Identification of silkworm hemocyte subsets and analysis of their response to BmNPV infection based on single-cell RNA sequencing

Feng, Mei; Xia, Junming; Fei, Shigang; Wang, Xiong; Zhou, Yaohong; Wang, Pengwei; Swevers, Luc; Sun, Jingchen

doi:10.1101/2020.10.18.344127

Cited by 2 publications

(2 citation statements)

References 67 publications

(100 reference statements)

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“…New developments in omics technologies [ e.g. , single-cell RNA sequencing ( Zhang et al 2015 ; Feng et al 2020 )] will be critical to assist on the complete genome assembly and annotation of the insect Hi5 cell line (incl. impact of cis and trans acting regulatory elements on transgene expression) and thus allow the use of these techniques for estimation of the copy number of inserted target cassette in the Hi5-RMCE master clone.…”

Section: Discussionmentioning

confidence: 99%

Insect High Five™ cell line development using site-specific flipase recombination technology

Dias

Vidigal

Sequeira

et al. 2021

G3 Genes|Genomes|Genetics

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Insect Trichoplusia ni High FiveTM (Hi5) cells have been widely explored for production of heterologous proteins, traditionally mostly using the lytic baculovirus expression vector system (BEVS), and more recently using virus-free transient gene expression systems. Stable expression in such host cells would circumvent the drawbacks associated with both systems when it comes to scale-up and implementation of more efficient high-cell density process modes for the manufacturing of biologics. In this work, we combined Flipase (Flp) recombinase-mediated cassette exchange (RMCE) with fluorescence-activated cell sorting (FACS) for generating a stable master clonal Hi5 cell line with the flexibility to express single or multiple proteins of interest from a tagged genomic locus. The 3-step protocol herein implemented consisted of (i) introducing the RMCE docking cassette into the cell genome by random integration followed by selection in Hygromycin B and FACS (Hi5-tagging population), (ii) eliminating cells tagged in loci with low recombination efficiency by transfecting the tagging population with an eGFP-containing target cassette followed by selection in G418 and FACS (Hi5-RMCE population), and (iii) isolation of pure eGFP-expressing cells by FACS and expansion to suspension cultures (Hi5-RMCE master clone). Exchangeability of the locus in the master clone was demonstrated in small-scale suspension cultures by replacing the target cassette by one containing a single protein (i.e. iCherry, as an intracellular protein model) or two proteins (i.e. influenza HA and M1 for virus-like particles production, as an extracellular protein model). Overall, the stable insect Hi5 cell platform herein assembled has the potential to assist and accelerate biologics development.

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Section: Discussionmentioning

confidence: 99%

Insect High Five™ cell line development using site-specific flipase recombination technology

Dias

Vidigal

Sequeira

et al. 2021

G3 Genes|Genomes|Genetics

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“…Hemocytes play a vital role in immune responses against pathogen infection (Lavine & Strand, 2002). BmNPV infection causes multiple changes in gene expression and suppresses immune response in major hemocyte types of B. mori (Feng et al ., 2020). Metarhizium acridum infection causes a significant decrease in hemocytes, and diverse genes are differentially expressed in the hemocytes of Locusta migratoria and Dysdercus cingulatus after infection (Sahayaraj et al ., 2014; Zhang et al ., 2015).…”

Section: Discussionmentioning

confidence: 99%

Genome‐wide identification and characterization of basic helix‐loop‐helix transcription factors in Spodoptera litura upon pathogen infection

Zhang

Zhu

et al. 2021

Insect Science

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Basic helix‐loop‐helix (bHLH) transcription factors play an important role in a wide range of metabolic and developmental processes in eukaryotes, and bHLH proteins also participate in immune responses, especially in plants. However, their roles in insects upon entomopathogen infection are unknown. In this study, 54 bHLH genes in 41 families were identified in a polyphagous pest, Spodoptera litura, including a new bHLH gene in group B, which is specifically present in Lepidoptera and was thus named Lep. The conserved amino acids in the bHLH domain, structural architecture, and chromosomal distribution of bHLH genes in S. litura were analyzed. The bHLH genes in Plutella xylostella and Apis mellifera were also updated, and genome‐wide comparison and phylogenetic analysis of bHLH members in 5 holometabolous insects were performed. The expression profiles of S. litura bHLH (SlbHLH) genes in 3 tissues at different developmental stages and their responses to S. litura nucleopolyhedrovirus (SpltNPV), Nomuraea rileyi (Nr), and Bacillus thuringiensis (Bt) infection were investigated. More SlbHLHs in group B were expressed and differentially expressed during pathogen infections, and SlbHLHs tended to be downregulated in the midgut of S. litura larvae after B. thuringiensis treatment. Our study provides an overview of bHLH family members in S. litura and their responses to different pathogens used for pest biocontrol. These findings on bHLH members may contribute to uncovering the mechanism of host–pathogen interaction.

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Identification of silkworm hemocyte subsets and analysis of their response to BmNPV infection based on single-cell RNA sequencing

Cited by 2 publications

References 67 publications

Insect High Five™ cell line development using site-specific flipase recombination technology

Insect High Five™ cell line development using site-specific flipase recombination technology

Genome‐wide identification and characterization of basic helix‐loop‐helix transcription factors in Spodoptera litura upon pathogen infection

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