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Tools for Anopheles gambiae Transgenesis.

Volohonsky G, Terenzi O, Soichot J, Naujoks DA, Nolan T, Windbichler N, Kapps D, Smidler AL, Vittu A, Costa G, Steinert S, Levashina EA, Blandin SA, Marois E - G3 (Bethesda) (2015)

Bottom Line: Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects.This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis.Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects.

View Article: PubMed Central - PubMed

Affiliation: INSERM U963, CNRS UPR9022, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France.

No MeSH data available.


Related in: MedlinePlus

Transgenic reporter Anopheles gambiae lines. All pictures were taken with the 5× objective of a Zeiss Axiovert fluorescence microscope yielding an 80× visual magnification, unless otherwise noted. (A and B) ppo6-RFP fourth instar larva, central ventral abdominal region, and head. Note the red fluorescent hemocytes throughout the larval body. Green fluorescence reflects the activity of the 3xP3-YFP transgenesis selection marker. (C–E) Lp-RFP. (C) Composite of two pictures showing the entire body of a fourth instar larva. Fat body tissue is revealed by red fluorescence. (D) Fat body tissue dissected from a transgenic adult female, observed with a 10× objective. (E) Neonate larva showing initial Lp promoter activity in the intestine. (F–H) Vg-GFP adult blood-fed female mosquitoes. GFP expression in the fat body renders the whole abdomen fluorescent (F). (G and H) Dissected abdomen with GFP and (GFP + bright field) overlay showing the ovary. (I–M) actin5C-GFP mosquitoes with 3xP3-RFP transgenesis marker. (I–K) Larva pictured in merged, red, and green channels. (L) Pupa (merged channels). (M) Dissected intestine from an adult female. (N–R) G12-GFP, intestine dissected from adult females without blood feeding (N and O, merged bright field + green channels) or with blood feeding (P–R, merged bright field + green and split channels). (S and T) vas2-tdTomato neonate larva showing systemic red fluorescence inherited from maternal deposition in the oocyte in addition to endogenous gonad expression (arrowheads point to gonads). (U and V) tdTomato-expressing gonad in female (U) and male (V) fourth instar larvae. (W) Testis in dissected adult male abdomen. (X and Y) Ovary in dissected adult female abdomen. (U–X) Merged bright field and red channels. (Y) Red channel.
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fig6: Transgenic reporter Anopheles gambiae lines. All pictures were taken with the 5× objective of a Zeiss Axiovert fluorescence microscope yielding an 80× visual magnification, unless otherwise noted. (A and B) ppo6-RFP fourth instar larva, central ventral abdominal region, and head. Note the red fluorescent hemocytes throughout the larval body. Green fluorescence reflects the activity of the 3xP3-YFP transgenesis selection marker. (C–E) Lp-RFP. (C) Composite of two pictures showing the entire body of a fourth instar larva. Fat body tissue is revealed by red fluorescence. (D) Fat body tissue dissected from a transgenic adult female, observed with a 10× objective. (E) Neonate larva showing initial Lp promoter activity in the intestine. (F–H) Vg-GFP adult blood-fed female mosquitoes. GFP expression in the fat body renders the whole abdomen fluorescent (F). (G and H) Dissected abdomen with GFP and (GFP + bright field) overlay showing the ovary. (I–M) actin5C-GFP mosquitoes with 3xP3-RFP transgenesis marker. (I–K) Larva pictured in merged, red, and green channels. (L) Pupa (merged channels). (M) Dissected intestine from an adult female. (N–R) G12-GFP, intestine dissected from adult females without blood feeding (N and O, merged bright field + green channels) or with blood feeding (P–R, merged bright field + green and split channels). (S and T) vas2-tdTomato neonate larva showing systemic red fluorescence inherited from maternal deposition in the oocyte in addition to endogenous gonad expression (arrowheads point to gonads). (U and V) tdTomato-expressing gonad in female (U) and male (V) fourth instar larvae. (W) Testis in dissected adult male abdomen. (X and Y) Ovary in dissected adult female abdomen. (U–X) Merged bright field and red channels. (Y) Red channel.

Mentions: GoldenGate Cloning plasmids for transgenesis. Scheme of the pDSA plasmid series and an example of GoldenGate cloning. The final letter of the plasmid name indicates the identity of the transgenesis selection marker (T: mTurquoise2; G: eGFP; Y: YFPvenus; N: with NLS; R: DsRed2; P: puromycin acetyl transferase). The attB site mediates insertion at a genomic attP docking site. Constructs of interest are assembled between the two BsaI sites (sequence underlined), which cut, leaving the indicated cohesive ends. The example shows the assembly of the Lp promoter with the tdTomato reporter gene that yielded the reporter line shown in Figure 6C. Each component is provided by a donor plasmid carrying appropriate BsaI sites.


