Browsing by Author "Riehle, Michelle M."

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    A major genetic locus controlling natural Plasmodium falciparum infection is shared by East and West African Anopheles gambiae
    Riehle, Michelle M.; Markianos, Kyriacos; Lambrechts, Louis; Xia, Ai; Sharakhov, Igor V.; Koella, Jacob C.; Vernick, Kenneth D. (2007-07-06)
    Background Genetic linkage mapping identified a region of chromosome 2L in the Anopheles gambiae genome that exerts major control over natural infection by Plasmodium falciparum. This 2L Plasmodium-resistance interval was mapped in mosquitoes from a natural population in Mali, West Africa, and controls the numbers of P. falciparum oocysts that develop on the vector midgut. An important question is whether genetic variation with respect to Plasmodium-resistance exists across Africa, and if so whether the same or multiple geographically distinct resistance mechanisms are responsible for the trait. Methods To identify P falciparum resistance loci in pedigrees generated and infected in Kenya, East Africa, 28 microsatellite loci were typed across the mosquito genome. Genetic linkage mapping was used to detect significant linkage between genotype and numbers of midgut oocysts surviving to 7-8 days post-infection. Results A major malaria-control locus was identified on chromosome 2L in East African mosquitoes, in the same apparent position originally identified from the West African population. Presence of this resistance locus explains 75% of parasite free mosquitoes. The Kenyan resistance locus is named EA_Pfin1 (East Africa_ Plasmodium falciparum Infection Intensity). Conclusion Detection of a malaria-control locus at the same chromosomal location in both East and West African mosquitoes indicates that, to the level of genetic resolution of the analysis, the same mechanism of Plasmodium-resistance, or a mechanism controlled by the same genomic region, is found across Africa, and thus probably operates in A. gambiae throughout its entire range.
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    Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B
    Eggleston, Heather; Njoya, Kimani; Anderson, Cameron E.; Holm, Inge; Eiglmeier, Karin; Liang, Jiangtao; Sharakhov, Igor V.; Vernick, Kenneth D.; Riehle, Michelle M. (2022-12-13)
    Background Anopheles cell lines are used in a variety of ways to better understand the major vectors of malaria in sub-Saharan Africa. Despite this, commonly used cell lines are not well characterized, and no tools are available for cell line identification and authentication. Methods Utilizing whole genome sequencing, genomes of 4a-3A and 4a-3B ‘hemocyte-like’ cell lines were characterized for insertions and deletions (indels) and SNP variation. Genomic locations of distinguishing sequence variation and species origin of the cell lines were also examined. Unique indels were targeted to develop a PCR-based cell line authentication assay. Mitotic chromosomes were examined to survey the cytogenetic landscape for chromosome structure and copy number in the cell lines. Results The 4a-3A and 4a-3B cell lines are female in origin and primarily of Anopheles coluzzii ancestry. Cytogenetic analysis indicates that the two cell lines are essentially diploid, with some relatively minor chromosome structural rearrangements. Whole-genome sequence was generated, and analysis indicated that SNPs and indels which differentiate the cell lines are clustered on the 2R chromosome in the regions of the 2Rb, 2Rc and 2Ru chromosomal inversions. A PCR-based authentication assay was developed to fingerprint three indels unique to each cell line. The assay distinguishes between 4a-3A and 4a-3B cells and also uniquely identifies two additional An. coluzzii cell lines tested, Ag55 and Sua4.0. The assay has the specificity to distinguish four cell lines and also has the sensitivity to detect cellular contamination within a sample of cultured cells. Conclusions Genomic characterization of the 4a-3A and 4a-3B Anopheles cell lines was used to develop a simple diagnostic assay that can distinguish these cell lines within and across research laboratories. A cytogenetic survey indicated that the 4a-3A and Sua4.0 cell lines carry essentially normal diploid chromosomes, which makes them amenable to CRISPR/Cas9 genome editing. The presented simple authentication assay, coupled with screening for mycoplasma, will allow validation of the integrity of experimental resources and will promote greater experimental reproducibility of results.