The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system utilizes a prokaryotic RNA programmable nuclease that can introduce a double-strand break (DSB) at a specific site on a chromosome through heterologous expression of two components: Cas9 nuclease and cheap cialis uk a targeting single guide RNA (sgRNA). Target-specific DSBs introduced by the CRISPR/Cas9 system can be repaired by homologous recombination if a donor template is provided.
The CRISPR/Cas9 system has been shown to be highly efficient in other organisms for generating gene knock-in (KI), KO, or allelic replacements.
Click here for the description of the SERA1 gene-deletion mutant and an description of the CRISP/Cas9 constructs and technology used to delete the P. yoelii.
- 22 April 2014 - The database contains at this moment information on more than 950 different mutants
- 4 december 2013
1. New GeneDB gene models/IDs for P. yoelii
In RMgmDB we have now changed from using the previous designation of P. yoelii gene models (i.e. PYxx…) to the new systematic IDs as provided by GeneDB (i.e. PY17X_xx…..). This means:
- For all mutants only the new gene models/IDs will be visible in RMgmDB
- While the database can still be searched using old gene models, only new gene models will be displayed
For both rodent and human Plasmodium species there is an increasing depth and breadth of genome sequences available for different parasite isolates/lines. In GeneDB/PlasmoDB the (orthologous) genes from lines of the same species have been given different gene IDs, for example for the two laboratory lines, YM and 17X, of P. y. yoelii.
For all mutants in RMgmDB we standardly refer to ONLY the gene IDs of a SINGLE ‘reference line’ for each of the 3 different rodent malaria species:
- P. berghei ANKA (PBANKA_xx...)
- P. y. yoelii (PY17X_xx...)
- P. c. chabaudi (PCHAS_xx...)
In addition we link the rodent gene to the ID of the orthologous gene of P. falciparum 3D7 (PF3D7_xx...).
In the case of P. y. yoelii we provide ONLY the gene ID for P. y. yoelii 17X, whether the lowest price cialis mutants were generated in 17X or in YM. If a mutant was generated in the YM line, we mention the YM gene ID but only as free text in ‘Additional remarks phenotype’ section.
In future we propose to use the same strategy for genes targeted in different P. chabaudi genomes (AJ, CB isolates of P. chabaudi chabaudi and isolates of P. chabaudi adami) and in different P. berghei genomes (NK65, K173, SP11).
2. Unpublished malaria mutants in RMgmDB
Recently we have included in RMgmDB a number of unpublished malaria mutants (gene knock-outs, gene-taggings etc). This includes mutants that have been generated in the laboratories of Dr. Taco Kooij and Dr. Inga Siden-Kiamos.
Researchers are encouraged to submit information to RMgmDB on mutants that are unpublished and for which no (immediate) plans exist to publish their generation/analysis. For example data on Plasmodium mutants that were generated but did not exhibit a clear phenotype as well as information on unsuccessful attempts to disrupt a parasite gene. This information (while sometimes difficult to publish) is of significant value and viagra compare prices can also prevent an unnecessary duplication of effort (and consequently an unnecessary use of laboratory animals). The existence of null-mutants without a distinct phenotype might provide information about the functional redundancy of the target gene. Similarly, the lack of an observable phenotype might be the result of assays that are currently inadequate or, as yet, too insensitive to reveal a phenotypic effect of the genetic modification. Further (collaborative) analysis of such mutants in improved phenotype assays might reveal novel aspects of gene function. We provide a simple (excel) template for submitting to RMgmDB the information on unpublished mutants and negative trials to disrupt or mutate genes.
- 20 August 2013 - New information on genetically modified/transgenic parasites in the RMgmDB database (now contains information on >860 mutants)
A number of P. berghei gene-deletion or gene-tagging mutants of genes that encode (putative) palmitoyltransferases (s-acyl transferases), recently described in a paper published in Traffic (Frénal et al. 2013), have been added to the RMgm database.
