Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B
Abstract 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 Utili...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2022-12-01
|
| Series: | Parasites & Vectors |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13071-022-05590-3 |
| _version_ | 1797980842063036416 |
|---|---|
| author | Heather Eggleston Kimani Njoya Cameron E. Anderson Inge Holm Karin Eiglmeier Jiangtao Liang Igor V. Sharakhov Kenneth D. Vernick Michelle M. Riehle |
| author_facet | Heather Eggleston Kimani Njoya Cameron E. Anderson Inge Holm Karin Eiglmeier Jiangtao Liang Igor V. Sharakhov Kenneth D. Vernick Michelle M. Riehle |
| author_sort | Heather Eggleston |
| collection | DOAJ |
| description | Abstract 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. Graphical abstract |
| first_indexed | 2024-04-11T06:00:14Z |
| format | Article |
| id | doaj.art-d247c24983e44af08627f2d880ef1ff3 |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-04-11T06:00:14Z |
| publishDate | 2022-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-d247c24983e44af08627f2d880ef1ff32022-12-22T04:41:43ZengBMCParasites & Vectors1756-33052022-12-0115111310.1186/s13071-022-05590-3Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3BHeather Eggleston0Kimani Njoya1Cameron E. Anderson2Inge Holm3Karin Eiglmeier4Jiangtao Liang5Igor V. Sharakhov6Kenneth D. Vernick7Michelle M. Riehle8Department of Microbiology and Immunology, Medical College of WisconsinDepartment of Microbiology and Immunology, Medical College of WisconsinDepartment of Microbiology and Immunology, Medical College of WisconsinInstitut Pasteur, Université de Paris, CNRS UMR 2000, Unit of Insect Vector Genetics and Genomics, Department of Parasites and Insect VectorsInstitut Pasteur, Université de Paris, CNRS UMR 2000, Unit of Insect Vector Genetics and Genomics, Department of Parasites and Insect VectorsDepartment of Entomology, Virginia Polytechnic Institute and State UniversityDepartment of Entomology, Virginia Polytechnic Institute and State UniversityInstitut Pasteur, Université de Paris, CNRS UMR 2000, Unit of Insect Vector Genetics and Genomics, Department of Parasites and Insect VectorsDepartment of Microbiology and Immunology, Medical College of WisconsinAbstract 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. Graphical abstracthttps://doi.org/10.1186/s13071-022-05590-3Anopheles gambiaeAnopheles coluzziiHemocytesCell line authenticationReproducibility |
| spellingShingle | Heather Eggleston Kimani Njoya Cameron E. Anderson Inge Holm Karin Eiglmeier Jiangtao Liang Igor V. Sharakhov Kenneth D. Vernick Michelle M. Riehle Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B Parasites & Vectors Anopheles gambiae Anopheles coluzzii Hemocytes Cell line authentication Reproducibility |
| title | Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B |
| title_full | Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B |
| title_fullStr | Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B |
| title_full_unstemmed | Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B |
| title_short | Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B |
| title_sort | molecular characterization and genetic authentication assay for anopheles hemocyte like cell lines 4a 3a and 4a 3b |
| topic | Anopheles gambiae Anopheles coluzzii Hemocytes Cell line authentication Reproducibility |
| url | https://doi.org/10.1186/s13071-022-05590-3 |
| work_keys_str_mv | AT heathereggleston molecularcharacterizationandgeneticauthenticationassayforanopheleshemocytelikecelllines4a3aand4a3b AT kimaninjoya molecularcharacterizationandgeneticauthenticationassayforanopheleshemocytelikecelllines4a3aand4a3b AT cameroneanderson molecularcharacterizationandgeneticauthenticationassayforanopheleshemocytelikecelllines4a3aand4a3b AT ingeholm molecularcharacterizationandgeneticauthenticationassayforanopheleshemocytelikecelllines4a3aand4a3b AT karineiglmeier molecularcharacterizationandgeneticauthenticationassayforanopheleshemocytelikecelllines4a3aand4a3b AT jiangtaoliang molecularcharacterizationandgeneticauthenticationassayforanopheleshemocytelikecelllines4a3aand4a3b AT igorvsharakhov molecularcharacterizationandgeneticauthenticationassayforanopheleshemocytelikecelllines4a3aand4a3b AT kennethdvernick molecularcharacterizationandgeneticauthenticationassayforanopheleshemocytelikecelllines4a3aand4a3b AT michellemriehle molecularcharacterizationandgeneticauthenticationassayforanopheleshemocytelikecelllines4a3aand4a3b |