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BMC Biotechnology

BioMed Central

Open Access

Methodology article

A new generation of pPRIG-based retroviral vectors
Olivier Albagli-Curiel*1, Yann Lécluse1, Philippe Pognonec2,
Kim E Boulukos2 and Patrick Martin*2
Address: 1INSERM U790 and IFR54, Institut Gustave Roussy, PR1, 39 Rue Camille Desmoulins, 94805 Villejuif, France and 2CNRS UMR 6548,
Université de Nice, Parc Valrose, 06108 Nice, France
Email: Olivier Albagli-Curiel* - oalbagli@igr.fr; Yann Lécluse - ylecluse@igr.fr; Philippe Pognonec - POGNONEC@unice.fr;
Kim E Boulukos - BOULUKOS@unice.fr; Patrick Martin* - PMARTIN@unice.fr
* Corresponding authors

Published: 30 November 2007
BMC Biotechnology 2007, 7:85

doi:10.1186/1472-6750-7-85

Received: 13 September 2007
Accepted: 30 November 2007

This article is available from: http://www.biomedcentral.com/1472-6750/7/85
© 2007 Albagli-Curiel et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Retroviral vectors are valuable tools for gene transfer. Particularly convenient are
IRES-containing retroviral vectors expressing both the protein of interest and a marker protein
from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/
or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral
vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV
enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient
transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP
expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i)
the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked
by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).
Results: The convenience of pPRIG makes it a good basic vector to generate additional derivatives
for an extended range of use. Here we present several novel pPRIG-based vectors (collectively
referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames
3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or
3' of the MCS (« modular » PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puroR (« single color/resistance » PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS
of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (« dual color/selection
» PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background
which has been widely used in pluripotent cells.
Conclusion: These novel vectors allow for straightforward detection of any expressed protein
(3'HA PRIGs), for functional studies of chimeric proteins (« modular » PRIGs), for multiple
transductions and fluorescence analyses of transduced cells (« single color/resistance » PRIGs), or
for quantitative detection of studied proteins in independently identified/selected transduced cells
(« dual color/selection » PRIGs). They maintain the original advantages of pPRIG and provide
suitable tools for either transient or stable expression and functional studies in a large range of
experimental settings.

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Background
Retroviral vectors are widely used to stably express any
protein of interest. They potentially can transduce a large
variety of primary or immortalized cells types, either in
vitro, ex vivo or in vivo. Selection of transduced cells is generally accomplished by the co-expression of a fluorescent
protein, a membrane protein or a gene encoding a drugresistance. The more the expression of the two proteins
correlates, the more the relevant cells in the selected population will be enriched. One way to obtain coupled
expression in cells is to express both proteins from a single
bicistronic mRNA: the 5' cistron encodes the protein of
interest while translation of the 3' cistron is initiated by an
IRES (Internal Ribosome Entry Site) element and gives
rise to the selection protein. The pAP2 plasmid [1,2]
belongs to this kind of retroviral vector since it contains
eGFP as a selectable marker just downstream of the IRES
element from the EMCV (encephalomyocarditis virus).
Moreover, while many retroviral vectors harbor two complete LTR sequences, the U3 region of the pAP2 5' LTR has
been replaced by the CMV immediate early enhancer/promoter. Hence pAP2 allows a potent CMV-driven expression upon transient transfection while retrotranscription
(and integration) restores a complete, LTR-controlled,
pro-viral sequence. However, despite this improvement,
the pAP2 vector suffers from two major flaws: 1) its multicloning site (MCS) is limited; and 2) its IRES sequence is
weak as a result of the creation of a HindIII site in its 3' terminus, which corresponds to two point mutations with
respect to the original EMCV sequence. These mutations,
though widely found in IRES containing vectors, destroy
the last IRES ATG, normally used by the ECMV virus [2].
We thus constructed an improved derivative of pAP2
termed pPRIG (for plasmid Polylinker Retroviral IRES
GFP) which keeps the advantages of pAP2 while eliminating its shortcomings. First, the pAP2 MCS had been
replaced by a much more complete MCS which contains
unique sites for twenty different restriction enzymes. This
new MCS was optimized to allow directional cloning
whatever the orientation of the cDNA since sites for compatible enzymes (e.g. BamHI and BglII) have been
inserted quite symmetrically with respect to the center of
the MCS. A T7 and a SP6 sequence were introduced at
each extremity of the MCS to facilitate sequencing and
allow in vitro transcription. Second, the IRES sequence had
been changed to restore a wild type sequence, which
enhances the translation of eGFP about 10 fold without
affecting any other important parameters (e.g. viral titer or
expression of the 5' cDNA). Finally, three pPRIG derivatives, termed pPRIG HAa/b/c, were also created. Each of
these three derivatives contains an upstream HA tag
encoding sequence in one reading frame with respect to
that of the complete open ORF encoded by the pPRIG
MCS [2].

http://www.biomedcentral.com/1472-6750/7/85

Given these improvements, we reasoned that pPRIG is a
very convenient basic vector to generate new useful retroviral tools. We provide here 14 novel pPRIG derivatives,
collectively referred to as PRIGs, which keep the advantages of their founding member, but extend and facilitate
the possibility of their uses to a wide range of studies.

Results and Discussion
3'HA PRIGs
In addition to the basic pPRIG vector (Fig. 1), we had previously described three derivatives bearing the HA tag coding sequence just upstream of the 5' site (BamHI) of the
MCS [2]. The 5' HA coding sequence was inserted in the
three reading frames relative to the MCS, giving the pPRIG
HAa/b/c [2].

