Site-specific integrase-mediated transgenesis in mice
via pronuclear injection
Bosiljka Tasica, Simon Hippenmeyera, Charlene Wangb, Matthew Gamboab, Hui Zongc, Yanru Chen-Tsaib,1,
and Liqun Luoa,1
aThe Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, CA 94305;bTransgenic Facility, Stanford Cancer Center,
Stanford University School of Medicine, Stanford, CA 94305; andcInstitute of Molecular Biology, University of Oregon, Eugene, OR 97403
Edited* by Matthew P. Scott, Stanford University/The Howard Hughes Medical Institute, Stanford, CA, and approved March 16, 2011 (received for review
December 29, 2010)
Microinjection of recombinant DNA into zygotic pronuclei has
been widely used for producing transgenic mice. However, with
this method, the insertion site, integrity, and copy number of the
transgene cannot be controlled. Here, we present an integrase-
based approach to produce transgenic mice via pronuclear in-
jection, whereby an intact single-copy transgene can be inserted
into predetermined chromosomal loci with high efficiency (up to
40%), and faithfully transmitted through generations. We show
transgene cause profound silencing and expression variability of
the transgenic marker. Removal of these undesirable elements
leads to global high-level marker expression from transgenes
driven by a ubiquitous promoter. We also obtained faithful marker
expression from a tissue-specific promoter. The technique pre-
sented here will greatly facilitate murine transgenesis and precise
structure/function dissection of mammalian gene function and
regulation in vivo.
30 years. Although still the predominant method used to produce
transgenic mice, it has several limitations: the insertion site, in-
tegrity, and copy number of the transgene cannot be controlled.
this manner can be influenced by the local chromatin environment
(i.e., position effect) that can lead to transgene silencing or ectopic
expression (4–7). Moreover, transgenic DNA concatemerized into
a large array is subject to repeat-induced gene silencing (8).
Single-copy transgenesis in mice can be achieved with retro-
viruses (9) and transposons (10, 11), but these approaches in-
tegrate transgenes throughout the genome. As a result, the
transgenes are subjected to the local chromatin environment and
can cause endogenous gene disruption, although the mutagenic
properties of transposons can be desirable for particular appli-
cations (10). These problems can be overcome by targeting the
transgene to a specific chromosomal locus via homologous re-
combination in embryonic stem (ES) cells (12, 13). However, this
method is significantly more laborious and time-consuming, as it
involves creation of modified ES cells and mouse chimeras, as
well as eventual germline transmission of the transgene.
Integrase enzymes from a variety of sources have been used
to catalyze integration of transgenes in heterologous systems
(14, 15). Integrases catalyze irreversible recombination between
appropriate attB and attP sites (14, 16). ϕC31 integrase from
a Streptomyces phage has previously been used for transgene in-
tegration in flies (17–19). In mice, ϕC31 integrase has been used
to catalyze integration of circular DNA into pseudo-attP sites in
the genome for gene therapy (20) or low-efficiency transgenesis
(21), for transgenesis in mouse ES cells (22), and for removal of
undesirable transgene portions or reporter activation (23, 24).
Here, we describe an integrase-mediated method for site-
specific transgenesis in mice via pronuclear microinjection, with
integration efficiencies as high as 40%. We use ϕC31 integrase
to catalyze recombination between one or two attB sites in a
recombinant DNA with one or more tandem attP sites that we
previously inserted intospecific loci in the mouse genome (Fig. 1).
roduction of transgenic mice via microinjection of DNA into
zygotic pronuclei (1–3) has served mammalian genetics for
and nearby transgenic elements dramatically decrease expression
global, high-level transgene expression from a ubiquitous pro-
moter. Finally, we show that a promoter for the murine tran-
scription factor Hb9 integrated into one of the predetermined loci
drives proper tissue-specific marker expression.