See link: DHHC genes (18 mutants)
A number of P. berghei gene-deletion and gene-tagging mutants of genes that encode proteins that are part of the PTEX export machinery have been added; see the recent paper published in Mol Microbiol. (Matthews et al. 2013).
See link: PTEX genes (11 mutants)
Linking between RMgmDB and PlasmoDB and GeneDB
In PlasmoDB (www.plasmodb.org) and GeneDB (www.genedb.org) there are direct links from appropriate gene pages to the relevant genetically modified rodent malaria parasites in RMgmDB.
‘Batch searching’ RMgmDB for mutants using multiple gene ID’s.
It is possible to search and retrieve collated information related to multiple Plasmodium genes at once, which not only speeds up searches but also facilitates the cross-linking and mining of data from multiple sources.
The output for searches using multiple gene ID’s will include: Gene ID (rodent and P. falciparum); RMgm ID, the type of modification (disruption, mutation, tagging etc); if the generic viagra cheapest modification was (un)successful; life cycle stages with a phenotype and if it is different or not-different compared to wild type parasites. This data will be displayed in a table on the website or it can be downloaded as an Excel/spreadsheet compatible format (i.e. as a CSV file).
Linking between RMgmDB and PlasmoGEM
In the RMgm database the information on DNA constructs used to produce mutants made using PlasmoGEM vectors has been linked directly to the detailed information available at the SANGER PlasmoGEM database (http://plasmogem.sanger.ac.uk/).
- 31 May 2013 - The database contains at this moment information on more thany 800 different mutants
- 31 december 2012 - The database contains at this moment information on nearly 750 different mutants
- 26 september 2012 - We have introduced improved search possibilities on RMgmDB and it is now possible to ‘batch search’ using multiple gene models. Consequently, it is now possible to search and retrieve collated information related to multiple genes at once, which not only speeds up searches but also facilitates the cross-linking and mining of data from multiple sources.
The output for searches with multiple gene ID’s will include:
Gene ID (rodent and P. falciparum); RMgm ID, the type of modification (disruption, mutation, tagging etc); if the modification was (un)successful; life cycle stages with a phenotype and if it is different or not-different compared to wild type parasites.
This data will be displayed in a table on the website or it can be downloaded as an Excel/spreadsheet compatible format (i.e. as a CSV file).
For example, searching with multiple gene ID’s will allow the user to:
- Rapidly identify genes that have been targeted for disruption but did not result in selection of mutants, indicating an essential role of these genes in blood stage development
- Identify genes that result in phenotypes in different life cycle stages, such as blood stages, gametocytes, oocysts, liver stages etc
- identify genes that have been tagged (for example with fluorescent markers)
- identify gene for which no information based on mutant phenotypes currently exists
- 30 July 2012 - 2. We have incorporated a module that permits linking between genotype information in RMgmDB with information on DNA-constructs available in the SANGER PlasmoGEM database (http://plasmogem.sanger.ac.uk/). Information on DNA constructs of all mutants generated using PlasmoGEM vectors will be linked, in the future, directly to the information on the PlasmoGEM database.
- 27 July 2012 - We have now changed from using the previous P. falciparum gene models (i.e. PFxxx.. MALxxx.., etc) to the new systematic id’s as provided by GeneDB; i.e. PF3D7_xxx..). This means:
- For all mutants only the new gene models will be visible
- The database can still be searched using old gene models, however only new gene models will be displayed
- 1 July 2012 - The database contains at this moment information on more than 660 different mutants
- 2 February 2012 - Recently a number of new Plasmodium mutants have been published in the RMgmDB database
- P. berghei mutants lacking expression of a protein, GEST, a protein indentified as being involved in infectivity of both gametocytes and sporozoites. Localization studies of GFP-tagged GEST provides evidence that this proteins is secreted by gametocytes and by salivary gland sporozoites (Talman et al., 2011 Mol Microbiol).
- P. berghei mutants generated for the ’conditional knock-down’ of AMA1 and RON4 in the sporozoite stage. Analysis of these mutants provide evidence for independent roles of AMA1 and RON4 during hepatocyte invasion
To silence the AMA1 and RON4 expression, the FLP/FRT site-specific recombination system was used to specifically delete the 3’ regulatory sequences of ama1 and ron4 in sporozoites.(Giovannini et al., 2011, Cell Host Microbe).