As a first step to design novel PRIG vectors, we created
reciprocal constructs by inserting the HA coding sequence
just downstream of the last restriction site (MfeI) of the
basic pPRIG vector MCS in the three reading frames
(pPRIGp a/b/cHA, Fig. 2). Importantly, although the 5'
and 3' HA are symmetrically positioned relative to the
MCS in our two sets of PRIG vectors, they are not fully
equivalent. Indeed, given the scanning model ruling the
choice of the initating ATG by the ribosome, the 5' HA is
translated no matter what the sequence of the inserted
cDNA is, while the 3'HA is optional since it is not translated if a stop codon is present in the inserted cDNA. A 3'
tag may be especially useful if, for instance, the N-terminus of the protein of interest contains a cleavable signal
peptide.
"Modular" PRIGs
Expression of conditional alleles
The usual way to express a conditional version of many
proteins is to fuse them to the hormone binding domain
of the estrogen receptor. Consequently, the activity of the
resulting chimeric protein is usually dependent upon the
presence of ligand [3]. We thus cloned the hormone binding domain of the mouse ERα (ER, amino acids (aa)
281–599) downstream of the MCS of pPRIGp aHA, leading to pPRIGp ER (Fig. 3). This insertion therefore allows
for the generation of X-ER chimera (Fig. 3). The presence
of the G525R point mutation in the cloned ER sequence
abrogates its binding to 17β-estradiol while keeping its
full sensitivity to the synthetic ligand 4'hydroxytamoxifen
(4OHT) [4]. Thus pPRIGp ER makes possible the culture
of transduced cells expressing chimeric proteins in the
presence of natural ER agonists without triggering basal
activity [4]. Moreover, 4OHT does not activate the transactivating domain (termed AF2) of the cloned ER region
[4,5], which therefore minimizes the risk of altering the
function of the X protein moiety upon hormonal treatment. Although only one reading frame is available to
construct X-ER chimera, we underline the presence of

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Bg-X-As-B
IRES
CMVpro

RU5

*

eGFP

δgag

LTR
Amp

U3 RU5

pAP2
(6787 bp)

HA-B-P-E-X-A-C-Ag-N-S-Sp-St-Sh-Bs-Ss-As-Ac-Hd-Sa-Bg-M

IRES

T7

B = BamHI
P = PvuII
E = EcoRI
X = XhoI
A = AvaIII
C = ClaI
Ag = AgeI
N = NotI
S = SacII
Sp = SplI

St = StuI
Sh = SphI
Bs = BspMII
Ss = Sse8387I
As = AsuII
Ac = AccI
Hd = HinDII
Sa = SalI
Bg = BglII
M = MfeI

eGFP

SP6

pPRIG HAa/b/c

400 bp

(6159/60/61 bp)

Figure 1
Schematic representation of the pAP2 and pPRIG vectors
Schematic representation of the pAP2 and pPRIG vectors. The parental pAP2 retroviral vector harbors the CMV enhancer/promoter (CMVpro)
in place of the U3 region of the 5' LTR and eGFP downstream of an ECMV-derived IRES containing two point mutations (hence indicated as IRES*) which
strongly weakens its activity. The pPRIG HAa/b/c vectors improve pAP2 by: 1) replacing IRES* by the completely wild-type ECMV-derived IRES sequence
(IRES), which restores its full activity; 2) inserting a much more complete multicloning site (MCS) and T7 and SP6 phage promoter on each side of the new
MCS; and 3) adding a HA tag sequence in the three reading frames (a/b/c) 5' of the MCS. pPRIG HAa/b/c, as well as a pPRIG (without HA), were previously
described and are used here to generate new vectors. For each vector, the unique sites of the MCS are indicated above the MCS.

restriction sites for compatible enzymes in different reading frames within the PRIG MCS (e.g. StuI and HindII).
Thus, the pPRIGp ER MCS provides different reading
frames in a single sequence, provided that the 3'extremity
of the X cDNA is clonable in one of the shifted compatible

restriction sites. The same remark holds true for the
fusions with any open sequence cloned in only one reading frame just upstream the PRIG MCS (KRAB, VP16, fluorescent protein), although in these cases the compatible

B-P-E-X-A-C-Ag-N-S-Sp-St-Sh-Bs-Ss-As-Ac-Hd-Sa-Bg-M-HA

IRES

eGFP

Pa

Pa

pPRIGp a/b/cHA

Pa = PacI

400 bp

(6180/82/83 bp)

Figure 2
Schematic representation of the HA3' PRIG vectors
Schematic representation of the HA3' PRIG vectors. pPRIGp a/b/c HA are three pPRIG derivatives containing a HA tag sequence downstream of
the MCS. Each of these derivatives harbors the HA sequence in one of the three reading frames relative to the MCS (hence pPRIG a/b/cHA). The reading
frame is therefore open from and throughout the MCS to the stop codon located at the 3' end of the HA sequence. Moreover, the IRES-eGFP cassette is
flanked by two PacI sites (Pa) a very rare cutter, hence pPRIGpa/b/c HA, allowing for accurate removal of the cassette. For each vector, the unique sites of
the MCS are indicated above the MCS.

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B-E-A-C-Ag-N-S-Sp-St-Sh-As-Ac-Hd-Sa

ER

IRES

Pa

pPRIGp ER

eGFP

Pa

(7155 bp)

B-P-E-X-A-C-Ag-N-S-Sp-St-Bs-Ss-As-Ac-Hd-Sa-Bg-M-HA

VP16

pPRIGp VP16HA
(6474 bp)

B-P-E-X-A-C-Ag-N-S-Sp-St-Sh-Bs-Ss-As-Ac-Hd-Sa-Bg-M-HA

KRAB

pPRIGp KRABHA

400 bp

(6463 bp)

Figure 3
Schematic representation of the « modular » PRIG vectors
Schematic representation of the « modular » PRIG vectors. pPRIGp ER is a pPRIG derivative containing the C-terminal portion of the mouse
estrogen receptor bearing the G525V mutation. This mutant does not bind estradiol but still binds the synthetic ligand hydroxytamoxifene. The ER
sequence has been cloned at the 3' end of the MCS and the reading frame is thus open from and throughout the MCS to the stop codon ending the ER
sequence. In contrast, in pPRIGp VP16HA and pPRIGp KRABHA, the sequence coding the functional module (VP16 transactivating domain from human
herpes virus or KRAB transrepressing domain from human KOX1 protein) was cloned upstream of the MCS. The reading frame is thus open from the
start codon of the module to the stop codon of the in-frame HA sequence 3' of the MCS. Pa: site for PacI. The two PacI sites of pPRIGp VP16HA and
pPRIGp KRABHA are symbolized by vertical bars. For each vector, the unique sites of the MCS are indicated above the MCS.

