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

Volume 5 • Issue 1 • 1000140
Transcriptomics, an open access journal
ISSN: 2329-8936
ISSN: 2329-8936
Transcriptomics: Open Access
Guo et al., Transcriptomics 2017, 5:1
DOI: 10.4172/2329-8936.1000140
Short Communication OMICS International
Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human
Guo RL, Lee YT, Byrnes C, and Miller JL*
Molecular Genomics and Therapeutics Section, Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, Maryland, USA
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
proles 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 proles 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 proles and variations between puromycin selected populations
after transduction of the separate lentiviral vectors was manifested by signicant differences in the RNA detection
levels of less than 0.1%. However, puromycin selection after pac gene transduction caused signicant 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 proles.
*Corresponding author: Jeffery L Miller, Genetics of Development and Disease
Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, 10 Center Drive, Building 10, Room 9N311, Bethesda,
Maryland 20892. USA, Tel: 3014801908; E-mail:
Received April 01, 2017; Accepted April 27, 2017; Published May 01, 2017
Citation: Guo RL, Lee YT, Byrnes C, Miller JL (2017) Puromycin Selection
Confounds the RNA-Seq Proles of Primary Human Erythroblasts. Transcriptomics
5: 140. doi:10.4172/2329-8936.1000140
Copyright: © 2017 Guo RL, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Keywords: Puromycin; Erythropoiesis; Drug selection; Gene
transfer; RNA-seq
Short Communication
Puromycin, an amino nucleoside antibiotic, is biosynthesized by the
bacterium species Streptomyces alboniger. e biomolecular structure
of puromycin resembles that of an aminoacyl-tRNA molecule. Its main
mechanism of action involves blocking protein synthesis through the
interference with the peptidyl transfer procedure during translation
[1,2]. Its amino group disrupts the ester bond between the nascent
peptide and tRNA, allowing for the puromycin molecule to attach itself
to the C-terminus. It contains an amide bond instead of ester bond, thus
preventing the next aminoacyl-tRNA from appending itself. Premature
chain termination thus occurs, and protein synthesis is aborted [3].
e pac gene encodes puromycin N-acetyltransferase, thus
conferring puromycin resistance [4]. Puromycin N-acetyltransferase
acetylates the nitrogen atom of the amino group in the puromycin
tyrosinyl moiety, thus preventing it from breaking the ester bond in
the peptidyl-tRNA. Since the puromycin molecule is no longer able to
attach itself to the peptide chain, it becomes biologically inactive, and
protein synthesis continues [2].
Predictable resistance to puromycin-mediated cell death aer
pac gene transduction was identied 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 eects of transgene expression
in a variety of model systems using high-throughput RNA sequencing
(RNA-Seq) with hematopoietic stem and progenitor cells [6-8]. Despite
utilization of this experimental approach, the potential eect of the
bacterial puromycin N-acetyltransferase gene upon the RNA-Seq
proles remains vague.
In this study, we explored the potential eects of puromycin
selection upon RNA-Seq proles using cultured primary hematopoietic
cells. Human CD34(+) cells were cultured ex vivo from healthy adult
human donors as previously described in a 3-week serum free culture
system [9]. Prior to this study, written informed consent was obtained
from research subjects. Consent documents regarding studies using
primary erythroblasts and approval of the research protocol were
obtained from the National Institute of Diabetes and Digestive and
Kidney DiseasesInstitutional Review Board.
Figure 1: Puromycin titration. The percent of live cells at (A) culture day 7 and
(B) culture day 14.
Citation: Guo RL, Lee YT, Byrnes C, Miller JL (2017) Puromycin Selection Confounds the RNA-Seq Proles of Primary Human Erythroblasts.
Transcriptomics 5: 140. doi:10.4172/2329-8936.1000140
Page 2 of 3
Volume 5 • Issue 1 • 1000140
Transcriptomics, an open access journal
ISSN: 2329-8936
An initial puromycin titration experiment was performed on cells
from three donors using 0.1 µg/ml to 1.0 µg/ml dosage to determine
the killing concentration aer 3-day selection (puromycin selection on
culture days 4-6). Puromycin was purchased from Sigma Aldrich (St.