Strategy and Proof of Principle for Integrase-Mediated Site-Specific
Transgenesis. To generate embryos containing attP sites for ϕC31
integrase-mediated transgenesis, we used standard homologous
recombination-based methods in mouse ES cells (12, 25). We
inserted three shortened tandem ϕC31 integrase attP sites
(attPx3) or a single “full-length” attP site (14) into two loci: the
Rosa26 locus on mouse chromosome 6 (26) and an intergenic
Hipp11 (H11) locus on mouse chromosome 11 (27) (Fig. 1, Left,
and SI Appendix, Fig. S1). The Rosa26 locus supports global
expression of a single copy knock-in transgene driven by a com-
bination of the CMV enhancer and the chicken β-actin promoter
(pCA) (28, 29). Knock-in experiments confirmed that H11 sup-
ports high-level global gene expression from the pCA promoter
and a higher rate of mitotic (interchromosomal) recombination
compared with Rosa26 (27). The latter property suggested that
H11 might allow better access to ϕC31 integrase than Rosa26.
The modified ES cells were used to produce chimeric mice, and
mice with germline-transmitted alleles were used to establish
mouse colonies homozygous for the knock-in cassettes.
For most experiments, we integrated transgenes into the H11
locus because homozygous insertions into this locus are not pre-
dicted to disrupt any endogenous genes, and the resulting mice
are completely healthy and fertile (27). We injected embryos
homozygous for attP or attPx3 at the H11 locus (H11P or H11P3,
respectively) with ϕC31 mRNA together with attB-pCA-GFP,
a minicircle DNA that was generated by removal of the plasmid
backbone to enable proper transgene expression (as detailed
later). The attB-pCA-GFP minicircle contains a full-length attB
site (14) and the sequence for a thermotolerant GFP (30, 31)
driven by the ubiquitous pCA promoter (SI Appendix, SI Materials
and Methods). We obtained integration of the transgene (Fig. 1,
Top Right, and Table 1, rows 1–3; SI Appendix, Table S1, provides
As an alternative to insertion of DNA at a single attP site, we
also injected a plasmid in which pCA-GFP is flanked by two attB
Author contributions: B.T., H.Z., Y.C.-T., and L.L. designed research; B.T., S.H., C.W., and
M.G. performed research; B.T. and L.L. analyzed data; S.H. contributed new reagents/
analytic tools; and B.T. and L.L. wrote the paper.
Conflict of interest statement: Y.C.-T. is a consultant and co-founder of Applied
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS Open Access Option.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or lluo@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1019507108 PNAS Early Edition
| 1 of 6
sites (pattB-pCA-GFP-attB) into H11P3 homozygous embryos. In
principle, this approach should allow ϕC31 integrase to catalyze
a recombinase-mediated cassette exchange reaction (Fig. 1,
Bottom Right). We tested the integration of the circular pattB-
pCA-GFP-attB plasmid into H11P3 and obtained successful
cassette exchange as confirmed by PCR (Fig. 1, Bottom Right,
and Table 1, row 4). In these experiments, we never detected
insertion of a full plasmid.
The integrated transgenes were properly transmitted from
founders to progeny (SI Appendix, Table S2). Both strategies
resulted in broad and high-level GFP expression in pCA-GFP
transgenic mice (Fig. 1, Right, Fig. 2D, and Fig. S1C, Bottom).
These data provide the proof of principle for our site-specific
integrase-mediated transgenesis in mice. In the subsequent
sections, we describe in more detail the optimization process
that led to these results.
Transgenic Integrase Did Not Enable Site-Specific Integration. Our
original knock-in cassettes contained a mammalian codon-
a neomycin resistance gene, flanked by FRT5 sites (33) (SI Ap-
pendix, Fig. S1). This “NVϕ cassette” (i.e., Neo-VASA-ϕC31o) was
designed to provide the integrase in embryos in situ. We injected
mouse embryos homozygous for the H11P3NVϕ knock-in with
a plasmid containing the attB-pCA-GFP transgene (pattB-pCA-
GFP) but did not obtain any site-specific integration (0/32 F0s;
Table 1, row 5) despite occasional random integrations. However,
coinjecting pattB-pCA-GFP with ϕC31o mRNA into homozygous
H11P3NVϕ embryos produced site-specific integrations (Table 1,
row 6). Thus, the VASA promoter does not promote sufficient
ϕC31o expression in situ to enable site-specific insertions.
