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

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Volume 5 • Issue 1 • 1000140
Transcriptomics, an open access journal
ISSN: 2329-8936
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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
Erythroblasts
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
Abstract
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: jm7f@nih.gov
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.
CLS-NCG-8).
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-
regulated
No. Up-
regulated
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.
References
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:
3690-3695.
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.
ncbi.nlm.nih.gov/geo/) 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
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
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