restriction site has to be at the 5'end of the cDNA (see
below).
Expression of transactivating or transrepressing chimera
The functional study of transcription factors frequently
includes their fusion to potent transactivating or transrepressing domains. These fusions provide either dominant
negative or dominant positive alleles, and help to determine whether a given DNA binding protein acts through
transactivation, transrepression, or both [6,7]. To make
possible such studies in PRIG vectors, we next constructed
two derivatives by inserting the sequence encoding either
the transactivating domain of the human herpes simplex
virus VP16 protein [8] preceded by a short nuclear localization signal (KKKRK), or the N-terminal KRAB transrepressing domain of the human KOX1 protein [9] just

upstream of the MCS of the pPRIG cHA, generating
pPRIGp VP16HA or pPRIGp KRABHA, respectively (Fig.
3). Both domains are relatively short (corresponding to aa
413–490 or 1–89 of the original protein, respectively),
highly potent, transferable and widely used to generate
chimeric transregulatory proteins [10-12]. The unique
restriction sites of the MCS are entirely preserved, with the
exception of SphI in pPRIGp VP16HA (Fig. 3). These two
modular PRIG vectors provides an in-frame 3'HA coding
sequence (Fig. 3) and are therefore engineered to create
KRAB-X or VP16-X chimeras with or without a C-terminal
HA tag depending upon the absence or presence of a stop
codon in the inserted cDNA.

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« Single-color/resistance » PRIGs
"single color" PRIGs
Though eGFP is widely used to track transduced or, more
generally, genetically modified cells, it has also several yellow or blue shifted suitable derivatives [13]. Moreover,
highly performant red fluorescent proteins were generated
by directed evolution of mRFP, which is itself a monomeric derivative of the DsRed protein [13,14]. We reasoned that PRIGs harboring one of these fluorescent
proteins instead of eGFP may extend the number of issues
amenable by these vectors. Hence, three other PRIGs were
generated in which the eGFP of pPRIGp aHA has been
replaced by either eCFP containing the H148D mutation,
eYFP or mCherry, generating pPRICp aHA, pPRIYp aHA
and pPRIChp aHA, respectively (Fig. 4). eCFP is a blue
shifted eGFP derivative [14]. Introduction of the H148D
mutation (eCFPH148D, thereafter termed eCFP*)
increases two-fold its quantum yield and brightness without affecting its spectral properties [15]. eYFP is a bright
and widely used yellow shifted eGFP derivative. mCherry
is a monomeric ("m") red fluorescent protein which displays very similar spectral properties, but improved photostability, compared with its ancestor, mRFP [13].
Moreover, we also replaced eGFP by eCFP* in pPRIGp
bHA, leading to pPRICp bHA, which is required for
another derivative (see below). Each of these fluorescent
proteins is compatible (i.e. well spectrally separated) with
a distinct set of fluorochromes. Thus, these three novel
single-color PRIGs provide convenient tools to combine
an identification (or sorting) of transduced cells together
with other fluorescent-based analyses (e.g. expression of
markers). Moreover, eCFP* and eYFP can be well separated spectrally from each other, and mCherry is readily
distinguishable from eCFP*, eGFP and even from eYFP
because of its red-shifted spectral properties as compared
with DsRed [13,14,16]. The four single color PRIGs are
therefore also suitable for performing double or even triple transductions, which may be required in many situations to induce biological changes [17].
« Drug-selectable » PRIGs
In addition, since FACS-based selection of transduced
cells is efficient and rapid but requires expensive materials, we also constructed two other PRIGs that contain the
puro-resistance gene instead of eGFP in either pPRIG or
pPRIG HAa, leading to pPRIPu or pPRIPu HAa, respectively (Fig. 4). Puromycin inhibits translation elongation,
and is a relatively cheap drug for selecting transduced cells
and acts rapidly even at very low concentrations. pPRIPu
and pPRIPu HA vectors can also be used with any of the
fluorescent protein expressing vectors (or any combination of them) further extending the possibility of multiple
transductions with PRIGs.

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« Dual color/selection » PRIGs
« Double color » PRIGs
Cells transduced with any of the PRIG vectors described
above express a bicistronic cDNA, the IRES-controlled
translation of the 3' cistron giving rise to a fluorescent or
selectable protein. Although the expression of the IREScontrolled protein usually correlates with that encoded by
the 5' cistron, a direct detection of the protein of interest
in transduced cells can be valuable. The best way to make
this direct detection easy and quantitative is to express chimeric fluorescent proteins from the 5' cistron, keeping in
mind that the second cis-coupled fluorescent protein
(downstream of the IRES element) should be spectrally
separated from the first one. Both mCherry and eYFP have
been used as convenient tags in chimeric proteins [13,18].
Moreover, as stated above, eYFP and eCFP* on one hand,
and eGFP and mCherry on the other hand, constitute two
pairs of fluorescent proteins fitting the criteria of spectral
separability [13,14,16]. We thus created two double color
PRIGs by inserting either an open(i.e. without its stop
codon) mCherry or an open eYFP cDNA just upstream of
the BamHI site of either the pPRIGp bHA or pPRICp bHA,
generating pPRIGp mChHA and pPRICp eYFPHA, respectively (Fig. 5). In these two vectors, the 5'fluorescent proteins and the 3'HA tag are in-frame but separated by the
entire intact MCS. Thus, these two double color PRIGs are
designed to express a mCherry -X or a eYFP-X as a chimeric
protein of interest, containing or not a 3'HA tag, together
with a spectrally separated fluorescent protein, either
eGFP or eCFP*, respectively.
"Dual selection" PRIG
Finally, we wished to make possible the combination of
both a drug-based selection of transduced cells and a
FACS-based method to detect and quantitate the expression of the protein of interest in these cells. Thus, we further introduced an open eGFP cDNA just upstream of the
BamHI site of the intact MCS of the pPRIPu vector, leading to pPRIPu eGFP vector (Fig. 5) designed to express
eGFP-X chimeric proteins in transduced, puro-resistant
cells.
Versatility of the PRIG vectors
Double color PRIG vectors provide the possibility to identify transduced cells both on the basis of the IRES-controlled fluorescent proteins (thereafter referred to as XFPs), as
well as by the expression of the protein of interest fused to
a distinct and spectrally separable fluorescent protein.
This may be valuable if the fluorescence emitted by the
chimeric protein of interest is too faint (intrinsically or as
a result of inadapted laser or filters) for a FACS-based sorting but sufficient (or adapted) for microscopical studies.
However, the expression of the IRES-controlled XFP provides redundant information when both transduction and
expression of the protein of interest can be evidenced by