Because the NVϕ cassette did not perform as expected we re-
moved it from the H11P3NVϕ allele by FLP-mediated recom-
bination to generate the H11P3 allele (SI Appendix, Fig. S1, and
SI Appendix, SI Materials and Methods). Similarly, the H11P al-
lele was derived from H11PNVϕ. Coinjection of pattB-pCA-GFP
and ϕC31o mRNA into homozygous H11P or H11P3 embryos
produced site-specific integrants (Table 1, rows 8–10). Thus,
we coinjected integrase mRNA with DNA for all subsequent
specific ϕC31 integrase-mediated
transgenesis via pronuclear injec-
tion in mice. A single or three
tandem attP sites were knocked
into the Hipp11 (H11) or Rosa26
loci via homologous recombina-
tion in ES cells (Left; for details, see
Fig. S1). Mice homozygous for one
of the modified loci (H11 is shown
as an example) served as embryo
donors. A mix of DNA and in vitro
transcribed ϕC31 mRNA was injec-
ted into a single pronucleus of
each zygote. The integration of
plasmid bacterial backbone (BB)
that decreases the transgene ex-
pression was avoided either by
injecting a minicircle DNA with a single attB site (top branch; in this case, ϕC31 catalyzes a typical integration reaction), or by injecting plasmid DNA where the
gene of interest (e.g., GFP) was flanked by two attB sites (bottom branch; in this case, ϕC31 catalyzes a recombinase-mediated cassette exchange reaction).
Right, Middle: Green fluorescence of a representative transgenic F0 embryo and the corresponding PCR results that indicate site-specific insertion. The red
numbers for the PCR results correspond to the numbers on the H11P3-pCA-GFP transgene scheme below; the same PCR tests can be used for the transgene
above. The particular embryo shown was obtained by cassette exchange. Inset: Bright-field image of the same embryo.
Schematic summary for site-
Table 1. Efficiency of site-specific integration
RowDNA* DNA type
size, kbStrain Background F0 (n) SS F0 (n)Significance¶
attB-pCA-GFP, no RNA
NS vs. row 24.8
P < 0.05 vs. row 2
NS vs. row 710
NS vs. row 9
P < 0.05 vs. row 9
Abbreviations: F0, embryos or animals obtained from injections; SS, site-specific integration; R, random integration; mix, mixed background of 129, C57BL/6
and DBA2; FVB N4, mice of the mixed background were outcrossed for 4 generations to the FVB strain and then intercrossed.
*All DNA was coinjected with ϕC31o mRNA, except for row 5.
†Both FRT and non-FRT versions of attB-pCA-GFP were used.
‡F0s were analyzed only as E10 or E11 embryos.
§F0s were analyzed either as E10 or E11 embryos or as live pups.
kThe six founders listed contained pCA-GFP without the bacterial backbone; five more founders with cassette exchange contained only the bacterial
backbone. Therefore, the total number of founders with cassette exchange is 11 (29%).
¶Fisher’s exact test. NS, not significant.
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| www.pnas.org/cgi/doi/10.1073/pnas.1019507108Tasic et al.
Bacterial Backbone and the NVϕ Cassette Affect Proper Transgene
Expression. Despite proper transmission of the site-specifically
integrated transgenes produced from the pattB-pCA-GFP plas-
mid, the progeny of these transgenic founders exhibited a wide
range of GFP expression levels as seen in whole-mount embryos
(SI Appendix, Fig. S1C). Moreover, the GFP expression in the
progeny was mosaic in several internal tissues, including the
heart, brain, and particularly the liver (Fig. 2B and SI Appendix,
Fig. S2). We first observed this variable and mosaic expression
with H11P3NVϕ as the host. We suspected that the nearby
germline-specific VASA promoter and/or other elements within
the NVϕ cassette (e.g., the neomycin resistance gene) could af-
fect the expression of pCA-GFP inserted at H11. Indeed, trans-
genic embryos containing pCA-GFP inserted at H11P3, which
lack the NVϕ cassette, produced more uniform GFP expression
(Fig. 2C and SI Appendix, Fig. S2). However, considerable vari-
ability of pCA-GFP expression still persisted especially in the
livers of N1 or N2 animals derived from a number of transgenic
founders (Fig. 2C and SI Appendix, Fig. S2).
