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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: Setting-up transgenesis in non-model organisms is challenging due to the diversity of biological life traits and due to knowledge gaps in genomic information.Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects.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

Examples of transgenic larvae population analysis with COPAS. Axes represent fluorescence intensity in a logarithmic scale. (A) Mosquito population carrying three transgenic constructs behaving as different alleles at the X1 docking locus. The different constructs are labeled with CFP (fluorescence undetectable by the sorter), RFP, and YFP. The six detected populations correspond to larvae carrying CFP/CFP (lowest fluorescence level), CFP/YFP, CFP/RFP, YFP/RFP (intermediate fluorescence levels), and YFP/YFP, RFP/RFP (strongest fluorescence levels). Note the absence of populations containing more than two transgenes due to the diploid nature of mosquitoes and a single docking locus. (B) F2 progeny of a cross between a GFP and an RFP transgenic line at two different loci. The nine larval clouds represent all possible genotype combinations (from left to right and bottom to top: +/+; +/+. RFP/+ ; +/+. RFP/RFP; +/+. +/+; GFP/+. RFP/+; GFP/+. RFP/RFP; GFP/+. +/+; GFP/GFP. RFP/+; GFP/GFP. RFP/RFP; GFP/GFP.). The black arrow points to larvae homozygous for both transgenes. (C) F1 progeny of homozygous FK (GFP) females crossed to heterozygous C2S (RFP) males. Segregating RFP yields a cloud of red positive larvae and a cloud of red negative larvae. All larvae inherited an intact GFP-positive copy of FK. (D) The larvae gated in C (one copy of C2S, one copy of FK undergoing lox cassette excision in the germ line) were raised to adulthood and males were backcrossed to FK females. The progeny shows the level of fluorescence of a single FK copy, indicating that the copy inherited from the father has now lost the fluorescence-coding gene. A single female from the larvae gated in (D) was selected to found the XK line.
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fig4: Examples of transgenic larvae population analysis with COPAS. Axes represent fluorescence intensity in a logarithmic scale. (A) Mosquito population carrying three transgenic constructs behaving as different alleles at the X1 docking locus. The different constructs are labeled with CFP (fluorescence undetectable by the sorter), RFP, and YFP. The six detected populations correspond to larvae carrying CFP/CFP (lowest fluorescence level), CFP/YFP, CFP/RFP, YFP/RFP (intermediate fluorescence levels), and YFP/YFP, RFP/RFP (strongest fluorescence levels). Note the absence of populations containing more than two transgenes due to the diploid nature of mosquitoes and a single docking locus. (B) F2 progeny of a cross between a GFP and an RFP transgenic line at two different loci. The nine larval clouds represent all possible genotype combinations (from left to right and bottom to top: +/+; +/+. RFP/+ ; +/+. RFP/RFP; +/+. +/+; GFP/+. RFP/+; GFP/+. RFP/RFP; GFP/+. +/+; GFP/GFP. RFP/+; GFP/GFP. RFP/RFP; GFP/GFP.). The black arrow points to larvae homozygous for both transgenes. (C) F1 progeny of homozygous FK (GFP) females crossed to heterozygous C2S (RFP) males. Segregating RFP yields a cloud of red positive larvae and a cloud of red negative larvae. All larvae inherited an intact GFP-positive copy of FK. (D) The larvae gated in C (one copy of C2S, one copy of FK undergoing lox cassette excision in the germ line) were raised to adulthood and males were backcrossed to FK females. The progeny shows the level of fluorescence of a single FK copy, indicating that the copy inherited from the father has now lost the fluorescence-coding gene. A single female from the larvae gated in (D) was selected to found the XK line.

Mentions: It is widely recognized that docking-site transgenesis offers many advantages over transposons (Bischof et al. 2007; Meredith et al. 2013). First, transgenes land in a well-defined locus chosen to be homozygous viable and without fitness cost when loaded with exogenous sequences. Second, the phenotypes of different lines carrying a series of transgenes (e.g., encoding variants of a protein of interest) can be directly compared because all are inserted at the same locus and subjected to identical positional effects. Third, should a transgenic line be lost, it can be regenerated using the same docking line and plasmid. Fourth, different transgenic constructs inserted in the same docking site and labeled with distinct screening markers can be maintained floating within a single mosquito population. A given transgenic construct required for an experiment can then be extracted from this population when needed. Considering the large amount of space, handling, and resources required for maintaining mosquito lines, this strategy optimizes these budgets by a factor of four. We routinely maintain mixed populations carrying up to four different transgenic constructs inserted at the same locus and labeled with CFP, YFP, GFP, and RFP (Figure 4A). Extraction of the desired homozygous genotype when needed and occasional verification/readjustment of the frequency of all transgenes present in the population are greatly facilitated by the automated sorting technology discussed below.