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B-P-E-X-A-C-Ag-N-S-Sp-St-Sh-Bs-Ss-As-Ac-Hd-Sa-Bg-M-HA

IRES

pPRICp aHA

*

eCFP

Pa

Pa

(6180 bp)

IRES

eYFP

pPRIYp aHA
(6180 bp)

B-E-X-A-C-Ag-N-S-Sp-Sh-Bs-As-Ac-Sa-Bg-M-HA

IRES

mCherry

pPRIChp aHA
(6171 bp)

HA-B-P-E-X-A-C-Ag-N-Sh-Ss-As-Ac-Hd-Sa-Bg-M

IRES

Puro-R

pPRIPu HAa
(6102 bp)

B-P-E-X-A-C-Ag-N-Sh-Ss-As-Ac-Hd-Sa-Bg-M

IRES

Puro-R

pPRIPu

400 bp

(6060 bp)

Figure 4
Schematic representation of the « single color/resistance » PRIG vectors
Schematic representation of the « single color/resistance » PRIG vectors. pPRICp aHA, pPRIYp aHA and pPRIChp aHA and pPRIPu vectors are
pPRIG derivatives in which the eGFP sequence from the pPRIG has been replaced by eCFP*, eYFP, mCherry or Puro-R coding sequence, respectively.
eCFP* is a derivative of eCFP bearing the H148D mutation which enhances its brightness. pPRICp aHA, pPRIYp aHA and pPRIChp aHA contain an inframe 3' HA sequence (frame « a ») with respect to the MCS. They also contain the two PacI sites flanking the IRES-XFP cassette (shown as vertical bars
in pPRIYp aHA and pPRIChp aHA). By contrast, pPRIPu is devoid of both HA sequence and PacI sites. For each vector, the unique sites of the MCS are
indicated above the MCS. Unique sites of the pPRIYp aHA MCS are not listed since they are the same ones as in the immediately above vector.

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B-P-E-X-A-C-Ag-N-S-Sp-St-Sh-Bs-Ss-As-Ac-Hd-Sa-Bg-M-HA

eYFP

IRES

*

eCFP

Pa

pPRICp eYFPHA

Pa

(6911 bp)

B-E-X-A-C-Ag-N-S-Sp-Sh-Bs-As-Ac-Sa-Bg-M-HA

mCherry

IRES

eGFP

pPRIGp mChHA
(6903 bp)

B-P-E-X-A-C-Ag-N-Sh-Ss-As-Ac-Hd-Sa-Bg-M

eGFP

IRES

Puro-R

400 bp

pPRIPu eGFP
(6790 bp)

Figure 5
Schematic representation of the « dual color/selection » PRIG vectors
Schematic representation of the « dual color/selection » PRIG vectors. pPRICp eYFPHA is a pPRICp aHA derivative in which the eYFP sequence
has been cloned upstream of the MCS. pPRIGp mChHA is a pPRIGp aHA derivative in which mCherry sequence has been cloned upstream of the MCS.
pPRIPu eGFP is a pPRIPu derivative in which the eGFP sequence has been cloned upstream of the MCS. The reading frame is open from the start codon of
the upstream fluorescent protein to the stop codon of the HA sequence (for pPRICp eYFPHA and pPRIGp mChHA) or to the in-frame stop codon 3' to
the MCS (for pPRIPu eGFP). pPRICp eYFPHA and pPRIGp mChHA, but not pPRIPu eGFP, harbor the two flanking PacI sites. For each vector, the unique
sites of the MCS are indicated above the MCS.

the signal emitted by the upstream, fused, fluorescent protein. In this case, the presence of the IRES-XFP cassette
both unnecessarily increases the size of the vector and
may complicate a further fluorescent characterization of
the transduced cells because of the spectral overlap
between XFP signal and several usual fluorochromes (e.g.
eGFP/FITC). Thus, to increase the versatility of our vectors, we created the possibility to delete the IRES-XFP cassette. To this end, we inserted a site for the PacI restriction
enzyme just downstream of the stop codon of the 3'HA
coding sequence, and added another PacI site just
upstream of the 3' LTR. Consequently, the IRES-XFP cassette is flanked by two PacI sites, not present elsewhere in
PRIGs, and is thus readily and precisely removable from
any of the double color PRIGs. Given that PacI is a very
rare cutter, deletion of the IRES-XFP sequence will most

likely be possible even when a cDNA is already cloned
into these vectors. Interestingly, the PacI deletion of our
two double color PRIGs generates two novel vectors
which maintain spectrally separated fluorescent proteins
(eYFP and mCherry), therefore remaining suitable for coexpression studies. Moreover, not only do the two double
color PRIGs harbor the two PacI sites flanking the IRESXFP cassette, but all other PRIGs designed with a « p » suffix in their name (defining the PRIGp sub-family, Table
1), namely the 3'HA PRIGs(pPRIGp a/b/cHA), the modular PRIGs(pPRIGpER, pPRIGp VP16HA, pPRIGp KRABHA), and the novel single color PRIGs (pPRICp aHA;
pPRIYp aHA; pPRIChp aHA). Accordingly, once cloned in
any of the PRIGp vectors, the chimeric and/or tagged
cDNA will be both suitable for a trackable (fluorescent)
transfection/transduction, as well as for a transient expres-

