Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.
Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.
Representative Results
Figure 1. Untransfected healthy A. stephensi MSQ43 (A) and A. gambiae SUA5B (B) cells at approximately 80% confluence.
Figure 2. A. stephensi MSQ43 (A) and A. gambiae SUA5B (B) cells transfected with positive control GFP expressing plasmid.
Figure 3. Flow cytometry data confirming conversion of BFP to GFP via mutagenesis of a single nucleotide following transfection with BFP-specific locked nucleic acid-modified oligonucleotides (LNA-ONs). SUA5B cells were transfected with a GFP expressing plasmid (pGFP) as positive control, with a BFP expressing plasmid (pBFP) as a negative control, or with pBFP and increasing concentrations of BFP-specific LNA-ONs. (A) Gating strategy for flow cytometry data showing from left to right: forward versus side scatter of live cells, propidium iodide (PI) negative cells, and GFP positive cells five days following transfection. (B) Graph of percentage of GFP positive cells shown in (A) following transfection with pBFP and increasing concentrations of LNA-ONs.
Table I. Transfection conditions.
Table II. Transfection conditions used in Figure 1.
Here we present an in vitro method and evidence for targeted conversion of a single nucleotide in an episomal gene target following transfection of LNA-ONs into A. stephensi and A. gambiae cells. LNA-ON-mediated gene conversion requires induction of a site-specific DNA repair response; our data indicate that mosquito cells can support this mechanism for site-specific mutagenesis. While the method presented here is based on conversion of an episomal target, our data suggest that LNA-ON-mediated conversion can be optimized for chromosomal targets in mosquito cells as it has been in other systems. Gene conversion, therefore, would facilitate the generation of stably transformed mosquito cell lines without complex protocols and lengthy drug selection regimes. Further, existing technology based on the use of mRNA-specific fluorescent tags in live cells can be leveraged to sort and enrich mosquito cells with converted chromosomal gene targets for biological studies 5-7.
The success of this method critically depends on the use of healthy cells at a density of 70-80%. In addition, at all steps during the transfection process it is important to determine that neither the plasmid nor LNA-ONs have precipitated out of solution by checking the solution visually for precipitate. One possible modification of this protocol is to plate the cells 24 hours prior to beginning the transfection process. This is suggested for slow growing or sensitive cell lines. We found that the optimal ratio of plasmid DNA to LNA-ONs was 1:5 but it will be important to determine the ideal ratio for each new gene and cell line of interest.
A variety of studies have identified mosquito loci and genes that confer resistance to malaria parasite infection 8-10. If these targets are amenable to manipulation – via the enhancement of or redirection of expression timing – they can provide a basis for genetic engineering as a strategy to control malaria parasite transmission in natural vector populations. The methodology discussed here is a novel and effective technique for introducing mutations into malaria resistance genes in the laboratory. Through these manipulations of genes of interest, we can determine specifically how gene products alter cellular anti-parasite responses in vitro, a critical step to understanding control of malaria parasite development in vivo.
The authors have nothing to disclose.
We would like to thank Abbie Spinner at the California National Primate Research Center for her assistance with flow cytometry. This study was supported by funding from the National Institute of Allergy and Infectious Diseases (NIAID) National Institutes of Health (NIH) AI073745, AI080799, and AI078183.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Effectene transfection reagent | Qiagen | 301425 | ||
GFP control plasmid (pcDNA6.2-GW/EmGFP-miR-neg) | Invitrogen | K4935-00 | ||
BFP plasmid (pcDNA5/FRT/TO) | Gift from Dr. Concordet3 | |||
LNA-ONs | Gift from Dr. Concordet3 | |||
MEM, Earle’s w/ glutamine | Invitrogen | 11095 | ||
Fetal calf serum (FCS) | Invitrogen | 16000 | ||
Schneider’s Drosophila medium | Invitrogen | 11730 | ||
Non-essential amino acids | Invitrogen | 11140 | ||
10% D-glucose | Sigma | G5767 | ||
Penicillin-streptomycin antibiotic | Invitrogen | 15140 | ||
6-well culture plates | Corning | 3516 |