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Maternal germline-specific genes in the Asian malaria mosquito Anopheles stephensi: characterization and application for disease control.

Biedler JK, Qi Y, Pledger D, James AA, Tu Z - G3 (Bethesda) (2014)

Bottom Line: Promoters from two of these candidates, vitellogenin receptor and nanos, were used in independent transgenic cassettes for the expression of artificial microRNAs against suspected mosquito maternal-effect genes, discontinuous actin hexagon and myd88.Additionally, we demonstrate maternal-specific delivery of mRNA and protein to progeny embryos.We discuss the application of this system of maternal delivery of mRNA/miRNA/protein in research on mosquito reproduction and embryonic development, and for the development of a gene drive system based on maternal-effect dominant embryonic arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Fralin Life Science Institute, Virginia Tech, Blacksburg, Virginia 24061 jbiedler@vt.edu jaketu@vt.edu.

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Constructs used in this study to generate transgenic lines. (A) Selected features of the transgenic cassette. 3XP3 hsp70, Pax-6/Drosophila hsp70 promoter; DsRed, DsRed fluorescent protein; SV40, SV40 termination/polydadenylation signal; PM, maternal promoter; Luc, luciferase reporter cassette. mRNA. Shown between the maternal promoter and luciferase cassette is the Ae. aegypti bZip1 Intron containing three modified miRNAs targeting the 5′ UTR of either myd88 or dah. Dark blue rectangle upstream of intron represents the first exon of Ae. aegypti bZip1. Block arrows indicate piggyBac arms necessary for transposition. Drawing is not to scale. (B) Native fold of the Ae. aegypti bZip1 intron that contains three miRNA hairpins that were modified to target three unique targets in the 5′ UTR of either myd88 or dah. Fold was generated using RNAfold (Gruber et al., 2008). Color key shows base pairing probabilities given by RNAfold. Arrows indicate strand and orientation of the native mature miRNA sequences.
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fig5: Constructs used in this study to generate transgenic lines. (A) Selected features of the transgenic cassette. 3XP3 hsp70, Pax-6/Drosophila hsp70 promoter; DsRed, DsRed fluorescent protein; SV40, SV40 termination/polydadenylation signal; PM, maternal promoter; Luc, luciferase reporter cassette. mRNA. Shown between the maternal promoter and luciferase cassette is the Ae. aegypti bZip1 Intron containing three modified miRNAs targeting the 5′ UTR of either myd88 or dah. Dark blue rectangle upstream of intron represents the first exon of Ae. aegypti bZip1. Block arrows indicate piggyBac arms necessary for transposition. Drawing is not to scale. (B) Native fold of the Ae. aegypti bZip1 intron that contains three miRNA hairpins that were modified to target three unique targets in the 5′ UTR of either myd88 or dah. Fold was generated using RNAfold (Gruber et al., 2008). Color key shows base pairing probabilities given by RNAfold. Arrows indicate strand and orientation of the native mature miRNA sequences.

Mentions: We designed gene cassettes that allow the simultaneous testing of maternal-specific promoters by luciferase reporter assay and expression/delivery of protein and miRNAs to the oocyte and embryo. As a stepping-stone toward engineering a Medea gene drive system in the mosquito An. stephensi, these cassettes were used to generate transgenic lines for the purpose of knocking down the mRNA of suspected maternal-effect genes myd88 and dah using amiRNAs that replaced the sequence of three natural miRNAs of the Ae. aegypti bZip1 intron (Li et al. 2009) (Figure 5). The remaining intron sequence and the first exon that is upstream of the miRNA-containing intron were retained in our constructs to avoid potential interference with processing. This Ae. aegypti gene is an ortholog to the An. stephensi bZip1 ASTEI01689 (accession JQ266222) identified in our 79 germline-specific genes. We had previously identified the gene in Ae. aegypti and determined that the intron was spliced when An. stephensi embryos were injected with a reporter gene cassette containing this intron (not shown). The maternal-effect gene myd88 was previously used as a target for the Drosophila Medea (Chen et al. 2007). The An. stephensi myd88 ASTEI05979 (same gene as ASTE008769 for An. stephensi SDA-500 strain) is identified as a 1:1 ortholog to the Drosophila myd88 based on Vectorbase. It has the highest RPKM values in 24-hr PBM ovary and in early embryos. Therefore, we reasoned myd88 was a good choice for a mosquito Medea target. We used the upstream sequence of An. stephensi nanos to drive expression of this cassette. Another cassette was designed to target dah using the vgr upstream sequence as the promoter. dah (ASTEI03515) is an ortholog to the Drosophila dah, a maternal-effect gene that is essential for cortical furrow formation during early embryonic development (Zhang et al. 1996; Zhang et al. 2000).