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Table 1: PRIG vector sub-series

PRIGps
Single colour/selection

Modular

Dual colour/selection

Puro-resistance PRIGs

Previous PRIGs

pPRIG a/b/cHA
pPRICp aHA
pRIYp aHA
pPRIChp aHA
pPRIGp
VP16HA
pPRIGp
KRABHA
pPRIGp ER
pPRIGp
mChHA
pPRICp
eYFPHA

pPRIPu
pPRIPu HAa

pPRIG
pPRIG HAa/b/c

PPRIPu eGFP

sion from a non-fluorescent CMV-driven vector. Finally,
the PacI-deleted PRIGps, except pPRIGp KRABHA, gain
another available cloning site in the MCS, HindIII, which
is otherwise not unique.
Modularity
In summary, the new PRIG vectors presented here combine the following elements: i) five 5'modules (KRAB,
VP16, mCherry, eYFP, eGFP); ii) four 3' modules (HA in
three reading frames and ER), all of them being designed
to tag and/or to functionally modify the protein of interest; iii) and four selectable markers of transduction
(eCFP*, eYFP, mCherry, PuroR) (Table 1). Clearly, we
have not constructed all the possible combinations. For
example, the three possible reading frames of the 3'HA tag
are present only in the pPRIG background, i.e. in IRESeGFP containing vectors. Yet, all the PRIG vectors are
identical in most of their sequences, which allows easy
swappings to design novel PRIGs. Moreover, the presence
of the two PacI sites in most of our vectors permits the
generation of new combinations even after the cloning of
the cDNA of interest. This modularity may be convenient
i) if a fluorescent protein distinct from eGFP is desired in
PRIGs containing any regulatory module (KRAB, VP16 or
ER); ii) if another pair of fluorescent proteins (e.g.
mCherry and eCFP*) is desired; or iii) to obtain the correct reading frame for the 3'HA in any single color PRIGs.
pMPI (pMigr MSCV) counterparts of the new PRIG vectors
pMigR is a retroviral vector [19] related to pPRIG since it
also contains an eGFP cDNA 3' of the wild-type (« strong
») EMCV IRES sequence. However, pMigR is distinct from
pPRIG in two aspects. First, it contains two LTR sequences,
while pPRIG harbors a CMV promoter replacing the U3
region of the 5' LTR; pMigR is therefore less adapted than
PRIGs for transient expression. Second, the LTR sequences
of pMigR are of MSCV/MESV (murine stem cell virus/
murine embryonic stem cell virus) origin while those of
pPRIG are of PCMV (PCC4-cell passaged myeloprolifera-

tive sarcoma virus [20]) origin. Consequently, pPRIG and
pMigR LTRs differ by several point mutations in the R and
U5 regions, while they are identical in the U3 region. In
addition, the two vectors also differ by several point mutations in the 5'part of the δgag region. pMigR vector has
been commonly used to transduce pluripotent cells especially hematopoietic stem cells and embryonic stem cells
[19-21]. Only four contiguous cloning sites are present in
the original pMigR MCS. We designed an improved
pMigR derivative, termed pMPI (previously termed
pMigR-ATG [2]), which harbors some features of the PRIG
vectors. Specifically, pMPI contains the MCS of PRIGs, as
well as the T7 and SP6 flanking sequences, but is otherwise identical to pMigR, notably in all the viral-derived
sequences (LTRs and δgag region). We also designed the
pMPI HAa/b/c vectors that contain the 5'HA tag coding
sequence in the three reading frames with respect to the
MCS. Either pMPI or its first 5'HA derivatives have been
previously used to transduce primary embryonic hematopoietic cells [22] or to transfect HEK293T (human
embryonic kidney) cells [2], respectively. While designing
the above described PRIG vectors, we also constructed the
pMPI counteparts for some of them, namely: i) three
derivatives bearing the 3' HA sequence in the three reading frames(pMPIp a/b/cHA); ii) four pMPI derivatives in
which the IRES-eGFP cassette has been replaced by either
the IRES-eCFP*, IRES-eYFP, IRES-mCherry or IRES-Puro
(termed pMPIpeC aHA, pMPIpeY aHA, pMPIpCh aHA,
pMPIPuro, respectively); and iii) a pMPIPuro derivative
containing an open eGFP sequence 5' of the MCS
(pMPIPu eGFP, corresponding to pPRIPu eGFP). Again,
the « p » suffix indicates that the IRES-XFP cassette is
flanked by two PacI sites.
Functional tests
To validate our novel PRIGs, transfection and transduction studies were performed. First, we tested the pPRIPu
eGFP derivative. To this end, mouse NIH3T3 cells were
transduced with this vector, and plated 48 hours later in

