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Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents.

ABSTRACT: Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfection, followed by flow cytometry of transfected HUVEC to determine the proportion of EGFP-expressing cells as a measure of transfection efficiency. Lipofectamine 2000 and Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) gave the highest transfection efficiencies of the reagents tested. Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays.

SUBMITTER: Hunt MA

PROVIDER: S-EPMC2884313 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents.

Hunt Michelle A MA Currie Margaret J MJ Robinson Bridget A BA Dachs Gabi U GU

Journal of biomolecular techniques : JBT 20100701 2

Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfec ...[more]

PMID: 20592869

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Project description:Human umbilical vein endothelial cells (HUVECs) are a widely-used model system to study many pathological and physiological processes associated with the cardiovascular system. An understanding of genes and proteins that are expressed in any cell type is a fundamental need which facilitates study of molecular changes in disease states and response to various stimuli. In this study, we employed next generation sequencing and mass spectrometry to profile the transcriptome and proteome of primary HUVECs. Analysis of 145 million paired-end reads from next generation sequencing confirmed expression of 12,186 protein-coding genes (FPKM>0.1), 439 novel long non-coding RNAs and revealed 6,089 novel isoforms that were not annotated in GENCODE. A comparison of the transcripts against human gene expression data for 53 tissues catalogued by the Genotype-Tissue Expression (GTEx) project revealed a number of HUVEC-specific genes. Proteomics analysis identified 6,477 proteins including confirmation of N-termini for 1,091 proteins and isoforms for 149 proteins for which transcriptomic evidence was also observed. Alternate translational start sites for seven proteins and alternate splicing in seven proteins were also identified. A database search to specifically identify other post-translational modifications provided evidence for a number of modification sites on 117 proteins which include ubiquitylation, lysine acetylation, mono, di- and tri-methylation. Based on the data from this study and a survey of other databases, we provide evidence for 11 “missing proteins,” which are proteins for which protein level evidence is lacking or insufficient. Proteogenomic analysis identified novel N-termini and exon-exon junctions at both RNA and protein levels by searching against a translated database created from alternate transcript assembly. Peptides supporting the novel events of expressed proteins are further validated by synthesis and spectral comparison. Alternative allelic expression of genes arising from transcription of two alleles of a gene in chromosome pairs is observed. By creating a custom database of proteins incorporated with sample specific single amino acid variations (SAAV) we identified alternative alleles for 207 expressed proteins with 245 peptides with SAAV. Overall, we believe that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.

Project description:Human umbilical vein endothelial cells (HUVECs) are a widely-used model system to study pathological and physiological processes associated with the cardiovascular system. An understanding of genes and proteins that are expressed in any cell type is a fundamental need which facilitates studies of molecular changes in disease states and response to various stimuli. In this study, we employed next generation sequencing and mass spectrometry to profile the transcriptome and proteome of primary HUVECs. Analysis of 145 million paired-end reads from next generation sequencing confirmed expression of 12,186 protein-coding genes (FPKM>0.1), 439 novel long non-coding RNAs and revealed 6,089 novel isoforms that were not annotated in GENCODE. A comparison of the transcripts against human gene expression data for 53 tissues catalogued by the Genotype-Tissue Expression (GTEx) project revealed a number of HUVEC-specific genes. Proteomics analysis identified 6,477 proteins including confirmation of N-termini for 1,091 proteins and isoforms for 149 proteins for which transcriptomic evidence was observed. Alternate translational start sites for seven proteins and alternate splicing in five proteins were also identified. A database search to specifically identify other post-translational modifications provided evidence for a number of modification sites on 117 proteins which included ubiquitylation, lysine acetylation and mono, di- and tri-methylation events. Based on the data from this study and a survey of other databases, we provide evidence for 11 “missing proteins,” which are proteins for which there was insufficient or no protein level evidence. Peptides supporting missing protein and novel events were validated by comparison of MS/MS fragmentation patterns with synthetic peptides. By creating a custom database of proteins containing sample-specific single amino acid variants (SAAV), we also identified 245 variant peptides derived from 207 expressed proteins. Overall, we believe that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.

Dataset Information

Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents.

Publications

Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents.

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