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The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies.

Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A - BMC Mol. Biol. (2009)

Bottom Line: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes.We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Imperial College London, Division of Cell and Molecular Biology, Imperial College Road, London, UK. p.papathanos@imperial.ac.uk

ABSTRACT

Background: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.

Results: We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae.

Conclusion: We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

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Maternally derived eGFP deposited in embryos of Vas2GFP transgenic mosquitoes. (A) Brightfield (upper panels) and fluorescent micrographs (lower panels) of embryos deriving from heterozygous transgenic Vas2GFP mothers (left) or Vas2GFP fathers (right) crossed to wild type. (B) RT-PCR analysis of eGFP transcript deposition in laid embryos deriving from transgenic Vas2GFP mothers or Vas2GFP fathers in a time course from 1 hour post-oviposition until 24 hours post-oviposition. C) Larvae deriving from either heterozygous Vas2GFP mothers crossed to wild type males or heterozygous Vas2GFP fathers crossed to wild type females scored for eGFP (maternal deposition) and RFP fluorescence (segregation of transgene). Scoring was performed in all hatched larvae immediately upon hatching (L1 larval stage) and 5 days later (L2 larval stage).
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Figure 4: Maternally derived eGFP deposited in embryos of Vas2GFP transgenic mosquitoes. (A) Brightfield (upper panels) and fluorescent micrographs (lower panels) of embryos deriving from heterozygous transgenic Vas2GFP mothers (left) or Vas2GFP fathers (right) crossed to wild type. (B) RT-PCR analysis of eGFP transcript deposition in laid embryos deriving from transgenic Vas2GFP mothers or Vas2GFP fathers in a time course from 1 hour post-oviposition until 24 hours post-oviposition. C) Larvae deriving from either heterozygous Vas2GFP mothers crossed to wild type males or heterozygous Vas2GFP fathers crossed to wild type females scored for eGFP (maternal deposition) and RFP fluorescence (segregation of transgene). Scoring was performed in all hatched larvae immediately upon hatching (L1 larval stage) and 5 days later (L2 larval stage).

Mentions: In Drosophila, vasa protein and mRNA are deposited into developing oocytes as maternal derived factors [27,29]. We observed that 72 hours post-blood-feeding, developing oocytes of Vas2GFP transformant female mosquitoes contained ubiquitous distribution of eGFP signal (data not shown). To investigate maternal deposition of the eGFP transgene by our vasa regulatory sequence, we mated Vas2GFP transgenic females to wild type males and, as a control, Vas2GFP transgenic males to wild type females. Maternal deposition of eGFP from Vas2GFP heterozygous females was readily detectable in uniformly fluorescent embryos and neonatal hatchlings but not in control embryos or hatchlings, where the transgene was transmitted from transgenic fathers (Figure 4A). The phenotype of nearly all larvae from heterozygous Vas2GFP maternal crosses was eGFP positive (uniformly staining the larvae), whilst the transgene, detected by the 3xP3-RFP marker, segregated to the expected 50% of the progeny. In progeny originating from crosses of Vas2GFP transgenic males to wild type females the eGFP phenotype (restricted to the gonads) was linked to the expression of the RFP marker (Figure 4C). We extracted total mRNA from embryos at several time points after oviposition and performed RT-PCRs to specifically amplify both the endogenous vasa and engineered eGFP transcript. Similarly to what was observed for vasa, the eGFP transcript could be detected up to 2 hours after oviposition (Figure 4B) thus showing that this promoter directs the transfer of both protein and mRNA from transgenic follicles to offspring of the next generation.


The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies.

Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A - BMC Mol. Biol. (2009)

Maternally derived eGFP deposited in embryos of Vas2GFP transgenic mosquitoes. (A) Brightfield (upper panels) and fluorescent micrographs (lower panels) of embryos deriving from heterozygous transgenic Vas2GFP mothers (left) or Vas2GFP fathers (right) crossed to wild type. (B) RT-PCR analysis of eGFP transcript deposition in laid embryos deriving from transgenic Vas2GFP mothers or Vas2GFP fathers in a time course from 1 hour post-oviposition until 24 hours post-oviposition. C) Larvae deriving from either heterozygous Vas2GFP mothers crossed to wild type males or heterozygous Vas2GFP fathers crossed to wild type females scored for eGFP (maternal deposition) and RFP fluorescence (segregation of transgene). Scoring was performed in all hatched larvae immediately upon hatching (L1 larval stage) and 5 days later (L2 larval stage).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713240&req=5

Figure 4: Maternally derived eGFP deposited in embryos of Vas2GFP transgenic mosquitoes. (A) Brightfield (upper panels) and fluorescent micrographs (lower panels) of embryos deriving from heterozygous transgenic Vas2GFP mothers (left) or Vas2GFP fathers (right) crossed to wild type. (B) RT-PCR analysis of eGFP transcript deposition in laid embryos deriving from transgenic Vas2GFP mothers or Vas2GFP fathers in a time course from 1 hour post-oviposition until 24 hours post-oviposition. C) Larvae deriving from either heterozygous Vas2GFP mothers crossed to wild type males or heterozygous Vas2GFP fathers crossed to wild type females scored for eGFP (maternal deposition) and RFP fluorescence (segregation of transgene). Scoring was performed in all hatched larvae immediately upon hatching (L1 larval stage) and 5 days later (L2 larval stage).
Mentions: In Drosophila, vasa protein and mRNA are deposited into developing oocytes as maternal derived factors [27,29]. We observed that 72 hours post-blood-feeding, developing oocytes of Vas2GFP transformant female mosquitoes contained ubiquitous distribution of eGFP signal (data not shown). To investigate maternal deposition of the eGFP transgene by our vasa regulatory sequence, we mated Vas2GFP transgenic females to wild type males and, as a control, Vas2GFP transgenic males to wild type females. Maternal deposition of eGFP from Vas2GFP heterozygous females was readily detectable in uniformly fluorescent embryos and neonatal hatchlings but not in control embryos or hatchlings, where the transgene was transmitted from transgenic fathers (Figure 4A). The phenotype of nearly all larvae from heterozygous Vas2GFP maternal crosses was eGFP positive (uniformly staining the larvae), whilst the transgene, detected by the 3xP3-RFP marker, segregated to the expected 50% of the progeny. In progeny originating from crosses of Vas2GFP transgenic males to wild type females the eGFP phenotype (restricted to the gonads) was linked to the expression of the RFP marker (Figure 4C). We extracted total mRNA from embryos at several time points after oviposition and performed RT-PCRs to specifically amplify both the endogenous vasa and engineered eGFP transcript. Similarly to what was observed for vasa, the eGFP transcript could be detected up to 2 hours after oviposition (Figure 4B) thus showing that this promoter directs the transfer of both protein and mRNA from transgenic follicles to offspring of the next generation.

Bottom Line: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes.We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Imperial College London, Division of Cell and Molecular Biology, Imperial College Road, London, UK. p.papathanos@imperial.ac.uk

ABSTRACT

Background: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.

Results: We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae.

Conclusion: We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

Show MeSH
Related in: MedlinePlus