Louis, MO). At culture day 7, ow cytometry analysis was performed to
determine the percent of live cells with a total of 10,000 events recorded
using a BD FACSAria I ow cytometer (BD Biosciences, San Jose, CA).
e cells were then transferred to phase II media without puromycin
for all conditions for an additional 7 days. On culture day 14, ow
cytometry was again performed to determine if any cells survived the
puromycin selection. e most eective concentration of puromycin
for 3-day selection was found to be 0.7 µg/ml based on less than 2% live
cells on culture day 7 and no surviving cells on culture day 14 from the
average of three donors (Figure 1).
For puromycin selection experiments, lentiviral transduction
was performed on cells from six donors as previously described [10].
Briey, the cells were transduced on day 3 of culture with either
SHC002V (Sigma Aldrich) or CLS-NCG-8 (Qiagen, Valencia, CA).
e next day, puromycin was added at 0.7 µg/ml for an additional 3
days. At culture day 7, the cells were transferred to phase II media
without puromycin. On culture day 14, erythroblast dierentiation
was assessed using ow cytometry analysis with the BD FACSAria I
ow cytometer to determine if maturation of the transduced cells was
aected as compared to control. e cells were stained with antibodies
directed against transferrin receptor, CD71 (Invitrogen, Carlsbad, CA)
and glycophorin A (Invitrogen). ere was no observed dierence in
cell dierentiation between the control and transduced cells (Figure 2).
To explore potential eects of puromycin selection on the RNA-Seq
proles of human primary erythroblasts, live cells from six healthy adult
human donors were sorted on culture day 14, and RNA was extracted
using the miRNeasy mini kit (Qiagen). Globin messages were depleted
from 1.0 μg of total RNA using the GLOBINclear Human Kit and rRNA
was depleted using the Ribo-Zero Gold rRNA Removal Kit (Human/
Mouse/Rat). Aer polyA-selection and depletion, the total RNA was
used for cDNA library generation. An Illumina HiSeq 2000 (version
3 chemistry) was utilized to sequence the pooled libraries on multiple
lanes. At least 40 million 101 base pair reads were achieved.
e Illumina sequence reads were aligned against human genome
build hg19 via Illumina sequencing soware, Real-Time Analysis
version 1.13.48, CASAVA version 1.8.2 and Ecient Large-Scale
Alignment of Nucleotide Databases (ELAND) mapping algorithm. e
separate lane reads were merged into BAM les, which were loaded
into the SamtoFastq tool to generate FASTQ les. Each FASTQ le was
processed with FASTQ-Trimmer module and FASTQ-Masker module
of FASTX-Toolkit (version 0.0.14) to trim 21 base pair from the end
and mask any sequence base with a quality score less than 30 with an
N to generate a nal 80 base pair FASTQ le for aligning against the
Human Genome hg19 build with Spliced Transcripts Alignment to a
Figure 2: Flow cytometry proles. Representative ow dot plots of control cells compared to cells transduced with either SHC002V or CLS-NCG-8 analyzed on culture
day 14.
Figure 3: Venn diagram comparisons. Venn diagrams comparing the gene proles of control (blue), SHC002V (red), and CLS-NCG-8 (yellow). (A) and (B) compare
the control with each transduced sample. Genes in the blue area were signicantly down-regulated by the lentivirus, genes in the red or yellow area were signicantly
up-regulated, and genes in the overlapped area were insignicantly differentially expressed. (C) compares the two transduced groups with each other (SHC002V vs.
Comparison No. of Genes Insignicantly Differentially
Expressed (FDR-adjusted p-value >0.01)
No. of Genes Signicantly Differentially
Expressed (FDR-adjusted p-value <0.01)
No. Down-
No. Up-
Between Conditions Control versus SHC002V 24803 1560 898 662
Control versus CLS-NCG-8 24949 1414 811 603
SHC002V versus CLS-NCG-8 26358 5 3 2
Inter-donor Variation Control versus Control 26359 42 2
SHC002V versus SHC002V 26363 0 0 0
CLS-NCG-8 versus CLS-NCG-8 26361 2 1 1
Table 1: Comparison of RNA-Seq identied genes.
