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: 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.

Keywords: Anopheles coluzzii; Anopheles gambiae; Cell line authentication; Hemocytes; Reproducibility.

Fig. 1

Fixed variants present in 4a-3A and 4a-3B cells. A Of the 289,485 fixed indels identified in 4a-3A and 4a-3B cells, 72% of variants are present in both cell lines, 23% are unique to 4a-3B, and the remaining 5% are unique to 4a-3A. B Of the 1,244,056 fixed SNPs identified in 4a-3A and 4a-3B, 74% of variants are present in both cell lines, 21% are unique to 4a-3B, and the remaining 5% are unique to 4a-3A

Fig. 2

Genetic variants differentiating 4a-3A and 4a-3B cells are clustered on the 2R chromosome. A The distribution of fixed indels (left) and SNPs (right) is distinct from the total genome composition of the chromosome arms, all pairwise χ² P-values < 0.0001. Distinct GT: variants have a distinction between the 4a-3A and 4a-3B cell lines. Same GT: variants have the same genotype in the 4a-3A and 4a-3B cell lines but each are distinct from the An. gambiae AgamP4 reference genome assembly. B Distribution of cell line Distinct indels (top) and SNPs (bottom) across the An. gambiae AgamP4 reference genome assembly. Window size of 10,000 bp with a step size of 2500 bp. *Position of the three indels used in the diagnostic assay described in Fig. 4. C The distribution of fixed indels (left) and SNPs (right) is distinct from the overall composition across the 2R chromosome arm, all pairwise χ² P-values < 0.0001. D Molecular diagnostic for the 2Rb (top) and 2Rj (bottom) chromosome inversions using published methods. Lane 1: 100-bp ladder, lane 2: 4a-3A, lane 3: 4a-3B. Expected size for 2R + ^j is 494 bp and for 2Rj is 253 bp (47) and for 2R + ^b is 630 bp and for 2Rb is 429 bp

Fig. 3

Molecular and in silico species typing of Anopheles cell lines indicates 4a-3A and 4a-3B are primarily An. coluzzii in origin. A The S200 X6.1 assay [49] molecular species diagnostic was run on genomic DNA isolated from cell lines. Lane 1 contains a 100-bp ladder; lanes 2 and 3 show amplified products from known An. gambiae and An. coluzzii samples, respectively. Lane 4 is amplified product from an equal volume mixture of the PCR template used in lanes 2 and 3 and thus represents a species hybrid. Lanes 5–8 show amplified product from 4a-3B, 4a-3A, Ag55 and Sua4.0 genomic DNA, respectively. Expected band sizes are 479 bp for An. coluzzii and 249 bp for An. gambiae. Lane 9 is a no template control, and lane 10 contains GeneRuler 1-kb Plus DNA Ladder. (B) In silico species typing performed following a previously published method [51]. SNP names follow the naming scheme provided by Lee et al. 2014 [51] with the last five digits of the AGAP gene name followed by the position of the SNP in the coding sequence of the gene (e.g. 01706-129 is a SNP at position 129 in AGAP001706). The second column of the table gives the proportion of An. gambiae ancestry ranging from 0 to 1 with 0 indicating complete An. coluzzii ancestry and 1 indicating complete An. gambiae ancestry. Gray shading indicates the degree of An. gambiae ancestry; dark gray 100% An. coluzzii, light gray 100% An. gambiae and medium gray species hybrids. In silico species typing was also done for the AgamP4 genome assembly; however, this may be an over- or underestimate of An. gambiae ancestry because genome assembly lacks heterozygosity

Fig. 4

Molecular assay to differentiate 4a-3A, 4a-3B, Ag55 and Sua4.0 cell lines. A Molecular fingerprints of three PCRs amplifying indel regions in 4a-3A (lanes 2–4), 4a-3B (lanes 5–7), Ag55 (lanes 8–10), Sua4.0 cells (lanes 11–13) and no PCR template controls (lanes 14–16); 100-bp ladder (lane 1), indel 2R.25670547 (lanes 2, 5, 8, 11, 14), indel 3R.11788474 (lanes 3, 6, 9, 12, 15), indel 3R.11809836 (lanes 4, 7, 10, 13, 16) and 1-kb Plus DNA Ladder (lane 17). B Table of expected PCR product sizes for 2R.25670547, 3R.11788474 and 3R.11809836 for each of the cell lines based on Sanger sequencing results. *Predicted size based on whole genome sequencing was 532 bp for 3R.11788474 in 4a-3A genomic DNA; however, upon Sanger sequencing an additional indel of 204-bp size was discovered following the predicted 148-bp indel resulting in an amplicon size of 736 bp. All sizes were verified by Sanger sequencing. C Detection of contamination by indel assay. The 3R.11788474 indel assay is able to detect contaminating genomic DNA present at 10% of the total PCR template input. Lane 1 GeneRuler 1-kb Plus DNA Ladder; lane 2: 20 ng 4a-3B gDNA; lane 3: 18 ng 4a-3B gDNA and 2 ng 4a-3A gDNA; lane 4: 16 ng 4a-3B gDNA and 4 ng 4a-3A gDNA; lane 5: 13.3 ng 4a-3B gDNA and 6.6 ng 4a-3A gDNA; lane 6: 10 ng 4a-3B gDNA and 10 ng 4a-3A gDNA; lane 7: 6.6 ng 4a-3B gDNA and 13.3 ng 4a-3A gDNA; lane 8: 4 ng 4a-3B gDNA and 16 ng 4a-3A gDNA; lane 9: 2 ng 4a-3B gDNA and 18 ng 4a-3A; lane 10: 20 ng 4a-3A gDNA, lane 11: control, no PCR template, lane 12: 100-bp ladder

Fig. 5

4a-3A and Sua4.0 cells are diploid. Representative images of chromosome preparations depicting the 2 autosomes (2 and 3) and the X chromosome. Both cell lines examined are female in origin and > 95% diploid. Chromosomes are shown in gray color. Scale bar: 1 µm