It has been reported that plasmid bacterial backbone can de-
crease the expression of integrated and episomal transgenes (34–
37). To test if this is the case in our system, we developed an in
vitro method to produce minicircle DNA: circular DNA con-
taining desired transgene elements, but devoid of the bacterial
backbone (SI Appendix, Fig. S3 and SI Appendix, SI Materials and
Methods). We injected the minicircle DNA into H11P3 embryos
to produce transgenic animals (SI Appendix, Fig. S1, and Table
1). For simplicity, we designate hereafter the transgenic alleles
derived from integration of the entire pCA-GFP plasmid (which
contains the bacterial backbone) as pCA-GFP-BB (Fig. 2C), and
the mice derived from integration of the pCA-GFP minicircle as
pCA-GFP (Fig. 2D). Transgenic animals derived from the pCA-
GFP minicircle exhibited higher and more uniform expression in
embryos and all adult tissues examined (Fig. 2D and SI Appendix,
Fig. S2). The removal of bacterial backbone from the pCA-GFP-
BB transgene by crossing to our newly generated GFP-FLPo
transgenic mice (SI Appendix, SI Materials and Methods) also
resulted in elevated transgene expression (SI Appendix, Fig. S4).
These data demonstrate that pCA-GFP at the H11 locus can
express GFP ubiquitously in the absence of the NVϕ cassette and
the bacterial backbone.
Because the greatest variability was observed in the liver (Fig.
2 and SI Appendix, Fig. S2), we used it as a model to determine
the relative contributions of the NVϕ cassette and the bacterial
backbone to GFP expression variability in these transgenic mice.
In addition, to test for the possible differences between a single
attP site and attPx3, we analyzed transgenic mice obtained by
insertion into the H11P allele (Table 1, rows 1 and 8). Finally, to
probe the effect of genetic background, we analyzed transgenes
inserted into the H11P3 allele in mice that had been outcrossed
to the FVB inbred strain for four generations (Table 1, rows 3
and 10). We compared the total GFP fluorescence of liver sec-
tions from different transgenic animals under identical con-
ditions. We observed no statistically significant differences in
GFP fluorescence between transgenes that differed only in the
number of attP copies or in the genetic background of the mouse
strain used for transgenesis (SI Appendix, Fig. S5). Therefore, we
grouped all data according to the presence of the NVϕ cassette
and/or the bacterial backbone (Fig. 2E). We found that, in the
introduced by ϕC31 integrase-mediated transgenesis. (A–D) Representative
fluorescence images from embryonic day 11 embryos and adult livers, hearts,
and cerebella of N1 or N2 transgenic animals corresponding to the geno-
types and schematics of transgenes shown (Upper). Embryos or same tissues
were imaged under identical conditions, except that “5×-exp” designates
fivefold longer exposure time than for the rest of the images in the same
column. Whole-mount embryos were imaged for GFP fluorescence; corre-
sponding bright-field images of each embryo are also shown (Insets). The
livers and hearts are represented by epifluorescence images of 10-μm sec-
tions stained only by DAPI (blue). The green signal is GFP fluorescence. The
cerebella are represented by confocal images of sections stained by anti-GFP
antibody (green), anti-calbindin (red) for Purkinje cells, and DAPI (blue). Two
Purkinje cells labeled by asterisks appear negative for GFP. ϕC31 attL and
attR are the product of recombination of an attP (black arrows) and attB site
(purple) and are therefore half black and half purple. Half circles represent
FRT5 sites. Half white/half black triangle represent λ-integrase attB site
created during minicircle production. pSV40, SV40 promoter. pVASA, VASA
promoter. U, unique sequence. pCA, CMV enhancer and β-actin promoter. G,
GFP. pA, polyA signal. BB, plasmid bacterial backbone. (Scale bars: 1 mm for
embryos, 100 μm for tissue sections.) (E) Average fluorescence in arbitrary
GFP expression in animals carrying site-specific pCA-GFP transgenes
units (AU) in the GFP channel for liver sections from animals of genotypes
shown below (no int., no integration; represents wt, H11P3NVϕ/wt, H11P3/
wt, and H11P/wt genotypes). Each dataset is represented by mean ± SD. The
numbers of individual animals and founders analyzed for each genotype are
listed below the genotypes. When samples from multiple founders were
combined to obtain an average, each founder was represented by the same
number of animals except in the case labeled by a spade. The fluorescence
intensities differ significantly among the groups by one-way ANOVA [F(3,
50) = 84.09, P < 0.0001]. Tukey’s post-hoc test was used for pair-wise com-
parisons (ns, not significant; *P < 0.05 and ***P < 0.001).
