Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

Lanza, Amanda M.; Kim, Do Soon; Alper, Hal S.

doi:10.1002/biot.201200364

Cited by 44 publications

(31 citation statements)

References 46 publications

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“…Also, puromycin itself may have effects on the cells that are not reversed by pac gene expression. Importantly, analogous effects from puromycin selection have been reported previously [5]. Other reports suggest that lentiviral transduction conferring puromycin resistance may lead to a misfolded protein response in human cell lines [12].…”

supporting

confidence: 76%

“…Predictable resistance to puromycin-mediated cell death after pac gene transduction was identified as a selection strategy for gene manipulation in eukaryotic cells [5]. More recently, lentiviral transduction followed by puromycin resistance has become an established procedure for exploring the effects of transgene expression in a variety of model systems using high-throughput RNA sequencing (RNA-Seq) with hematopoietic stem and progenitor cells [6][7][8].…”

mentioning

confidence: 99%

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Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human Erythroblasts

Rl¹,

Yt²,

Byrnes³

et al. 2017

Transcriptomics

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Lentiviral transduction followed by puromycin selection is a well-recognized procedure for gene transfer and expression experiments using a variety of cell types including human hematopoietic stem and progenitor cells. Despite its widespread application, research regarding the potential effects of bacterial puromycin N-acetyltransferase (pac) gene expression in mammalian cell cultures is incomplete. Here the potential for puromycin selection to affect transcriptome profiles was examined using a well-studied model for human erythropoiesis. Experiments were performed using primary CD34(+) cells from six adult healthy human donors transduced with two commercially available pac-encoding lentiviral vectors and compared to non-transduced control cells. RNA-Seq gene expression profiles were generated at the proerythroblast stage of differentiation, then differential gene expression was analyzed with DEseq2 in R-Studio software. Inter-donor variation in the gene expression profiles and variations between puromycin selected populations after transduction of the separate lentiviral vectors was manifested by significant differences in the RNA detection levels of less than 0.1%. However, puromycin selection after pac gene transduction caused significant changes in over 5% of the mRNA when compared to non-transduced controls. The results suggest that consideration should be given for the potential of puromycin selection to confound the interpretation of RNA-Seq transcriptome profiles.

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supporting

confidence: 76%

mentioning

confidence: 99%

Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human Erythroblasts

Rl¹,

Yt²,

Byrnes³

et al. 2017

Transcriptomics

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“…TLR4 has been examined mostly for its broad contribution to immune-related disorders. Evidences suggest that TLR4 not only contributes to cellular pathophysiology, but also plays a vital role in facilitating neurodegenerative diseases such as ischemic stroke, Alzheimer's disease and multiple sclerosis (Lanza et al 2013). However, most of the evidence for role of TLR4 is observational, and little is known about its molecular mechanism.…”

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confidence: 99%

PPARγ ameliorated LPS induced inflammation of HEK cell line expressing both human Toll-like receptor 4 (TLR4) and MD2

et al. 2015

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TLR4 is transmembrane pattern-recognition receptor that initiates signals in response to diverse pathogen-associated molecular patterns especially LPS. Recently, there have been an increasing number of studies about the role of TLRs in the pathogenesis of several disorders as well as the therapeutic potential of TLR intervention in such diseases. Peroxisome proliferator-activated receptorgamma (PPARc) is a ligand-activated transcription factor with numerous biological effects. PPARc has been shown to exert a potential anti-inflammatory effect through suppression of TLR4-mediated inflammation. Therefore, PPARc agonists may have a potential to combat inflammatory conditions in pathologic states. The current study aims to show the decrease of inflammation by overexpression of PPARc in a cell reporter model. To reach this goal, recombinant pBudCE4.1 (?) containing encoding sequences of human TLR4 and MD2 was constructed and used to transfect HEK cells. Subsequently, inflammation was induced by LPS treatment as control group. In the treatment group, overexpression of PPARc prior to inflammation was performed and the expression of inflammatory markers was assessed in this condition. The expression of inflammatory markers (TNFa and iNOS) was defined by quantitative real time PCR and the amount of phosphorylated NF-jB was measured by western blot. Data indicated expression of TNFa and iNOS increased in LPS induced inflammation of stably transformed HEK cells with MD2 and TLR4. In this cell reporter model overexpression of PPARc dramatically prevented LPSinduced inflammation through the blocking of TLR4/ NF-jB signaling. PPARc was shown to negatively regulate TLR4 activity and therefore exerts its antiinflammatory action against LPS induced inflammation.

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“…The product of Zeocin R binds to Zeocin in a one-to-one ratio and prevents the DNA cleavage. Zeocin R was used as the selection marker owing to its relatively smaller size (375 bp versus 1 kb for the hygromycin resistance gene) and greater reliability in stable cell clone selection (19). PQR linkers were cloned in frame between all of the encoding genes to split them into discrete proteins.…”

Section: Resultsmentioning

confidence: 99%

Generating stable cell lines with quantifiable protein production using CRISPR/Cas9-mediated knock-in

Greben

Chen

2017

BioTechniques

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Cell lines expressing foreign genes have been widely used to produce a variety of recombinant proteins. However, generating recombinant protein-expressing cell lines is usually a lengthy process and the resulting protein expression levels are often inconsistent. Here, we describe an efficient method for making stable cell lines expressing any recombinant protein of interest in a controllable and quantifiable manner. We integrate transgenes into specific genomic loci using CRISPR/Cas9 such that transgene expression is driven by endogenous promoters to ensure consistent and predictable expression of the recombinant protein. Expression levels can be predetermined by selecting promoters from genes with the desired level of expression. To quantify recombinant protein expression, a protein quantitation reporter (PQR) is incorporated between the endogenous and foreign genes. The PQR allows equimolar production of the endogenous protein, the recombinant protein, and a fluorescent reporter. As a result, expression levels of both the endogenous and recombinant proteins can be continuously monitored using fluorescence.

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Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

Cited by 44 publications

References 46 publications

Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human Erythroblasts

Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human Erythroblasts

PPARγ ameliorated LPS induced inflammation of HEK cell line expressing both human Toll-like receptor 4 (TLR4) and MD2

Generating stable cell lines with quantifiable protein production using CRISPR/Cas9-mediated knock-in

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