Citation: Guo RL, Lee YT, Byrnes C, Miller JL (2017) Puromycin Selection Confounds the RNA-Seq Proles of Primary Human Erythroblasts.
Transcriptomics 5: 140. doi:10.4172/2329-8936.1000140
Page 3 of 3
Volume 5 • Issue 1 • 1000140
Transcriptomics, an open access journal
ISSN: 2329-8936
other primary tissues or cancer cell lines. In this preliminary study,
mechanisms responsible for the dramatic change in the transcriptome
proles were not identied. We speculate that lentiviral transduction
of the pac gene may have o target eects that indirectly aect RNA
transcription or stability. Also, puromycin itself may have eects on
the cells that are not reversed by pac gene expression. Importantly,
analogous eects 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]. us, adequate controls should be
incorporated in experimental designs in other model systems to more
clearly interpret or compare high-throughput gene expression proles
in puromycin selected cells.
1. De la Luna S, Ortín J (1992) [33] pac gene as efcient dominant marker and
reporter gene in mammalian cells. Methods Enzymol. 216: 376-385.
2. Vara J, Perez-Gonzalez JA, Jimenez A (1985) Biosynthesis of puromycin by
Streptomyces alboniger: Characterization of puromycin N-acetyltransferase.
Biochemistry 27: 8074-8081.
3. Palmer M, Chan A, Dieckmann T, Honek J (2012) Biochemical Pharmacology.
John Wiley & Sons, Inc., Hoboken 271-273.
4. Lacalle RA, Pulido D, Vara J, Zaiacaín M, Jiménez A (1989) Molecular analysis
of the pac gene encoding a puromycin N-acetyl transferase from Streptomyces
alboniger. Gene 2: 375-380.
5. Lanza AM, Kim DS, Alper HS (2013) Evaluating the inuence of selection
markers on obtaining selected pools and stable cell lines in human cells.
Biotechnol J 7: 811-821.
6. Lee YT, de Vasconcellos JF, Yuan J, Byrnes C, Noh S, et al. (2013)
LIN28B-mediated expression of fetal hemoglobin and production of fetal-like
erythrocytes from adult human erythroblasts ex vivo. Blood 6:1034-1041.
7. Ali N, Karlsson C, Aspling M, Hu G, Hacohen N, et al. (2009) Forward RNAi
screens in primary human hematopoietic stem/progenitor cells. Blood 16:
8. Sims D, Mendes-Pereira AM, Frankum J, Burgess D, Cerone MA (2011) High-
throughput RNA interference screening using pooled shRNA libraries and next
generation sequencing. Genome Biology 10: 104.
9. de Vasconcellos JF, Lee YT, Byrnes C, Tumburu L, Miller JL (2016)
HMGA2 moderately increases fetal hemoglobin expression in human adult
erythroblasts. PLoS ONE 11: 0166928.
10. Lee YT, de Vasconcellos JF, Byrnes C, Kaushal M, Miller JL (2015) Erythroid-
specic expression of LIN28A is sufcient for robust gamma-globin gene and
protein expression in adult erythroblasts. PLoS ONE 12:0144977.
11. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C (2013) STAR: Ultrafast
universal RNA-seq aligner. Bioinformatics 29: 15-21.
12. Moran DM, Shen H, Maki CG (2009) Puromycin-based vectors promote a
ROS-dependent recruitment of PML to nuclear inclusions enriched with HSP70
and Proteasomes. BMC Cell Biology 10: 32.