Tasic et al. PNAS Early Edition
| 3 of 6
presence of both the NVϕ cassette and the bacterial backbone,
GFP expression was detectable in the liver in a small number of
cells and at a very low level (Fig. 2B), but total fluorescence was
statistically indistinguishable from negative controls (Fig. 2E,
second column vs. first column). In other organs analyzed (heart
and brain), GFP expression was apparent but mosaic (Fig. 2B
and SI Appendix, Fig. S2). When the NVϕ cassette was removed
but the bacterial backbone was still present, average GFP fluo-
rescence intensity became significantly higher (Fig. 2E, third
column vs. second column). Finally, when the bacterial backbone
was removed, average GFP fluorescence intensity became even
higher (Fig. 2E, fourth column vs. third column). Thus, both the
NVϕ cassette and the bacterial backbone significantly reduced
transgene expression. The reduction of total fluorescence in-
tensity could be caused by low-level of expression in every cell,
absence of expression in a subset of cells, or a combination of the
two. As is evident from Fig. 2 A–D and SI Appendix, Fig. S2, both
mechanisms contributed to the reduced level of transgene ex-
pression in the presence of the NVϕ cassette and/or the bacte-
H11 Can Support Tissue-Specific Expression. To test if a tissue-spe-
cific promoter can provide appropriate expression using our
transgenesis method, we integrated pattB-pHb9-GFP-FRT5 into
H11P3 (Table 1, row 11). This plasmid contains an approxi-
mately 9-kb promoter fragment from the murine transcription
factor Hb9 gene that has been shown to be sufficient to direct
appropriate tissue- and cell-specific expression in transgenic
animals (38). We examined tissue-specific marker expression
before and after removal of the bacterial backbone by using the
GFP-FLPo transgene (SI Appendix, SI Materials and Methods and
Fig. 3A). In agreement with the reported expression pattern (38),
we observed GFP expression in motor neurons in the ventral
spinal cord and the tail tip (Fig. 3B). In this case, the removal of
bacterial backbone did not appear to affect the expression level
of the transgene (Fig. 3C). Double-labeling with endogenous
Hb9 protein confirmed the motor neuron-specific expression of
the transgene (Fig. 3C). These experiments indicate that our
integrase-based strategy can be used for faithful tissue-specific
expression of transgenes.
Integration Efficiency. We compared the integration efficiency for
attP-modified loci, expressed as the percentage of F0 animals with
site-specific integrations obtained from the total number of F0s
(Table 1; SI Appendix, Table S1, provides more details). Although
in pooled data (SI Appendix, Table S3), H11P3 (three copies of
shortened attP) appeared somewhat more efficient than H11P
(one copy of the full-length attP), the efficiencies of site-specific
insertions into these two loci were statistically indistinguish-
able (SI Appendix, Table S3, compare rows 1 vs. 2; and Table 1,
compare rows 1 vs. 2, 6 vs. 7, and 8 vs. 9). In contrast, outcrossing
the H11P3 mice to the FVB strain for four generations (FVB N4)
significantly increased the integration efficiency to approximately
40% (Table 1, compare rows 2 vs. 3 and 9 vs. 10; and SI Appendix,
Table S3, compare rows 2 vs. 3). This efficiency is comparable to
or better than the efficiency of traditional transgenesis with
random integration. Circular DNAs with sizes from 3 to 6 kb
appeared to have similar efficiencies of integration (Table 1), but
larger DNA (14 kb) showed decreased integration efficiency
(∼3%; Table 1, row 11). The efficiency of cassette exchange by
using H11P3 is approximately 30%, but because identical attB
sites in the plasmid and identical attP sites in the genome were
used, cassette exchange could result in either integration of the
transgene of interest or the bacterial backbone. Therefore, only
half of the cassette-exchange insertions (∼16%) contained GFP
and the other half contained the plasmid backbone (Table 1,
Although we used circular DNA for injections, we also ob-
served insertions at locations other than our intended attP sites
(Table 1). In 20 of 23 founders that transmitted their site-specific
transgenes to the progeny, the site-specific integrants contained
a single-copy transgene and did not contain a second random
insertion as judged by PCR and quantitative PCR (SI Appendix,
SI Materials and Methods). In rare cases, when site-specific and
random integration occurred in the same transgenic founder, the
two distinct transgene integrations could be readily segregated in
the N1 progeny.
