Takuya Tsubota¹, Keiro Uchino¹, Takao K. Suzuki¹, Hiromitsu Tanaka², Takumi Kayukawa³, Takaaki Daimon³, Tetsuro Shinoda³, Hideki Sezutsu¹

¹Transgenic Silkworm Research Unit, ²Insect-Microbe Research Unit, ³Insect Growth Regulation Research Unit

［Abstract］

We identified a novel strong and ubiquitous promoter in the silkworm. Using this promoter, screening of the transgenic silkworm in the embryonic stage has become very efficient. We also developed a novel technique to integrate exogenous gene into the targeted chromosome via genome editing. Efficient selection of knock-in individuals is now possible by using these techniques.

［Keywords］

Keywords: promoter, genome editing, knock-in, gene functional analysis, useful material production

［Background］

Silkworm is frequently used for the production of useful materials according to its high ability to generate recombinant proteins. However, the efficiency to produce transgenic silkworm is not high and a novel technique to select transgenic silkworm easily at an early developmental stage has been required. In addition, the amount of recombinant protein expressed using transgenic silkworm is not high enough. Gene knock-in is a promising technique to increase protein expression level but this technique has not been available in the silkworm. Thus, we attempted to improve the screening technique and develop novel knock-in technology.

［Results and Discussion］

1. The hsp90 gene was found to be expressed in a strong and ubiquitous pattern in all developmental stages (embryo, larva, pupa and adult) of silkworm. In addition, the promoter fragment responsible for such an expression pattern was isolated.
   
2. Transgenic silkworm expressing the Green Fluorescent Protein (GFP) gene using hsp90 promoter showed strong and ubiquitous GFP expression in various developmental stages (Fig. 1). In particular, GFP expression could be detected for at least five days during the embryonic stage suggesting that hsp90 promoter can be utilized for the easy screening of transgenic silkworm embryo.
   
3. Novel knock-in technique was developed using this promoter. Although homologous recombination-mediated knock-in was very difficult in the silkworm, utilization of programmable nucleases and MMEJ repair mechanism succeeded in the efficient integration of hsp90 promoter and GFP into the silkworm urate granule formation gene (Fig. 2).
   
4. The exogenous gene was inserted in a very precise manner suggesting that this gene knock-in technique is a very versatile tool.

1. Screening of the transgenic silkworm has become very easy using the hsp90 promoter. This promoter also enables the detailed analysis of each gene function to facilitate the study for efficient production of useful materials or the development of novel insecticides.
   
2. Integration of exogenous genes into the highly-active endogenous genomic locus will also be possible using the novel knock-in technique to facilitate the production of large amount of useful materials. In addition, this technique will also contribute to the development of novel high-performance silk.

Fig. 1. Induction of GFP expression by hsp90 promoter in the embryo, larva, pupa and adult of transgenic silkworm. The left panel of each figure indicates the bright-field image and the right shows GFP-fluorescence. GFP is expressed in green-colored region. hsp90 promoter is ubiquitously active in all of the developmental stages. Scale bar is 0.3mm in the embryo, 1mm in the larva and 3mm in the pupa and adult. Partially modified from G3:Genes, Genomes, Genetics doi:10.1534/g3.114.011643.

 

Fig. 2. Development of a novel knock-in technology. (A) Comparison of the knock-in efficiency between the conventional and novel method. Around 100 fold increase was achieved using the novel technique. (B) Images of the knock-in silkworm. The left panel indicates the bright-field image and the right panel shows GFP-fluorescence. The upper and lower individuals depict the normal and knock-in larva, respectively. In the knock-in larva the oily skin phenotype can be observed according to the disruption of urate granule formation gene. In addition, the larva shows ubiquitous green fluorescence due to the insertion of GFP gene. Partially modified from Nature Communications doi:10.1038/ncomms6560.

 

[Collaborators]

Shota Nakade, Yuto Sakane, Satoshi Kume, Naoaki Sakamoto, Masanobu Obara, Takashi Yamamoto, Tetsushi Sakuma, Ken-ichi T. Suzuki (Hiroshima University)

[Reference]

1. Tsubota T, Uchino K, Suzuki T.K, Tanaka H, Kayukawa T, Shinoda T, Sezutsu H (2014) Identification of a novel strong and ubiquitous promoter/enhancer in the silkworm Bombyx mori G3:Genes, Genomes, Genetics 4 (7):1347-1357
   
2. Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T, Suzuki K.T (2014) Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9 Nature Communications 5:5560
   
3. International publication #WO2015/022971