Reference (STAR) soware [11]. Quantitation of 26,363 named gene
transcripts was performed using HTseq. Dierential gene expression
analysis was performed with DESeq2 based on applying negative
binomial generalized linear models in R package and R-Studio to
compare the expression levels of each transcript between SHC002V,
CLS-NCG-8, and control samples. Aer applying the Benjamini-
Hochberg procedure, genes were considered signicantly up- or down-
regulated if the adjusted p-value of the test statistics was less than the
false discovery rate (FDR<0.01) with log2 of ≥1 for up-regulated or
≤-1 for down-regulated genes and baseMean value above 25. e same
bioinformatics procedure was then repeated to quantify the eects of
inter-donor variation by comparing multiple donors within SHC002V,
CLS-NCG-8, and control. RNA-Seq Bam les from each experiment
were deposited in Gene Expression Omnibus (GEO, https://www. and released to the public: SRP097005 (Adult
CD34+), SRP097630 (Cord CD34+), SRP096196 (CLS-NCG-8), and
SRP098089 (SHC002V).
e results showed that SHC002V signicantly aected expression
of 1560 genes (898 down-regulated, 662 up-regulated), comprising
approximately 5.9% of the identied transcripts. Whereas, CLS-
NCG-8 had a total of 1414 signicantly dierentially expressed genes
(representing approximately 5.4% of the total prole), with 811 down-
regulated and 603 up-regulated. Each of these vectors has a dierent
recombinant lentivirus encoding the puromycin N-acetyltransferase
gene, a 600-nucleotide fragment originating from Lactobacillus
harbinensis. Consequently, comparisons between SHC002V and CLS-
NCG-8 revealed only 5 genes to be signicantly dierentially expressed
(less than 0.1% of the total gene IDs), suggesting that both lentiviruses
had similar eects on the transcriptome proles of the samples.
To determine the eects of inter-donor variation upon the RNA-
Seq proles, donors within the same condition were compared. e
inter-donor variation tests between the control, SHC002V, and CLS-
NCG-8 revealed only 4, 0, and 2 signicantly dierentially expressed
genes, respectively, which is less than 0.1% of the total number of genes
identied. us, neither dierences in cell maturation, nor inter-donor
variation in RNA-Seq proles likely caused the signicant dierences
between the gene proles of puromycin selected and non-transduced
samples (Table 1 and Figure 3).
ese novel and unexpected results suggest that puromycin
selection may cause broad and signicant changes in the RNA-Seq
proles of cultured primary hematopoietic cells. erefore, selection
aer expression of puromycin N-acetyltransferase can potentially
be a confounding variable for experimental design, interpretation,
and comparison of high-throughput sequencing data. While our
experiments were limited to eects in primary human erythroblasts,
similar comparisons should be considered in alternate models including
Guo RL, Lee YT, Byrnes C, Miller JL (2017) Puromycin Selection
Confounds the RNA-Seq Proles of Primary Human Erythroblasts.
Transcriptomics 5: 140. doi:10.4172/2329-8936.1000140
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... Furthermore, the exosomes were prepared for electrophoresis after first being stored at À20˚C, instead of immediately after isolation, which might be critical for the detection of phospho-protein detection in exosomes. Inclusion of untransduced B16-F10 controls could have also indicated if unintended changes occurred in the stable cell lines utilized, especially given the potential impact puromycin selection can have on transcriptome profiles (Guo et al., 2017), and should be considered in the experimental design of future studies. The original study reported Met and pMet expression by Western blot in B16-F10 exosomes; however, the level of knockdown achieved in shMet exosomes was not reported. ...
... Additionally, others were unknown or not easily controlled for. These include variables such as cell-intrinsic changes in Met expression and the associated gene expression profiles (Adachi et al., 2016), cell line genetic drift (Ben-David et al., 2018;Hughes et al., 2007;Kleensang et al., 2016), non-random genetic/transcriptional drift in the heterogeneous stable cells (Guo et al., 2017;Shearer and Saunders, 2015), genetic heterogeneity of mouse inbred strains (Casellas, 2011), the microbiome of recipient mice (Macpherson and McCoy, 2015), housing temperature in mouse facilities (Kokolus et al., 2013), and lot variability and quality of key reagents, such as the lentiviral particles (Leek et al., 2010). Environmental differences such as husbandry staff, bedding type and source, light levels, and other intangibles, all of which, by necessity, differed between the studies also affect experimental outcomes with mice (Howard, 2002;Jensen and Ritskes-Hoitinga, 2007;Nevalainen, 2014;Sorge et al., 2014). ...