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medium alone or in medium containing puromycin. We
observed that transduced cells readily grew in medium
containing up to 10 μg/ml of puromycin while control
NIH3T3 cells died in approximately 24 hours under the
same conditions. After 5 to 7 days, no difference was
observed between transduced cells exposed to 2 or 10 μg/
ml of puromycin and transduced cells grown in medium
without puromycin with respect to cell viability and morphology (data not shown). This indicates that pPRIPu
eGFP indeed confers resistance to puromycin. As
expected, FACS analyses showed that all transduced cells
exposed to puromycin express eGFP, while some cells are
eGFP-negative in the population not treated with puromycin (Fig. 6A and 6B), confirming that puromycin efficiently eliminates rare untransduced cells. The same
results were obtained upon transduction of NIH3T3 cells
with pMPIPu eGFP (data not shown). Expression of puroresistance and eGFP were also confirmed by fluorescence
microscopy for pPRIPu eGFP transfected HEK293T cells
(data not shown). Finally, the expression of the puroresistance gene from the pPRIPu vector was verified in
transiently transfected human HEK293T cells using
microscopy (Fig. 7A).
Next we verified that each single color PRIG vector indeed
gives the expected fluorescence in transduced cells. By
FACS analyses we observed that pPRIYp aHA-, pPRICp
aHA-, pPRIChp aHA- transduced cells emit yellow, blue
and red fluorescence respectively, which are readily separated on a LSRII cytometer equiped with the lasers and filters described in the Methods section (Fig. 6C). As
expected, the pPRIG HAb-transduced cells are heavily positive for eGFP expression (Fig. 6C). The expression of each
single color PRIGs was also confirmed in transfected
HEK293T cells and in transduced primary rat osteoblasts
using fluorescence microscopy (Fig. 7B and 7C).
We next took advantage of the clear separation of each fluorescent signal in FACS analyses to demonstrate the feasability of either double (either pPRIYp aHA plus pPRICp
aHA or priG-HAb plus pPRIChp aHA) or triple (pPRIYp
aHA plus pPRICp aHA plus pPRIChp aHA) co-transductions. These experiments show that cells exposed to the
mixtures of viral supernatants can be characterized for
each signal whose origin is unambigously ascribed to one
of the fluorescent proteins (Fig. 6D and 6E). Thus, single
color PRIGs are potentially suitable to simultaneously
express up to three genes in transduced cells, with the possibility to identify the subpopulations expressing any of
them or any combination (including all) of them. For all
these experiments, similar results were obtained using the
pMPI counterparts of these single color PRIGs (data not
shown). Moreover, pPRIPu can be used together with any
of the single color PRIGs, further extending the possibilities of multiple transductions. Again, the same results

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were obtained upon transduction with the pPMI counterparts of the single color PRIGs, namely pMPIpeC aHA,
pMPIpeY aHA, pMPIpCh aHA, and MPI HAa (data not
shown). It is worth noting, however, that in order to
improve the balance between the different fluorescent signals, we had to correct for the relatively weak eCFP* and
mCherry fluorescences compared to eYFP or eGFP, by
exposing the cells to greater amounts of pPRICp aHA or
pPRIChp aHA viral supernatants (500 μl instead of 50 μl
for pPRIYp aHA or pPRIG HAb (Fig. 6D and 6E)). The difference in fluorescence intensities is probably due to
either the intrinsically low brightness of eCFP* compared
to eGFP or eYFP, or to the non-optimal excitation of
mCherry by our 488 nm laser [13,15]. Indeed, when
translated from the same mRNA in double color PRIG
transduced cells, the fluorescent signals exhibit similar
differential intensities (see below) ruling out that the differences observed among single color PRIG-transduced
cells stem from differential viral titer.
Finally, we show that the vast majority of NIH3T3 cells
transduced with one of the double color PRIGs (either
pPRIGp mChHA or pPRICp eYFPHA) is positive for the
two expected fluorescences (Fig. 6F). In these cases, it is
impossible to experimentally balance the fluorescent signals emitted by the proteins of each couple, since the two
proteins are coded by a single vector. Consequently,
though most cells are doubly positive, eGFP appears
stronger than mCherry upon transduction with pPRIGp
mChHA and eYFP appears stronger than eCFP* upon
transduction with pPRICp eYFPHA.
During the course of these experiments, we also observed
that mouse NIH3T3 fibroblasts or rat osteoblasts transduced with pPRIGp mChHA display a predominant cytoplasmic localization of the red signal ("pitted mCherry "),
while eGFP exhibit a normal, uniform, distribution (data
not shown). This unexpected mCherry localization is also
observed in HEK293T cells transfected with this vector
(Fig. 7D, left), but not in pPRIpCh aHA-transduced or
transfected cells (Fig. 7B and data not shown), which display an uniform mCherry localization. We strongly suspect that the disturbed localization of mCherry
originating from the pPRIGp mChHA is caused by its
fusion to the MCS. Indeed, in one reading frame, the MCS
encodes a short peptide in its 5' part which somehow disturbs the localization of mCherry. Accordingly the same
effect, although less pronounced, is observed for the 5' fluorescent proteins of pPRICp eYFPHA and for pPRIPu
eGFP which fuse eYFP or eGFP, respectively, to the same
reading frame of the MCS (data not shown). Moreover,
the insertion of mCherry 5' of the MCS in the two other
reading frames leads to an uniform mCherry distribution.
Finally, mCherry expressed from the pPRIGp mChHA
recovers a normal homogeneous localization upon the in-

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Figure 6
FACS analyses of NIH3T3 cells transduced with different PRIG vectors
FACS analyses of NIH3T3 cells transduced with different PRIG vectors. Cells were analysed using a LSRII cytometer (Becton-Dickinson) 5 to 7
days after exposure to the viral supernatant(s). For each point, 104 events were recorded. A) Non-transduced NIH3T3 cells we analysed for cyan (eCFP*)
yellow/green (eYFP/eGFP) or red (Cherry) fluorescences using the lasers and filters described in the Methods section to determine the basal level for each
signal. B) Functional test of the pPRIPu eGFP vector. 48 hours after the exposure to a pPRIPu eGFP viral supernatant, cells were plated in a fresh medium
containing (right panel) or not (left panel) 10 μg/ml of puromycin, and analysed for eGFP expression after 7 days of culture. All the selected and almost all
the non-selected cells strongly express eGFP. Note that at this dose of puromycine, control untransduced NIH3T3 cells died in approximately 24 hours
(not shown). C) Functional test of the single color vectors. Cells transduced with the indicated vector (at the bottom of each panel) were analyzed for
each fluorescence. Note that under these conditions of transduction and detection, each fluorescent protein (eCFP*, eYFP/eGFP, mCherry) is mainly, if
not exclusively, detected in its appropriate channel. D) Feasability of double transductions. Cells were transduced with the indicated (right hand side) mixture of two viral supernatants. In the cases of double transductions, untransduced cells, doubly transduced cells and cells transduced with either one of the
two single color vectors can be discriminated from each other. E) Feasability of triple transductions. Cells were transduced with the indicated mixture of
the three viral supernatants. Again, because of the minimal overlapping between the three signals, the transduction status of any cell with respect to each
vector can be determined. Note however, that for D and E, the quantity of each viral supernatant was not equal in the mixture to correct for either relatively inefficient detection of the signal in our conditions (mCherry) or relatively intrinsic weakness of the protein (eCFP*) (see text). F) Functional test of
the doublecolor vectors. Cells were transduced with the indicated vector (at the bottom of the panel). In each case, the vast majority of the cells are positive for the two expected signals.