- RMgmDB contains several P. berghei mutants expressing tagged RON2 and RON4 proteins and also reports unsuccessful attempts to disrupt ama1, ron2 and ron4 in blood stages, indicating their essential nature for asexual blood stage proliferation
- P. berghei mutants expressing P. vivax proteinsIn these mutants the dhfr-ts gene of P. berghei is replaced with (pyrimethamine-resistant) dhfr-ts genes of P. vivax (Somsak et al., 2011 Malar J)
- P. berghei mutants lacking expression MSP7 and/or plasmepsin4, which show (virulence) attenuation during blood stage proliferation
- RMgmDB contains information on three independent P. berghei mutants lacking MSP7. All show a slightly slower blood stage proliferation, most probably due to a more severe restriction in reticulocyte invasion compared to wild type parasites. Rodent malaria parasites have three MSP7-related genes, all adjacently located on the same chromosome. The exact orthology with the 6 MSP7-related genes of P. falciparum, (MSP7 (PF13_0197) and the want to buy cialis at cheap price five MSP7-related proteins (MSRP1-5), is not clear.
- P. berghei mutants lacking expression of SMAC, a protein exported into the red blood cell; this protein is shown to be involved in CD36-mediated sequestration of P. berghei schizont (Fonager et al., 2011 J Exp Med)
In this study an additional 18 gene deletion mutants has been reported, as well as the unsuccessful attempt to disrupt a further 11 genes (therefore likely to be essential in proliferation of asexual blood stages). All mutants have been included in RMgmDB.
- Plasmodium kinases
RMgmDB contains >70 entries/P. berghei mutants generated for the analysis of Plasmodium protein kinases (see also Tewari et al., 2010 Cell Host & Microbe). All mutants have now been updated with information on the functional analysis of the P. falciparum orthologs by gene targeting (as published in Solyakov et al., 20011 Nat Commun).
RMgmDB contains a number of entries/mutants for functional analysis of members of the ETRAMP family of both P. yoelii and synthroid for sale P. berghei. ETRAMPs belong to a conserved Plasmodium family of proteins. These small (frequently less than 200 aa) integral membrane proteins contain a signal peptide plus a transmembrane domain and are frequently located within the parasitophorous vacuolar membrane (PVM). The ETRAMP/SEP proteins of P. yoelii and P. berghei (8-11 genes) show homology to members of the ETRAMP family of proteins of P. falciparum (14 genes) but the orthologous relationship of the different members is not completely resolved. Functional analysis of the different P. yoelii/berghei ETRAMPs by gene-tagging and gene-deletion indicate some are essential while others are not essential for asexual proliferation and demonstrate expression in both blood stages and/or liver stages, PVM location and export into the red blood cell cytoplasm (in punctuate, vesicle-like structures).(Mackellar et al., 2011. Cell Microbiol; Currà et al., 2011. Traffic).
- P. berghei parasites lines that are ’deleter lines’ for conditional mutagenesis.