Maternal germline-specific genes in the Asian malaria mosquito Anopheles stephensi: characterization and application for disease control.

Biedler JK, Qi Y, Pledger D, James AA, Tu Z - G3 (Bethesda) (2014)

Constructs used in this study to generate transgenic lines. (A) Selected features of the transgenic cassette. 3XP3 hsp70, Pax-6/Drosophila hsp70 promoter; DsRed, DsRed fluorescent protein; SV40, SV40 termination/polydadenylation signal; PM, maternal promoter; Luc, luciferase reporter cassette. mRNA. Shown between the maternal promoter and luciferase cassette is the Ae. aegypti bZip1 Intron containing three modified miRNAs targeting the 5′ UTR of either myd88 or dah. Dark blue rectangle upstream of intron represents the first exon of Ae. aegypti bZip1. Block arrows indicate piggyBac arms necessary for transposition. Drawing is not to scale. (B) Native fold of the Ae. aegypti bZip1 intron that contains three miRNA hairpins that were modified to target three unique targets in the 5′ UTR of either myd88 or dah. Fold was generated using RNAfold (Gruber et al., 2008). Color key shows base pairing probabilities given by RNAfold. Arrows indicate strand and orientation of the native mature miRNA sequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321024&req=5

fig5: Constructs used in this study to generate transgenic lines. (A) Selected features of the transgenic cassette. 3XP3 hsp70, Pax-6/Drosophila hsp70 promoter; DsRed, DsRed fluorescent protein; SV40, SV40 termination/polydadenylation signal; PM, maternal promoter; Luc, luciferase reporter cassette. mRNA. Shown between the maternal promoter and luciferase cassette is the Ae. aegypti bZip1 Intron containing three modified miRNAs targeting the 5′ UTR of either myd88 or dah. Dark blue rectangle upstream of intron represents the first exon of Ae. aegypti bZip1. Block arrows indicate piggyBac arms necessary for transposition. Drawing is not to scale. (B) Native fold of the Ae. aegypti bZip1 intron that contains three miRNA hairpins that were modified to target three unique targets in the 5′ UTR of either myd88 or dah. Fold was generated using RNAfold (Gruber et al., 2008). Color key shows base pairing probabilities given by RNAfold. Arrows indicate strand and orientation of the native mature miRNA sequences.
Mentions: We designed gene cassettes that allow the simultaneous testing of maternal-specific promoters by luciferase reporter assay and expression/delivery of protein and miRNAs to the oocyte and embryo. As a stepping-stone toward engineering a Medea gene drive system in the mosquito An. stephensi, these cassettes were used to generate transgenic lines for the purpose of knocking down the mRNA of suspected maternal-effect genes myd88 and dah using amiRNAs that replaced the sequence of three natural miRNAs of the Ae. aegypti bZip1 intron (Li et al. 2009) (Figure 5). The remaining intron sequence and the first exon that is upstream of the miRNA-containing intron were retained in our constructs to avoid potential interference with processing. This Ae. aegypti gene is an ortholog to the An. stephensi bZip1 ASTEI01689 (accession JQ266222) identified in our 79 germline-specific genes. We had previously identified the gene in Ae. aegypti and determined that the intron was spliced when An. stephensi embryos were injected with a reporter gene cassette containing this intron (not shown). The maternal-effect gene myd88 was previously used as a target for the Drosophila Medea (Chen et al. 2007). The An. stephensi myd88 ASTEI05979 (same gene as ASTE008769 for An. stephensi SDA-500 strain) is identified as a 1:1 ortholog to the Drosophila myd88 based on Vectorbase. It has the highest RPKM values in 24-hr PBM ovary and in early embryos. Therefore, we reasoned myd88 was a good choice for a mosquito Medea target. We used the upstream sequence of An. stephensi nanos to drive expression of this cassette. Another cassette was designed to target dah using the vgr upstream sequence as the promoter. dah (ASTEI03515) is an ortholog to the Drosophila dah, a maternal-effect gene that is essential for cortical furrow formation during early embryonic development (Zhang et al. 1996; Zhang et al. 2000).

Bottom Line: Promoters from two of these candidates, vitellogenin receptor and nanos, were used in independent transgenic cassettes for the expression of artificial microRNAs against suspected mosquito maternal-effect genes, discontinuous actin hexagon and myd88.Additionally, we demonstrate maternal-specific delivery of mRNA and protein to progeny embryos.We discuss the application of this system of maternal delivery of mRNA/miRNA/protein in research on mosquito reproduction and embryonic development, and for the development of a gene drive system based on maternal-effect dominant embryonic arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Fralin Life Science Institute, Virginia Tech, Blacksburg, Virginia 24061 jbiedler@vt.edu jaketu@vt.edu.

Show MeSH
Related in: MedlinePlus