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Figure 7
Fluorescence microscopy analyses of cells transfected or transduced with PRIG vectors
Fluorescence microscopy analyses of cells transfected or transduced with PRIG vectors. A. pPRIPu confers puro-resistance. HEK293T cells
were transfected with either peGFP-N1 + pPRIPu (left) or peGFP-N1 (Clontech) + pRK5 (irrelevant carrier DNA) (right). 24 hours after transfections,
cells were further grown for 30 hours in the presence of 10 μg/ml of puromycin. eGFP serves as a control for transfection efficiency. pPRIPu + peGFP-N1
transfected cells are living while most mock-transfected cells are dying. B. Single color PRIGs are expressed in HEK293T transfected cells. HEK293T cells
were transfected with the indicated single color PRIG vector. Fluorescence microscopic analyses were performed 24 hours later. The signal emitted by
each fluorescent protein was observed and photographed using the appropriate filter. Note that the mCherry signal is relatively weak probably due to the
suboptimal excitation light delivered by our filter (546/12 nm). Moreover, a weak portion of the eGFP signal is detectable in the cyan filter, and conversely
a residual portion of the eCFP* signal is detectable in the green filter. By contrast, the eYFP (green filter) and mCherry (red filter) signals are only detected
in their appropriate filter. C. Single color PRIGs are expressed in transduced rat primary osteoblasts. Rat primary osteoblasts were transduced by the indicated single color PRIG vector and observed using a fluorescent microscope 48 hours later. Signals were photographed using the appropriate filter for
each fluorescent protein. D. mCherry localization in HEK293T transfected cells. Left: HEK293T cells were transfected with pPRIGp mChHA. The red signal appears to be predominantly cytoplasmic, which is not observed in pPRIChp transfected cells, where mCherry shows an uniform distribution (see B).
Right: HEK293T cells were transfected with a pPRIGp mChHA derivative containing an in-frame deletion of the 5' part of the MCS (PvuII/StuI). This deletion completely restores a normal (uniform) distribution of mCherry. Note that in cells transfected with pPRIGp mChHA (left) and with its PvuII/StuI
deleted derivative (right), the eGFP (green, encoded by the 3' cistron) appears normally and uniformly distributed throughout the cell. The regions of
interest were enlarged for a better visualization.

frame PvuII/StuI deletion of the 5' part of the MCS (or
upon the almost entire MCS in-frame XhoI/SalI deletion),
but not upon the in-frame ClaI/AsuII deletion (Fig. 7D,
right and data not shown). Thus, users may want to delete
the 5' part of the MCS (between the PvuII site and the ClaI
site and encoding the sequence LEFSRSLCI) upon cloning
when this reading frame is in phase with their cDNA.

Conclusion
We describe here a new generation of PRIG vectors. They
keep the original improvements of the pPRIG vector with
respect to its ancestor pAP2, i.e. a far more complete and
convenient MCS and a wild type strong EMCV IRES element, and provide several modifications to extend their
use to a wide variety of experimental settings or biological
issues, as detailed above. Moreover, we introduced some
other changes to enhance their versatility, and duplicated
eight of them in a pMigR (MSCV) background for their

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use in stem cells. All constructs were generated by restriction and modification enzymes and all cloning modifications were verified by sequencing. In addition, the viral
parts (i.e. from the 5' end of the 5' LTR to the 3' end of the
3' LTR) of the ancestor pAP2 vector and of two of its final
descendents (pPRIGp aHA and pPRIYp aHA) were
entirely sequenced and show no discrepancy with respect
to the expected sequence. Finally, their reliability was further demonstrated by the experimental validations we
show here. All these vectors are made freely available to
the scientific community and all the maps and sequences
will be posted on our website in the near future [23].

Methods
Constructs
All the DNA constructs were made using standard procedures. Restriction and modification enzymes were purchased from Fermentas, New England Biolabs and
Invitrogen. Plasmids were prepared using CsCl procedure
or Purelink Midiprep kits (Invitrogen). The VP16
sequence was taken from the pVP16 vector (Clontech, a
gift from R. Galien). It is worth mentioning that this vector contains the « full-length » (aa 410 to 490 of the HSV1
protein) VP16 acidic transactivation domain (plus an Nterminal nuclear targeting domain) and not the 411–455
subdomain as described by the supplier. The KRAB
domain of the human KOX1 protein was isolated from a
PAX3-KRAB encoding vector ([11], a gift from Drs
Rauscher and Hongzuang, Wistar Institute, Philadelphia,
USA). eGFP and eYFP cDNAs were isolated from the
peGFP-C1 and peYFP-C1, respectively (Clontech).
mCherry [13] cDNA was a gift from Dr Tsien (San Diego,
USA). The sequence encoding the ER hormone binding
domain containing the G525R mutation was provided by
D. Monté [4]. The eCFP cDNA bearing the H148D mutation (eCFP*) was a gift from Dr Graihle (Pasteur Institute,
Seoul, South Korea).
Verification of the constructs
Detailed cloning strategies, maps and sequences of all vectors are available upon request, but some points should
be underlined. First, we did not use PCR. All of our constructs were built using restriction and modification
enzymes. Second, we have extensively sequenced all the
constructs presented here. All the viral portions of pAP2
and pMigR plasmids have been sequenced and several differences were found compared to the theoretical
sequence, and at least all the module and/or fluorescent/
selectable proteins in each construct have also been
sequenced. In summary, we are confident that the entire
nucleotide sequence of each construct posted on the web
site [23] is error free.

