Improved Honeybee Germline Transformation

Ari Yasunaga is an Associate Scientist at Verily Life Sciences. MORE ABOUT AUTHOR

Otte et al (2018) build upon their previous work (Schulte et al. 2014) in this new publication and demonstrate a twofold improved efficiency at generating transgenic honeybees using the piggyBac transposon system. 

The inability to generate high transformation rates has been a significant hurdle for functional genetic studies in honeybees. A lack of reliable genetic tools has hindered genome manipulation in honeybees and limited research to genomic studies, until Schulte et al (2014) introduced a robust tool using transposable elements.

In their latest work, Otte et al. implemented three improvements that significantly increase the rate of generating transformed queen honeybees with transgenic offspring; a major bottleneck for genetic studies as each queen must be maintained in a separate colony. The authors reported an increased transformation rate from 19% to 43%.

Apis mellifera

The first improvement introduced was generating a honeybee codon-optimized hypersensitive transposase gene within the piggyBac transposon system. The authors incorporated mutations from a hyperactive variant of piggyBac transposase protein (Yusa et al. 2011), and confirmed hyperactivity of this new variant (hyPBaseapis) compared with the original iPBase using an excision assay in Sf21 cells . The injection with hyPBaseapis provided a 3% increase in the queens with the transgenic offspring, compared to iPBase, when they were injected with the same picogram (pg) amount at the same injection site.

The second improvement was to increase the amount of injected transposase mRNA from 90 pg to 240 pg (Figure 1). This 2.7 fold increase in the injection amount of hyPBaseapis led to 12% increase in the queens with transgenic offspring compared to iPBase, when eggs were injected at the posterior end.

Making transgenic honeybees. from commentary by Ben-Shahar PNAS 2014 on Schulte et al (2014) Note that Otte et al (2018) alter the site of injection

The third improvement was interesting, the authors shifted the injection site from the posterior side, a commonly used insect injection site, to the anterior first third of the embryo , in order to “target the first nuclei”.

Collectively, the change in the injection site combined with the increase in mRNA led to 24% increase in the queens with transgenic offspring when injected with hyPBaseapis compared to iPBase injected controls.

Overall, this paper showed a promising increase in the transformation efficiency of the honey bee with implementation of all three simple improvements. This more efficient transformation technique leads to more manageable and less laborious screening efforts, and also has a great potential to successfully improve transformation rates in other insect systems.

Otte, M. Netschitailo, O., Kaftanoglu, O., Wang, Y., Page Jr., R. E and M. Beye. Improving genetic transformation rates in honeybees Scientific Reports 8, Article number: 16534 https://www.nature.com/articles/s41598-018-34724-w (2018)

Schulte, C., Teilenberg, E., Muller-Borg, M., Gempe, T. & Beye, M. Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera). Proc Natl Acad Sci USA 111, 9003–9008, https://doi.org/10.1073/pnas.1402341111 (2014).

Yusa, K., Zhou, L., Li, M. A., Bradley, A. & Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci USA 108, 1531–1536, https://doi.org/10.1073/pnas.1008322108 (2011).

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