scholarly journals Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium

2006 ◽  
Vol 34 (5) ◽  
pp. e39-e39 ◽  
Author(s):  
J. A. M. Braks
Keyword(s):  
Reverse Genetics ◽  
Selectable Marker ◽  
Marker System ◽  
Negative Selectable Marker

scholarly journals Development of a Negative Selectable Marker for Entamoeba histolytica

10.3791/2410 ◽  
2010 ◽  
Author(s):  
Mayuresh M Abhyankar ◽  
Sarah M Haviland ◽  
Carol A Gilchrist ◽  
William A Petri, Jr.
Keyword(s):  
Entamoeba Histolytica ◽  
Selectable Marker ◽  
Negative Selectable Marker

scholarly journals Genetic Mapping Reveals that Sinefungin Resistance in Toxoplasma gondii Is Controlled by a Putative Amino Acid Transporter Locus That Can Be Used as a Negative Selectable Marker

2014 ◽  
Vol 14 (2) ◽  
pp. 140-148 ◽  
Author(s):  
Michael S. Behnke ◽  
Asis Khan ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Genetic Mapping ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Map ◽  
Selectable Marker ◽  
Stop Codon ◽  
Whole Genome ◽  
Content Type ◽  
Negative Selectable Marker

ABSTRACTQuantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasiteToxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDRrand type 10 VAND-SNFr. The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of theSNR1gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies inT. gondii.

scholarly journals Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta

2020 ◽  
Author(s):  
David S. Booth ◽  
Nicole King
Keyword(s):  
Genome Editing ◽  
Reverse Genetics ◽  
Selectable Marker ◽  
Model System ◽  
Genetic Screen ◽  
Reverse Genetic ◽  
Genetic Screens ◽  
Multicellular Development ◽  
Forward Genetic Screen ◽  
Salpingoeca Rosetta

AbstractIn a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.

A novel, negative selectable marker for gene disruption in Dictyostelium

Gene ◽  
1997 ◽  
Vol 202 (1-2) ◽  
pp. 171-176 ◽  
Author(s):  
Alastair Morrison ◽  
Rolf Marschalek ◽  
Theo Dingermann ◽  
Adrian J Harwood
Keyword(s):  
Gene Disruption ◽  
Selectable Marker ◽  
Negative Selectable Marker
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