Tools for Anopheles gambiae Transgenesis.

Volohonsky G, Terenzi O, Soichot J, Naujoks DA, Nolan T, Windbichler N, Kapps D, Smidler AL, Vittu A, Costa G, Steinert S, Levashina EA, Blandin SA, Marois E - G3 (Bethesda) (2015)

Transgenic reporter Anopheles gambiae lines. All pictures were taken with the 5× objective of a Zeiss Axiovert fluorescence microscope yielding an 80× visual magnification, unless otherwise noted. (A and B) ppo6-RFP fourth instar larva, central ventral abdominal region, and head. Note the red fluorescent hemocytes throughout the larval body. Green fluorescence reflects the activity of the 3xP3-YFP transgenesis selection marker. (C–E) Lp-RFP. (C) Composite of two pictures showing the entire body of a fourth instar larva. Fat body tissue is revealed by red fluorescence. (D) Fat body tissue dissected from a transgenic adult female, observed with a 10× objective. (E) Neonate larva showing initial Lp promoter activity in the intestine. (F–H) Vg-GFP adult blood-fed female mosquitoes. GFP expression in the fat body renders the whole abdomen fluorescent (F). (G and H) Dissected abdomen with GFP and (GFP + bright field) overlay showing the ovary. (I–M) actin5C-GFP mosquitoes with 3xP3-RFP transgenesis marker. (I–K) Larva pictured in merged, red, and green channels. (L) Pupa (merged channels). (M) Dissected intestine from an adult female. (N–R) G12-GFP, intestine dissected from adult females without blood feeding (N and O, merged bright field + green channels) or with blood feeding (P–R, merged bright field + green and split channels). (S and T) vas2-tdTomato neonate larva showing systemic red fluorescence inherited from maternal deposition in the oocyte in addition to endogenous gonad expression (arrowheads point to gonads). (U and V) tdTomato-expressing gonad in female (U) and male (V) fourth instar larvae. (W) Testis in dissected adult male abdomen. (X and Y) Ovary in dissected adult female abdomen. (U–X) Merged bright field and red channels. (Y) Red channel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478545&req=5

fig6: Transgenic reporter Anopheles gambiae lines. All pictures were taken with the 5× objective of a Zeiss Axiovert fluorescence microscope yielding an 80× visual magnification, unless otherwise noted. (A and B) ppo6-RFP fourth instar larva, central ventral abdominal region, and head. Note the red fluorescent hemocytes throughout the larval body. Green fluorescence reflects the activity of the 3xP3-YFP transgenesis selection marker. (C–E) Lp-RFP. (C) Composite of two pictures showing the entire body of a fourth instar larva. Fat body tissue is revealed by red fluorescence. (D) Fat body tissue dissected from a transgenic adult female, observed with a 10× objective. (E) Neonate larva showing initial Lp promoter activity in the intestine. (F–H) Vg-GFP adult blood-fed female mosquitoes. GFP expression in the fat body renders the whole abdomen fluorescent (F). (G and H) Dissected abdomen with GFP and (GFP + bright field) overlay showing the ovary. (I–M) actin5C-GFP mosquitoes with 3xP3-RFP transgenesis marker. (I–K) Larva pictured in merged, red, and green channels. (L) Pupa (merged channels). (M) Dissected intestine from an adult female. (N–R) G12-GFP, intestine dissected from adult females without blood feeding (N and O, merged bright field + green channels) or with blood feeding (P–R, merged bright field + green and split channels). (S and T) vas2-tdTomato neonate larva showing systemic red fluorescence inherited from maternal deposition in the oocyte in addition to endogenous gonad expression (arrowheads point to gonads). (U and V) tdTomato-expressing gonad in female (U) and male (V) fourth instar larvae. (W) Testis in dissected adult male abdomen. (X and Y) Ovary in dissected adult female abdomen. (U–X) Merged bright field and red channels. (Y) Red channel.
Mentions: GoldenGate Cloning plasmids for transgenesis. Scheme of the pDSA plasmid series and an example of GoldenGate cloning. The final letter of the plasmid name indicates the identity of the transgenesis selection marker (T: mTurquoise2; G: eGFP; Y: YFPvenus; N: with NLS; R: DsRed2; P: puromycin acetyl transferase). The attB site mediates insertion at a genomic attP docking site. Constructs of interest are assembled between the two BsaI sites (sequence underlined), which cut, leaving the indicated cohesive ends. The example shows the assembly of the Lp promoter with the tdTomato reporter gene that yielded the reporter line shown in Figure 6C. Each component is provided by a donor plasmid carrying appropriate BsaI sites.

Bottom Line: Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects.This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis.Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects.

View Article: PubMed Central - PubMed

Affiliation: INSERM U963, CNRS UPR9022, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France.

No MeSH data available.


Related in: MedlinePlus