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)

Examples of transgenic larvae population analysis with COPAS. Axes represent fluorescence intensity in a logarithmic scale. (A) Mosquito population carrying three transgenic constructs behaving as different alleles at the X1 docking locus. The different constructs are labeled with CFP (fluorescence undetectable by the sorter), RFP, and YFP. The six detected populations correspond to larvae carrying CFP/CFP (lowest fluorescence level), CFP/YFP, CFP/RFP, YFP/RFP (intermediate fluorescence levels), and YFP/YFP, RFP/RFP (strongest fluorescence levels). Note the absence of populations containing more than two transgenes due to the diploid nature of mosquitoes and a single docking locus. (B) F2 progeny of a cross between a GFP and an RFP transgenic line at two different loci. The nine larval clouds represent all possible genotype combinations (from left to right and bottom to top: +/+; +/+. RFP/+ ; +/+. RFP/RFP; +/+. +/+; GFP/+. RFP/+; GFP/+. RFP/RFP; GFP/+. +/+; GFP/GFP. RFP/+; GFP/GFP. RFP/RFP; GFP/GFP.). The black arrow points to larvae homozygous for both transgenes. (C) F1 progeny of homozygous FK (GFP) females crossed to heterozygous C2S (RFP) males. Segregating RFP yields a cloud of red positive larvae and a cloud of red negative larvae. All larvae inherited an intact GFP-positive copy of FK. (D) The larvae gated in C (one copy of C2S, one copy of FK undergoing lox cassette excision in the germ line) were raised to adulthood and males were backcrossed to FK females. The progeny shows the level of fluorescence of a single FK copy, indicating that the copy inherited from the father has now lost the fluorescence-coding gene. A single female from the larvae gated in (D) was selected to found the XK line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Examples of transgenic larvae population analysis with COPAS. Axes represent fluorescence intensity in a logarithmic scale. (A) Mosquito population carrying three transgenic constructs behaving as different alleles at the X1 docking locus. The different constructs are labeled with CFP (fluorescence undetectable by the sorter), RFP, and YFP. The six detected populations correspond to larvae carrying CFP/CFP (lowest fluorescence level), CFP/YFP, CFP/RFP, YFP/RFP (intermediate fluorescence levels), and YFP/YFP, RFP/RFP (strongest fluorescence levels). Note the absence of populations containing more than two transgenes due to the diploid nature of mosquitoes and a single docking locus. (B) F2 progeny of a cross between a GFP and an RFP transgenic line at two different loci. The nine larval clouds represent all possible genotype combinations (from left to right and bottom to top: +/+; +/+. RFP/+ ; +/+. RFP/RFP; +/+. +/+; GFP/+. RFP/+; GFP/+. RFP/RFP; GFP/+. +/+; GFP/GFP. RFP/+; GFP/GFP. RFP/RFP; GFP/GFP.). The black arrow points to larvae homozygous for both transgenes. (C) F1 progeny of homozygous FK (GFP) females crossed to heterozygous C2S (RFP) males. Segregating RFP yields a cloud of red positive larvae and a cloud of red negative larvae. All larvae inherited an intact GFP-positive copy of FK. (D) The larvae gated in C (one copy of C2S, one copy of FK undergoing lox cassette excision in the germ line) were raised to adulthood and males were backcrossed to FK females. The progeny shows the level of fluorescence of a single FK copy, indicating that the copy inherited from the father has now lost the fluorescence-coding gene. A single female from the larvae gated in (D) was selected to found the XK line.
Mentions: It is widely recognized that docking-site transgenesis offers many advantages over transposons (Bischof et al. 2007; Meredith et al. 2013). First, transgenes land in a well-defined locus chosen to be homozygous viable and without fitness cost when loaded with exogenous sequences. Second, the phenotypes of different lines carrying a series of transgenes (e.g., encoding variants of a protein of interest) can be directly compared because all are inserted at the same locus and subjected to identical positional effects. Third, should a transgenic line be lost, it can be regenerated using the same docking line and plasmid. Fourth, different transgenic constructs inserted in the same docking site and labeled with distinct screening markers can be maintained floating within a single mosquito population. A given transgenic construct required for an experiment can then be extracted from this population when needed. Considering the large amount of space, handling, and resources required for maintaining mosquito lines, this strategy optimizes these budgets by a factor of four. We routinely maintain mixed populations carrying up to four different transgenic constructs inserted at the same locus and labeled with CFP, YFP, GFP, and RFP (Figure 4A). Extraction of the desired homozygous genotype when needed and occasional verification/readjustment of the frequency of all transgenes present in the population are greatly facilitated by the automated sorting technology discussed below.

Bottom Line: Setting-up transgenesis in non-model organisms is challenging due to the diversity of biological life traits and due to knowledge gaps in genomic information.Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects.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