Identification of Genomic Safe Harbors in the Anhydrobiotic Cell Line, Pv11

Abstract

Genomic safe harbors (GSHs) provide ideal integration sites for generating transgenic organisms and cells and can be of great benefit in advancing the basic and applied biology of a particular species. Here we report the identification of GSHs in a dry-preservable insect cell line, Pv11, which derives from the sleeping chironomid, Polypedilum vanderplanki, and similar to the larvae of its progenitor species exhibits extreme desiccation tolerance. To identify GSHs, we carried out genome analysis of transgenic cell lines established by random integration of exogenous genes and found four candidate loci. Targeted knock-in was performed into these sites and the phenotypes of the resulting transgenic cell lines were examined. Precise integration was achieved for three candidate GSHs, and in all three cases integration did not alter the anhydrobiotic ability or the proliferation rate of the cell lines. We therefore suggest these genomic loci represent GSHs in Pv11 cells. Indeed, we successfully constructed a knock-in system and introduced an expression unit into one of these GSHs. We therefore identified several GSHs in Pv11 cells and developed a new technique for producing transgenic Pv11 cells without affecting the phenotype.

Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives

“…To transition to development of genetic control mechanisms, there is now a pressing need for the development of efficient methods for creating gain-of-function mutants via gene integration. One limitation to deploying these technologies is the fact that genomic safe harbors for insect genomes are just beginning to emerge ( Miyata et al, 2022 ). Genomic safe harbors are genome target sites that allow for the stable expression of transgenes without phenotypic consequences for the organism.…”

A sodium-dependent trehalose transporter contributes to anhydrobiosis in insect cell line, Pv11

“…To analyze the changes in the morphology of live cells during anhydrobiosis, AcGFP1-positive Pv11 cell lines were analyzed. We used Strt1 −/− cells and, as a control, an AcGFP1 + /ZeoR + cell line in which the exogenous genes AcGFP1 and ZeoR were inserted into a safe harbor region of the genome (chromosome 1: intergenic region between g1212124 and g1212125 ) ( 48 ). The AcGFP1 + / ZeoR + cells showed comparable desiccation tolerance to WT ( 48 ).…”

“…The donor vectors pCR4-121-AcGFP1_ Strt1 and pCR4-121-ZeoR_ Strt1 ( Dataset S2 ) were constructed using PCR, a HiFi Assembly kit (New England BioLabs) and a TOPO cloning kit (Thermo Fisher Scientific) as previously described ( 17 , 46 , 48 ). Briefly, AcGFP1 and ZeoR expression units were amplified from pPv121-AcGFP1-Pv121-ZeoR ( 77 ) as a PCR template using specific primers (Oligonucleotide set 2 in SI Appendix , Table S1 ); these amplicons contained the microhomology regions and the gRNA-target site for Strt1 .…”

A Sodium-dependent Trehalose Transporter Contributes to Anhydrobiosis in Insect Cell Line, Pv11

“…To analyze the changes in the morphology of live cells during anhydrobiosis, AcGFP1-positive Pv11 cell lines were analyzed. We used Strt1 −/− cells and, as a control, an AcGFP1 + / ZeoR + cell line in which the exogenous genes AcGFP1 and ZeoR were inserted into a safe harbor region of the genome (chromosome 1: intergenic region between g1212124 and g1212125) (55). The AcGFP1 + / ZeoR + cells showed comparable desiccation tolerance to WT (55).…”

“…The donor vectors pCR4-121-AcGFP1_ Strt1 and pCR4-121-ZeoR_ Strt1 (Supplemental Dataset S2) were constructed using PCR, a HiFi Assembly kit (New England BioLabs) and a TOPO cloning kit (Thermo Fisher Scientific) as previously described (16, 17, 55). Briefly, AcGFP1 and ZeoR expression units were amplified from pPv121-AcGFP1-Pv121-ZeoR (15) as a PCR template using specific primers (Oligonucleotide set 2 in Supplemental Table S1); these amplicons contained the microhomology regions and the gRNA-target site for Strt1 .…”