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... 30 Apart from differences in the expression levels of endogenous gene(s) rendering puromycin resistance, unmatched intracellular levels of transgenic puromycin N-acetyltransferase and/or puromycin itself might distinctly affect cell transcriptomes. 31 Applying more restrictive rules for the design of control-and genespecific shRNAs might limit off-target effects. The problem, however, is quite complex due to the fact that the processing of the shRNA molecules, the choice of the guide strand from the siRNA duplex, and profiles of siRNA-mRNA interactions are not fully predictable. ...
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... doi: bioRxiv preprint overexpressing multidrug resistance gene(s) coding for ABC transporters able to expel puromycin from the cells (Theile, Staffen, & Weiss, 2010). Apart from the differences in the expression levels of endogenous gene(s) rendering puromycin resistance also unmatched intracellular levels of transgenic puromycin N-acetyltransferase and/or puromycin itself may distinctly affect cell transcriptomes (Guo, Lee, Byrnes, & Miller, 2017). ...
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Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating nascent chains, blocking further extension and resulting in premature termination of translation. It is most commonly known as a selection marker for cell lines genetically engineered to express a resistance transgene, but its additional uses as a probe for protein synthesis have proven invaluable across a wide variety of model systems, ranging from purified ribosomes and cell-free translation to intact cultured cells and whole animals. Puromycin is comprised of a nucleoside covalently bound to an amino acid, mimicking the 3′ end of aminoacylated tRNAs that participate in delivery of amino acids to elongating ribosomes. Both moieties can tolerate some chemical substitutions and modifications without significant loss of activity, generating a diverse toolbox of puromycin-based reagents with added functionality, such as biotin for affinity purification or fluorophores for fluorescent microscopy detection. These reagents, as well as anti-puromycin antibodies, have played a pivotal role in advancing our understanding of the regulation and dysregulation of protein synthesis in normal and pathological processes, including immune response and neurological function. This manuscript reviews the current state of puromycin-based research, including structure and mechanism of action, relevant derivatives, use in advanced methodologies and some of the major insights generated using such techniques both in the lab and the clinic.
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Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30-40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts.
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Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and β-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of γ-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype.
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Motivation: Accurate alignment of high-throughput RNA-seq data is a challenging and yet unsolved problem because of the non-contiguous transcript structure, relatively short read lengths and constantly increasing throughput of the sequencing technologies. Currently available RNA-seq aligners suffer from high mapping error rates, low mapping speed, read length limitation and mapping biases. Results: To align our large (>80 billon reads) ENCODE Transcriptome RNA-seq dataset, we developed the Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure. STAR outperforms other aligners by a factor of >50 in mapping speed, aligning to the human genome 550 million 2 × 76 bp paired-end reads per hour on a modest 12-core server, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. Using Roche 454 sequencing of reverse transcription polymerase chain reaction amplicons, we experimentally validated 1960 novel intergenic splice junctions with an 80-90% success rate, corroborating the high precision of the STAR mapping strategy. Availability and implementation: STAR is implemented as a standalone C++ code. STAR is free open source software distributed under GPLv3 license and can be downloaded from
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RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.