These parasite lines can be used ‘to silence genes’ in the sporozoite/liver stage using the Flp/FRT site-specific recombination (SSR) system. These parasite lines have been used to silence expression of ama1, ron4, msp1 and pkg in sporozoites for the study of their role during sporozoite/liver stage development(Lacroix et al., 2011 Nat Protoc)
- ‘Promoter-swap’ mutants in which expression of a gene is specifically down regulated in gametocytes/ookinetes while expression is maintained in asexual blood stages
In the promoter-swap approach the promoter of a gene is replaced by an 'asexual blood stage-specific' promoter that is silent in gametocytes/ookinetes. 'Promoter swapping' is a powerful approach for functional analysis of proteins in gametocytes (and in subsequent stages), when the protein is normally essential during asexual blood-stage development. This approach has been used to study the function of the histone chaperone FACT in gametocytes and myosin A in ookinetes (Siden-Kiamos et al., Cell Microbiol 2011; Laurentino et al., Cell Microbiol. 2011)
- New reference P. berghei (ANKA) and P. yoelii (17XNL) ‘mother lines’ for the rapid introduction of transgenes free of a drug-selectable marker. The use of these lines (in combination with the GIMO transfection method), which greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and is far faster and requires fewer laboratory animals than existing protocols (Lin et al., 2011. Plos One)
A P. yoelii 17XNL reporter line, PyGFP-luccon, which is marker-free and purchase levitra 20 mg online expresses a GFP-luciferase fusion protein under control of the constitutive eef1α promoter. Similar P. berghei reporter lines have been used to visualize and quantify host parasite interactions in vivo, analysis of drug-susceptibility, and in vivo quantification of liver stage development (Lin et al., 2011. Plos One)
- 29 September 2011: A new version of genome of the P. falciparum 3D7 strain is now available on GeneDB, and in this new version all genes have been assigned new gene identifiers (gene ID’s; starting with PF3D7_xxxxxxx)
For users of RMgmDB this means:
• RMgmDB can now be searched using the new PF3D7_ID’s, however only the previous P. falciparum gene identifiers will be displayed.
• We do not intend to change from the ‘old’ P. falciparum ID’s to the new systematic ID’s as displayed in GeneDB until PlasmoDB have also adopted the new ID’s
• Links in GeneDB and PlasmoDB (i.e. phenotype information) to both existing and new mutants in RMgmDB are NOT affected
Currently, RMgmDB contains information on nearly 600 different parasite mutants
- 6 June 2011- Recently a number of new malaria mutant parasites have been published in RMgmDB that show phenotypes with potential relevance for drug/vaccine development.
P. berghei mutants expressing mutated circumsporozoite proteins
- P. berghei mutants expressing circumsporozoite proteins in which the natural H-2Kd restricted epitope is replaced with an H-2Kb restricted epitope. Phenotype analyses of these mutants indicate that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. (RMgm-613)
- P. berghei mutants expressing circumsporozoite proteins with mutated ‘region I’. Phenotype analyses of these mutants establish the link between CSP cleavage and hepatocyte invasion and demonstrates that efficient cleavage requires region I (RMgm-608).
P. berghei mutants expressing P. falciparum proteins
- P. berghei mutants expressing the Chloroquine Resistance Transporter (CRT) of the human parasite P. falciparum (RMgm-610, RMgm-611)
- P. berghei mutants expressing the Hexose Transporter 1 (HT1) protein of the human parasite P. falciparum. These mutants have been used to test the sensitivity PfHT1 in vivo to an inhibitor of HT1, the glucose analog C3361 (RMgm-604)
P. berghei mutants lacking expression of potential drug target proteins
P. berghei mutants that lack expression of ‘antioxidant enzymes’ (thioredoxin reducatase and glutathione reductase) demonstrating that these enzymes are not essential for blood stage development.(RMgm-586, RMgm-587, RMgm-403)
mutants with disrupted genes that show a developmental defect in sporozoite development and transition into liver stages
- P. berghei
mutants lacking genes encoding RNA binding proteins (Pumilio; Puf). Phenotype analyses indicate that the Puf2 protein is a key regulator of the transformation of sporozoites into liver stages (RMgm-514
- P. berghei
mutants lacking genes encoding members of the Cysteine Repeat Modular Protein family (CRMPs). Phenotype analyses show a role in sporozoite egress from oocysts and during liver stage development (RMgm-584
- 24 March 2011 - The database contains at this moment information on 540 different mutants
- 23 March 2011 - The RMgm database now also contains ~70 new entries created by piggyBac-mediated mutations/insertions into P. berghei genes (see Fonager, J et al., BMC Genomics (2011) 12:155). Using the search term ‘piggyBac’ in RMgmBD results in a list of ~70 genes which contain piggyBac inserts. The piggyBac inserts were identified using a TAIL PCR approach in DNA collected from blood stage parasites. For all mutated genes the site of integration is indicated as ~20 nucleotides of gene sequence on either site of the TTAA insertion site. Only piggyBac insertions have been included that were found in the open reading frame, 5’UTR (0-500bp from the start codon) or within introns of genes. These piggyBac insertions are expected to affect/disrupt expression of the gene. The presence of an insert may therefore provide indirect evidence that the gene is NOT essential for blood stage development. For this reason we have included this information in RMgmDB. However, neither expression of the target gene nor the phenotype of the mutant parasite containing the insert has been analyzed.