Site-Specific Integration into Rosa26. To test whether ϕC31-medi-
ated integration is applicable to other genomic loci, we injected
pattB-pCA-GFP into embryos homozygous for R26P3NVϕ
(attPx3+NVϕ integrated into the Rosa26 locus). We obtained
site-specific integrants (Table 1, row 12). We have removed the
NVϕ cassette from R26P3NVϕ by using Flpo, and have recently
created homozygous R26P3 mice to provide a second locus for
Here we describe an efficient method for producing transgenic
mice containing an intact, single-copy transgene integrated into
a predetermined locus via pronuclear injection. Our method is
considerably simpler than transgenesis using homologous recom-
bination in ES cells and offers many technical advantages com-
pared with the current method of random integration of trans-
genes via pronuclear injection (1–3). Transgenes produced from
number and chromosomal environment, and do not disrupt en-
dogenous genes (at least at the H11 locus). These properties will
facilitate many transgenesis-based experiments and will increase
their reliability and efficiency. For example, the relationships be-
carrying a single copy of pHb9-GFP
H11. (A) Schematic representation of
the generation of H11P3-pHb9-GFP al-
lele. After site-specific integration of
the plasmid pBT366 (SI Appendix, SI
Materials and Methods), the bacterial
backbone (BB) was removed by cross-
ing to the GFP-FLPo transgenic line (SI
Appendix, SI Materials and Methods).
The embryos that inherited only the
Hb9 allele but not the Flpo transgene
were tested for GFP expression. (B) GFP
expression in a whole-mount repre-
sentative embryonic day 11 embryo
containing the H11P3-pHb9-GFP allele.
A WT littermate is also shown (Right).
in blue. Insets: Magnified bottom left portions of each image containing Hb9-positive nuclei. (Scale bar, 100 μm.)
GFP expression in embryos
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| www.pnas.org/cgi/doi/10.1073/pnas.1019507108 Tasic et al.
tweenaminoacidsequencesordomainstructuresofa protein and
its in vivo biological functions can be more reliably compared if
a series of transgenes encoding different variants of a protein are
expressed at the same level. The regulatory elements that control
transgenes from the same integration site are compared. Subtle
be overwhelmed by positional effects and differences in copy
numbers in randomly integrated transgenes are more appropri-
ately compared by using site-specific integration of transgenes.
Recently, two other approaches for site-specific transgenesis
in mice using pronuclear injection were reported (39–41). One
approach relied on zinc-finger nucleases to create site-specific
double stranded breaks that were repaired by injected recombi-
nant DNA via homologous recombination (39, 41). The two
reports using this approach achieved transgene integration with
a frequency of approximately 2.5% (two events among 80 em-
bryos) into the Rosa26 locus (39), and approximately 5% (two of
yet to demonstrate germline transmission and proper adult ex-
for the same technique in rats was reported (41). The other ap-
proach used the Cre recombinase to catalyze cassette exchange in
the Rosa26 locus and a tissue-specific locus, H2-Tw3, at an aver-
age frequency of approximately 4.3%, with proper expression
and germline transmission of the transgenes (40). One drawback
of the Cre-based method is that it cannot be used to generate
Cre-activated transgenes (containing loxP-STOP-loxP), which are
frequently used for reporting Cre activity and perturbing gene
function in cells in which Cre is expressed. The integration fre-
quencies of these studies and the present study cannot be easily
compared, as the studies used different strains, loci, and con-
structs. However, our approach with the FVB strain and the H11
locus consistently produces higher integration efficiencies with
3- to 6-kb plasmids than either of the other two approaches.