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Promyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. Previous studies reported that misfolded proteins or proteins containing polyglutamine tracts form aggregates with PML, chaperones, and components of the proteasome, supporting a role for PML in misfolded protein degradation. In the current study, we have identified a reactive oxygen species (ROS) dependent aggregation of PML, small ubiquitin-like modifier 1 (SUMO-1), heat shock protein 70 (HSP70) and 20S proteasomes in human cell lines that have been transiently transfected with vectors expressing the puromycin resistance gene, puromycin n-acetyl transferase (pac). Immunofluorescent studies demonstrated that PML, SUMO-1, HSP70 and 20S proteasomes aggregated to form nuclear inclusions in multiple cell lines transfected with vectors expressing puromycin (puro) resistance in regions distinct from nucleoli. This effect does not occur in cells transfected with identical vectors expressing other antibiotic resistance genes or with vectors from which the pac sequence has been deleted. Furthermore, ROS scavengers were shown to ablate the effect of puro vectors on protein aggregation in transfected cells demonstrating a dependency of this effect on the redox state of transfected cells. Taken together we propose that puromycin vectors may elicit an unexpected misfolded protein response, associated with the formation of nuclear aggresome like structures in human cell lines. This effect has broad implications for cellular behavior and experimental design.
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The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.
Selection markers are common genetic elements used in recombinant cell line development. While several selection systems exist for use in mammalian cell lines, no previous study has comprehensively evaluated their performance in the isolation of recombinant populations and cell lines. Here we examine four antibiotics, hygromycin, neomycin, puromycin, and Zeocin, and their corresponding selector genes, using a green fluorescent protein (GFP) as a reporter in two model cell lines, HT1080 and HEK293. We identify Zeocin as the best selection agent for cell line development in human cells. In comparison to the other selection systems, Zeocin is able to identify populations with higher fluorescence levels, which in turn leads to the isolation of better clonal populations and less false positives. Further, Zeocin-resistant populations exhibit better transgene stability in the absence of selection pressure compared to other selection agents. All isolated Zeocin-resistant clones, regardless of cell type, exhibited GFP expression. By comparison, only 79% of hygromycin-resistant, 47% of neomycin-resistant and 14% of puromycin-resistant clones expressed GFP. Based on these results, we would rank Zeocin > hygromycin ∼ puromycin > neomycin for cell line development in human cells. Furthermore, this study demonstrates that selection marker choice does impact cell line development.
Nucleotide sequence of a 906-bp fragment of Streptomyces alboniger DNA containing the gene (pac), which encodes a puromycin N-acetyltransferase (PAC), has been determined. The pac gene contains a 600-nt open reading frame, starting with an ATG codon, which encodes a polypeptide of Mr 21,531; this is consistent with the 23 +/- 1.5 kDa size of the PAC enzyme. High-resolution S1 mapping indicates that transcription starts at or next to a C residue 35 bp upstream from the putative ATG start codon. A 263-bp DNA fragment from the 5' region of the pac gene has promoter activity in the promoter-probe plasmid pIJ486. Its -35 and -10 regions show significant structural homology to the corresponding regions of the hyg gene promoter, but they are different from the promoter sequences of other Streptomyces and Escherichia coli genes.
Puromycin N-acetyltransferase from Streptomyces alboniger inactivates puromycin by acetylating the amino position of its tyrosinyl moiety. This enzyme has been partially purified by column chromatography through DEAE-cellulose and Affigel Blue and characterized. It has an Mr of 23 000, as determined by gel filtration. In addition to puromycin, the enzyme N-acetylates O-demethylpuromycin, a toxic precursor of the antibiotic, and chryscandin, a puromycin analogue antibiotic. The Km values for puromycin and O-demethylpuromycin are 1.7 and 4.6 microM, respectively. The O-demethylpuromycin O-methyltransferase from S. alboniger, which apparently catalyzes the last step in the biosynthesis of puromycin [Rao, M. M., Rebello, P. F., & Pogell, B. M. (1969) J. Biol. Chem. 244, 112-118], also O-methylates N-acetyl-O-demethylpuromycin. The Km values of the methylating enzyme for O-demethylpuromycin and N-acetyl-O-demethylpuromycin are 260 and 2.3 microM, respectively. These findings suggest that O-demethylpuromycin, if present in S. alboniger, would be N-acetylated and then O-methylated to be converted into N-acetylpuromycin. It might even be possible that N-acetylation of the puromycin backbone takes place at an earlier precursor.