- 28 February 2011 - The database contains at this moment information on more than 460 different mutants. New mutants include mutants with a mutated Chloroquine Resistant Transporter and mutants with a mutated Circumsporozoite Protein.
- 22 October 2010 - The database contains at this moment information on more than 440 different mutants
- 22 October 2010 - Unsuccessful attempts to knock-out P. berghei genes based on the old gene models
The RMgmDB contains information on a large number of (un)published and unsuccessful attempts to knock-out Plasmodium genes. These unsuccessful attempts strongly indicate that these genes may have an essential role during blood stag development. The new P. berghei annotation, in GeneDB, has revealed that for several unsuccessful attempts to knock-out genes the primers for the 5’ and 3’ targeting regions are located in NOT ONE BUT IN TWO neighboring/adjacent genes. Therefore the integration of the DNA-construct would target TWO genes. The failure to disrupt/knock-out the target gene might, therefore, be due to the unintentional disruption of an additional gene. In RMgmDB we have indicated this new information on the unsuccessful attempts where it is now apparent that the targeting construct will disrupt two adjacent genes.
- 22 October 2010 - RMgmDB database now also contains 64 new entries/mutants generated for the analysis of Plasmodium protein kinases, details of this study have recently been published in Cell Host & Microbe (2010, 8:377-87).
- 19 July 2010 - A paper has been published in Trends in Parasitology which describes the (use of the) RMgm-database. Special emphasis is on standardization of mutant generation and describing mutant phenotypes.
- 26 March 2010 - In GeneDB there are direct links from appropriate gene pages to genetically modified rodent malaria parasites in the RMgm database.
- 5 March 2010 - We have now changed from using the previous P. berghei gene models (i.e. PBXXXXX.XX.X) to the new systematic id’s as provided by GeneDB; i.e. PBANKA_xxxxxx). This means:
- For all mutants only the new gene models will be visible
- The database can still be searched using old gene models, however only new gene models will be displayed
- The P. berghei gene models are now linked to GeneDB gene pages (and not to PlasmoDB, as it still uses the old gene models).
- Existing links in PlasmoDB out to the RMgm database are not affected. Information on new mutant entries will be linked to PlasmoDB once they have adopted the new gene models.
- 29 December 2009 - The database contains at this moment information on more than 300 different mutants.
- 23 July 2009 - In PlasmoDB there are direct links from appropriate gene pages to genetically modified rodent malaria parasites in the RMgm database.
- 16 June 2009 - The database contains at this moment information on more than 260 different mutants.
- 1 April 2009 - The database contains at this moment information on more than 200 different mutants.
- 28 January 2009 - The database contains information on approximately 150 different mutants.
- 6 November 2008 - This database is in its preliminary stages; we are still technically improving the database. At this moment we have information on less then 100 parasite mutant lines.
- 5 October 2008 - The new database, RMgmDB, online!
Pberghei.eu hosts the RMgmDB database containing information on genetically modified malaria parasites generated by many labs worldwide. It contains data of 3 rodent malaria parasites: Plasmodium berghei, P. yoelii and P. chabaudi. The aim of this database is to provide the research community access to detailed information on the generation (e.g. disruption, tagging, mutation, transgene expression) and phenotype of mutant malaria parasites. The database also provides information on (un)published mutants without a clear phenotype and negative trials to disrupt genes.
See two papers in Trends in Parasitology and Methods in Molecular Biology describing RMgmDB with special emphasis on standardization of mutant generation and describing mutant phenotypes.