The presentstudyalsorevealedthat plasmidbacterialbackbone
and a nearby transgenic cassette (NVϕ) have profound effects on
the expression reliability of the GFP transgenes driven by a ubiq-
uitous promoter. We did not observe any obvious change in HB9-
GFP transgene expression upon removal of the bacterial back-
bone; this observation could be the consequence of small numbers
backbone could have different effects on different promoters. The
effect of bacterial backbone has been reported for randomly in-
tegrated and episomal transgenes (34–37), and other native bac-
have been linked to variegation in transgenic animals (40, 42–44).
Our experiments based on site-specific integration enabled us to
systematically and quantitatively characterize these effects for
single-copy chromosomally integrated transgenes.
We have generated mice that allow integration at two defined
loci, the widely used Rosa26 locus (26) and the new Hipp11 locus
(27), which support high-level ubiquitous expression of integrated
transgenes. We describe three different approaches to create
transgenes devoid of the bacterial backbone: (i) use of minicircle
DNA for transgenesis, (ii) flanking the gene of interest with two
attB sites in a plasmid to enable cassette exchange, (iii) removing
the bacterial backbone from a transgene generated from a plas-
mid by crossing the transgenic mice to GFP-Flpo transgenic mice.
Although currently only half of the cassette exchange events are
desirable, the cassette exchange strategy removes the bacterial
backbone without the need to produce minicircle DNA or to
remove the plasmid backbone by subsequent crossing to GFP-
Flpo mice. Therefore, the cassette exchange may be the approach
of choice because of its combination of convenience and good
integration efficiency. A future improvement could use two mu-
tually noncompatible pairs of attB and attP sites to control for the
direction of insertion. In addition, to expand the application of
our method for producing transgenic mouse models, we are in
the process of introducing the attP sites into the frequently used
C57BL/6 genetic background. In summary, the present study fa-
cilitates murine transgenesis, highlights the requirements for
gene expression reliability in mammals, and provides an efficient
system for studies of gene expression and function in vivo.
Materials and Methods
Recombinant DNA. We used standard methods of recombinant DNA to
construct all plasmids used in this study. Construction details are described in
SI Appendix, SI Materials and Methods.
Gene Targeting in Mouse ES Cells. We used standard techniques to modify
mouse ES cells (45). SI Appendix, SI Materials and Methods, provides
Mouse Breeding and Maintenance. All experimental procedures were carried
out in accordance with the Administrative Panel on Laboratory Animal Care
protocol and the institutional guidelines by the Veterinary Service Center at
J females (stock no. 100006; Jackson Laboratories) were crossed to each other
intercrosses between homozygous animals. To outcross the mice to FVB
(Charles River), we started from a homozygous transgenic male and bred him
During the outcrossing, we preferentially selected transgenic mice of white
coat color. The fourth generation outcrossed mice (FVB N4) were crossed to
each other to make homozygous males and females that were subsequently
used to produce zygotes for microinjection. The FVB N4 homozygous line was
subsequently maintained by homozygous crosses. For testing transgenic
founders we crossed the founder (F0) animals to WT CD1 mice (Charles River)
to generate the N1 generation. For the N2 and N3 generations, we continued
crossing to CD1. We have generated homozygous mice from the founder E1
(SI Appendix, Table S2), and they were healthy and fertile.
Preparation of mRNA and DNA for Microinjection. Capped mRNAs for ϕC31o
and Flpo were generated by using a mMESSAGEmMACHINE in vitro tran-
scription kit (Ambion) according to the manufacturer’s instructions from
BamHI-digested pBT317 (SI Appendix, SI Materials and Methods) and BssHII-
digested pFlpo (23), respectively. The integrity of the RNA was assessed by
electrophoresis on a 1% agarose gel. Before loading on the gel, the RNA was
denatured by using the loading buffer provided in the Ambion kit according
to the manufacturer’s instructions.
