A combination of transposable elements and magnetic cell sorting provides a very efficient transgenesis system for chicken primary erythroid progenitors

Abstract

Background: Stable transgenesis is an undeniable key to understanding any genetic system. Retrovirus-based insertional strategies, which feature several technical challenges when they are used, are often limited to one particular species, and even sometimes to a particular cell type as the infection depends on certain cellular receptors. A universal-like system, which would allow both stable transgene expression independent of the cell type and an efficient sorting of transfected cells, is required when handling cellular models that are incompatible with retroviral strategies.

Modeling Neurodegenerative Spinocerebellar Ataxia Type 13 in Zebrafish Using a Purkinje Neuron Specific Tunable Coexpression System

“…To express this kcnc3a X12 isoform specifically in PCs, we inserted the cDNA of this variant (referred to hereafter as kcnc3a wt ) as well as its pathogenic allele carrying the R355H mutation (kcnc3a R335H ; Mock et al, 2010) into one of the MCS of our established pTol2-4xcpce-MCS vector. As additional control for transgene expression a cDNA encoding a nonfunctional truncated receptor of human CD4 (tCD4) lacking its cytoplasmic domain (Mejia-Pous et al, 2009) was used. Into the other MCS, a bicistronic transgene was inserted encoding two subcellular fluorescent reporters: membrane targeted Fyn-TagRFP-T to visualize Kcnc3a R335H affected PC morphologies, and nuclear localized H2B-GFP to quantify PC numbers.…”

Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts

“…Stably transfected clones, expressing a fluorescent reporter, were obtained as previously described [81]. Briefly, 6C2 cells were nucleofected in a transfection apparatus (Nucleofector™ II; Amaxa Nucleofector™ Technology) (T-16 program) using a commercial kit (Cell Line Nucleofector® Kit V; Lonza GmBH, Cologne, Germany) and a pT2.CMV- mCherry /pCAGGS-T2TP plasmid mix (ratio 5/1).…”

“…Briefly, 6C2 cells were nucleofected in a transfection apparatus (Nucleofector™ II; Amaxa Nucleofector™ Technology) (T-16 program) using a commercial kit (Cell Line Nucleofector® Kit V; Lonza GmBH, Cologne, Germany) and a pT2.CMV- mCherry /pCAGGS-T2TP plasmid mix (ratio 5/1). The pT2.CMV- mCherry plasmid was constructed using the same strategy as described for the pT2.CMV- hKO plasmid [81], except that the hKO reporter gene was replaced by mCherry , extracted from the pRSET-B plasmid (kindly provided by Dr Roger Tsien, University of California, San Diego, CA, USA). mRNA birth/death fluctuations constitute a major source of stochasticity in gene expression because many mRNA species are present at very low molecular counts within cells [58,70,82,83], thus we reduced this source of intrinsic noise by using the cytomegalovirus (CMV) promoter.…”

“…For each clone, the genomic reporter insertion sites were identified using a splinkerette PCR method as previously described [81], in order to select only clones with a single insertion site. Briefly, genomic DNA isolated from clones expressing the gene reporter was purified by phenol extraction and ethanol precipitation, before being digested for 16 hours at 65°C with Tai I, a restriction enzyme with a 4 bp recognition site.…”

Methods of Cell Purification: A Critical Juncture for Laboratory Research and Translational Science

“…The transposase inserts the reporter sequence randomly into the genome, allowing cell sorting based on reporter expression [Shibano et al, 2007;Mejia-Pous et al, 2009]. After cell sorting, another transient transfection with a transposase-encoding vector is used to remove the inserted reporter sequence.…”