Efficient transgene expression system using a cumate-inducible promoter and Cre-loxP recombination in avian cells

Abstract

ObjectiveTransgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells.MethodsAfter stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector.ResultsIn the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination.ConclusionTransgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression.

Functional analyses of miRNA-146b-5p during myogenic proliferation and differentiation in chicken myoblasts

“…For miR-146b-5p-expressing myoblast cells, we conducted co-transfection of the transgene expression vector, piggyBac CMV-GFP (control), or piggyBac CMV-GFP-miRNA-146b-5p with piggyBac transposase using Lipofectamine 3000 (Invitrogen; cat#L3000015) according to our previous report [22]. Transgene DNA-lipid complex consisting of 7.5 µL Lipofectamine 3000 reagent in 250 µL Opti-MEM (Invitrogen; cat#31985-062) and 10 µL P3000 reagent with 2.5 µg piggyBac transgene vector and piggyBac transposase plasmid in 250 µL Opti-MEM was added to each well according to our previous report [22].…”

“…However, additional research is required because it is not clear how miR-146b-5p affects myogenic differentiation, and there are no reports addressing its effects on chicken myogenesis. Thus, in this study, we designed and constructed a miRNA expression vector system to overexpress miR-146b-5p in pCM cells using the piggyBac transposon system, which previous studies have demonstrated to be an e cient transgene delivery system [22,23]. miR-146b-5p increased proliferation and decreased differentiation by regulating genes related to the cell cycle in chicken myoblasts.…”

Functional analysis of miRNA-146b during myotube differentiation in chicken myoblasts

“…Thus, in this study, we designed and constructed the microRNA (miRNA) expression vector system to overexpress the miR-146b in chicken myoblast cells. For functional genomics study, the piggyBac transposon system which previous study demonstrated as an efficient transgene delivery system was used (Park et al 2017;Park et al 2019).…”

Construction and Characterization of a Synthetic Baculovirus-inducible 39K Promoter

“…In general, the inducible promoter, similar to the transcriptional activator, exists in an inactive form and can be directly or indirectly activated by the corresponding signal. Currently, several technical methods supported by inducible promoters (se.g., Cre-loxp, Tet-On/Tet-Off, and ecdysone and pathogen inducible systems), are widely used in the field of animal and plant genetic engineering, including gene function identification or variety improvements (3-6). Insects are the largest group of organisms on earth.…”