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Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA.

Amdam GV, Simões ZL, Guidugli KR, Norberg K, Omholt SW - BMC Biotechnol. (2003)

Bottom Line: The vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees.Injection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages.To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Genetics and Department of Animal Science, Agricultural University of Norway, N-1432 Aas, Norway. gro.amdam@ihf.nlh.no

ABSTRACT

Background: The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees.

Results: The vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees. This allowed us to introduce dsRNA in preblastoderm eggs without affecting gene function during development. Of workers reared from eggs injected with dsRNA derived from a 504 bp stretch of the vitellogenin coding sequence, 15% had strongly reduced levels of vitellogenin mRNA. When dsRNA was introduced by intra-abdominal injection in newly emerged bees, almost all individuals (96%) showed the mutant phenotype. An RNA-fragment with an apparent size similar to the template dsRNA was still present in this group after 15 days.

Conclusion: Injection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages. To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages.

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Targeted vitellogenin disruption. (a) Individual samples of workers injected as embryo and (b) workers injected as adults. All bees were sampled as 7 days old. Total RNA loaded was 6 μg, and an equal 0.1 μl hemolymph volume was loaded for SDS-PAGE. (c) Visualization of ~500 bp fragment in a worker injected as adult. Lane 1: Control. Lane 2: 6 μg total RNA. Lane 3: 20 μg total RNA. (d) Pooled samples of 10 workers each enriched with small RNA fragments. Two oligonucleotides (27 and 50 bp) were included as markers. Lane 1: Control. Lane 2: Adult workers injected with dsRNA at the preblastoderm stage. Lane 3: Intra-abdominally injected workers.
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Figure 2: Targeted vitellogenin disruption. (a) Individual samples of workers injected as embryo and (b) workers injected as adults. All bees were sampled as 7 days old. Total RNA loaded was 6 μg, and an equal 0.1 μl hemolymph volume was loaded for SDS-PAGE. (c) Visualization of ~500 bp fragment in a worker injected as adult. Lane 1: Control. Lane 2: 6 μg total RNA. Lane 3: 20 μg total RNA. (d) Pooled samples of 10 workers each enriched with small RNA fragments. Two oligonucleotides (27 and 50 bp) were included as markers. Lane 1: Control. Lane 2: Adult workers injected with dsRNA at the preblastoderm stage. Lane 3: Intra-abdominally injected workers.

Mentions: We found that 15 % (N = 70) of the adult bees reared from eggs injected with dsRNA had strongly reduced levels of vitellogenin mRNA. The disruption appeared to be incomplete in some individuals (Figure 2a), but the RNAi effect was detectable at emergence and persistent over 15 days (not shown). In the intra-abdominally injected group, 96 % (N = 30) showed loss of vitellogenin mRNA expression when sampled at 7 days old (Figure 2b). Furthermore, an unambiguous fragment with an apparent size similar to the template dsRNA (504 bp) was visualized in all adult injected bees with a mutant phenotype (Figure 2c). Samples enriched with short RNA show that vitellogenin mRNA fragments as small as 25 bp could be detected (Figure 2d). For all mutants, the level of vitellogenin in the hemolymph was almost undetectable (Figure 2a,2b). Reduced levels of vitellogenin mRNA was not encountered in any of the control individuals assayed in this manner (N = 96).


Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA.

Amdam GV, Simões ZL, Guidugli KR, Norberg K, Omholt SW - BMC Biotechnol. (2003)

Targeted vitellogenin disruption. (a) Individual samples of workers injected as embryo and (b) workers injected as adults. All bees were sampled as 7 days old. Total RNA loaded was 6 μg, and an equal 0.1 μl hemolymph volume was loaded for SDS-PAGE. (c) Visualization of ~500 bp fragment in a worker injected as adult. Lane 1: Control. Lane 2: 6 μg total RNA. Lane 3: 20 μg total RNA. (d) Pooled samples of 10 workers each enriched with small RNA fragments. Two oligonucleotides (27 and 50 bp) were included as markers. Lane 1: Control. Lane 2: Adult workers injected with dsRNA at the preblastoderm stage. Lane 3: Intra-abdominally injected workers.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC149360&req=5

Figure 2: Targeted vitellogenin disruption. (a) Individual samples of workers injected as embryo and (b) workers injected as adults. All bees were sampled as 7 days old. Total RNA loaded was 6 μg, and an equal 0.1 μl hemolymph volume was loaded for SDS-PAGE. (c) Visualization of ~500 bp fragment in a worker injected as adult. Lane 1: Control. Lane 2: 6 μg total RNA. Lane 3: 20 μg total RNA. (d) Pooled samples of 10 workers each enriched with small RNA fragments. Two oligonucleotides (27 and 50 bp) were included as markers. Lane 1: Control. Lane 2: Adult workers injected with dsRNA at the preblastoderm stage. Lane 3: Intra-abdominally injected workers.
Mentions: We found that 15 % (N = 70) of the adult bees reared from eggs injected with dsRNA had strongly reduced levels of vitellogenin mRNA. The disruption appeared to be incomplete in some individuals (Figure 2a), but the RNAi effect was detectable at emergence and persistent over 15 days (not shown). In the intra-abdominally injected group, 96 % (N = 30) showed loss of vitellogenin mRNA expression when sampled at 7 days old (Figure 2b). Furthermore, an unambiguous fragment with an apparent size similar to the template dsRNA (504 bp) was visualized in all adult injected bees with a mutant phenotype (Figure 2c). Samples enriched with short RNA show that vitellogenin mRNA fragments as small as 25 bp could be detected (Figure 2d). For all mutants, the level of vitellogenin in the hemolymph was almost undetectable (Figure 2a,2b). Reduced levels of vitellogenin mRNA was not encountered in any of the control individuals assayed in this manner (N = 96).

Bottom Line: The vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees.Injection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages.To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Genetics and Department of Animal Science, Agricultural University of Norway, N-1432 Aas, Norway. gro.amdam@ihf.nlh.no

ABSTRACT

Background: The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees.

Results: The vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees. This allowed us to introduce dsRNA in preblastoderm eggs without affecting gene function during development. Of workers reared from eggs injected with dsRNA derived from a 504 bp stretch of the vitellogenin coding sequence, 15% had strongly reduced levels of vitellogenin mRNA. When dsRNA was introduced by intra-abdominal injection in newly emerged bees, almost all individuals (96%) showed the mutant phenotype. An RNA-fragment with an apparent size similar to the template dsRNA was still present in this group after 15 days.

Conclusion: Injection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages. To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages.

Show MeSH
Related in: MedlinePlus