Third, eGFP cDNA from pMigr/pMPI vectors differs from
that of pAP2 and of its first descendents pPRIG and pPRIG

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HA a/b/c in two nucleotides and one amino acid (V164 in
pAP2 type eGFP, A in pMigr/pMPI-type, if the first Met residue is 1). pPAP2-type and pMigr/pMPI-type eGFPs however exhibit indistinguishable fluorescent properties. Due
to the phylogeny of our new PRIG vectors (reflecting the
cloning strategy designed to introduce the distal PacI site),
all the herein described pPRIG vectors (as well as all pMPI
vectors) contain the pMigr/pMPI-type eGFP, except the
pPRIPu eGFP whose 5'eGFP originates from peGFP-C1
which is identical to the pAP2-type eGFP.
Fourth, we previously reported that the efficiency of the
EMCV IRES is strongly dependent upon the integrity of its
last ATG, which should be used as start codon for the
downstream eGFP cDNA. Thus, in each construct where
eGFP was replaced by another cDNA (eCFP*, eYFP,
mCherry, Puro-R), we kept the last IRES ATG intact and
in-frame with the natural start codon of the cDNA. The
high efficiency of the IRES in all our constructs is confirmed by strong expression of the fluorescent proteins or
Puro-resistance of transduced cells (see text).
Transfection, transduction, FACS and
immunofluorescence analyses
HEK293T (a HEK293 subline containing the simian virus
40 (SV40) T antigen) were cultured in DMEM medium
supplemented with 10% FCS and antibiotics, mouse
NIH3T3 fibroblasts in a mixture of 3/4 of DMEM and 1/4
of F12 medium (Invitrogen) supplemented with 10% FCS
and antibiotics. Primary rat osteoblasts were obtained and
grown according to ref [24].

For NIH3T3 cells, all the retroviral supernatants were prepared by transfecting human 5 × 105 HEK293T in 6 well
dishes cells by 0.4 μg of pCMV-intron gag-pol, 0.4 μg of
the FB-Mo-Salf vector encoding the ecotropic MLV env
protein (two gifts from F.L. Cosset, CNRS Lyon, France
[25]) together with 0.8 μg of the PRIG, pMPI or pMPI
derivatives using Fugene 6 (Roche) as transfection reagent. 24 hours later, the indicated volume of viral supernatant(s) were filtered on 0.45 μm filters and added to 105
NIH3T3 mouse fibroblasts in the presence of 5 μg/ml of
polybrene. 5 to 7 days later, NIH3T3 cells were analysed
for the expression of the fluorescent protein through FACS
analyses using LSRII (Becton Dickinson). eCFP* (eCFP
containing the H148D substitution) was excited by a 405
nm laser and its emission was collected by a 440/40 nm
filter. eGFP, eYFP and mCherry were excited by a 488 nm
laser, emission of both eGFP and eYFP was collected by a
510/20 nm filter while mCherry emission was collected
by a 610/20 nm filter. 104 events were recorded.
For HEK293T cells and rat primary osteoblasts, transfections and transductions were performed according to the
protocol described in ref [2]. Fluorescence microscopy

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was performed using a Axiovert 40 CFL microscope
(Zeiss) at 20× magnification. The eGFP and eYFP signals
were observed using the filterset 44 (Zeiss; excitation:
475/40; emission: 530/50; beamsplitter: 455, "green filter"), mCherry signal using the Filterset 15 (Zeiss; excitation: 546/12; emission: LP 590; Beamsplitter: 580, "red
filter") and eCFP* signal was observed using the HE 47 filter (Zeiss; excitation: 436/25; emission: 480/40; Beamsplitter: 455, "cyan filter").

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6.
7.

8.

9.

Abbreviations
eCFP/eGFP/eYFP: enhanced cyan/green/yellow fluorescent protein. IRES: Internal Ribosome Entry Site. LTR:
Long Terminal Repeat. ECMV: encephalomyocarditis
virus. MSCV: murine stem cell virus. PCMV: PCC4-cell
passaged myeloproliferative sarcoma virus. ER: hormone
binding domain of the mouse estrogen receptor alpha.
VP16: viral protein 16. KRAB: krüppel-associated box.
Puro-R : puromycine resistance gene. FACS : fluorescenceactivated cell-sorting.

10.
11.

12.

13.

Authors' contributions
PM and OAC designed and constructed the vectors. OAC
wrote the paper, PM helped in the writing of the paper.
PM made the figures with the help of OA. OAC and YL
performed the FACS analyses, including NIH3T3 cell
transduction. PM, PP and KEB performed the fluorescence
analyses, including rat osteoblast transduction and
HEK293T cell transfection. KEB edited the manuscript. All
authors read and approved the final manuscript.

14.
15.
16.

17.

Acknowledgements
We thank R. Graihle (Institut Pasteur, Seoul, South Korea) for the gift of
the eCFPH148D cDNA, R. Tsien (Howard Hughes Medical Institute, San
Diego, USA) (via P.A. Defossez, CNRS, Paris, France) for the mCherry vector, F. Rauscher and P. Hongzuang (Wistar Institute, Philadelphia, USA) for
the KOX1 KRAB domain, D. Monté for the ERG525R containing vector and
R. Galien for the pVP16 vector. We are also grateful to H. Pelczar for critical reading of our manuscript. This work was supported by fundings from
INSERM (Institut National de la Santé et de la Recherche Médicale) and
CNRS (Centre National de la Recherche Scientifique).

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