Plasmid DNA was prepared using a modified Qiagen miniprep procedure
and was subsequently extracted with phenol/chloroform (SI Appendix, SI
Materials and Methods). The DNA was diluted to 6 ng/μL by sterile micro-
injection TE buffer (0.1 mM EDTA, 10 mM Tris, pH 7.5) and was kept at −80 °C
until the injection. The DNA was tested to be RNase-free by incubation with
an in vitro transcribed RNA at 37 °C for 1 h and then by analyzing the mix
on a 1% agarose gel. Before loading on the gel, the RNA was denatured as
described earlier. SI Appendix, SI Materials and Methods provides details on
preparation of minicircle DNA.
Microinjection for Generation of Site-Specific Integrants. Microinjection was
performed with an established setup at the Stanford Transgenic Facility.
Superovulated homozygous attP-containing females were bred to corre-
sponding males to generate homozygous attP-containing zygotes. A DNA/
mRNA mix of interest was microinjected into a single pronucleus and cyto-
plasm of each zygote by using a continuous flow injection mode. The sur-
viving zygotes were implanted into oviducts of pseudopregnant CD1
(Charles River) recipient mothers. All injection mixes contained 3 ng/μL DNA
and 48 ng/μL of in vitro transcribed ϕC31o mRNA in microinjection TE buffer
(0.1 mM EDTA, 10 mM Tris, pH 7.5). The injection mixes were prepared fresh
before each injection by mixing equal volumes of 6 ng/μL DNA solution and
96 ng/μL mRNA solution.
PCR. To test F0 animals for site-specific and random insertions, we performed
three PCRs: one for the 5′-end junction, one for the 3′-end junction, and one
internal to the transgene. These PCRs cannot detect random insertions that
occurred in mice with site-specific insertions. For that purpose, see PCR
analysis of N1 generation below. For testing integration into H11P or H11P3
alleles, we used PCR1, PCR2, and PCR6 (SI Appendix, SI Materials and
Methods). To test if a particular site-specific insertion into H11P or H11P3
originated from appropriate cassette exchange or minicircle insertion, we
used PCR3 and PCR4 or PCR4′ (SI Appendix, SI Materials and Methods). These
PCRs demonstrated that all injections of minicircle DNA produced only
Tasic et al.PNAS Early Edition
| 5 of 6
minicircle insertions, suggesting that contamination of minicircle preps with Download full-text
full-length plasmid was negligible.
To test germline transmission of both site-specific and random insertions to
N1 animals, we performed three PCRs on the N1 progeny: one for the 5′-end
of 100% between the GFP-specific and site-specific integration PCRs on DNA
from N1 animals suggested that the corresponding F0 founder most likely
contained only a single site-specific insertion. This conclusion was reinforced by
quantitative PCR (SI Appendix, SI Materials and Methods) for GFP to show that
a selected number of N1 animals indeed had a single-copy transgene.
SI Appendix, SI Materials and Methods (46, 47), provides additional in-
formation on PCR procedures used in this study, and SI Appendix, Table S4
provides primer sequences.
Tissue Preparation and Immunohistochemistry. The procedures were per-
formed essentially as described (29). SI Appendix, SI Materials and Methods,
includes further details.
Quantification of GFP Fluorescence in Liver Sections. At least three individual
images were taken from randomly chosen 10-μm sections for each liver by
a camera connected to a fluorescence microscope (Nikon) with a 20× ob-
jective. The regions of interest were consistently chosen to contain minimal
number of large blood vessels, so that the majority of every image would be
covered by hepatocytes. All images were taken with the same exposure time
(5 ms), same gain, and during two consecutive days of imaging. At this
condition, even the samples with brightest fluorescence had no saturated
pixels. Total fluorescence for each image was calculated by using ImageJ.
Averaged total fluorescence from all images of the same liver was plotted
on a graph (SI Appendix, Fig. S2). The fluorescence images shown in figures
represent the same fields that were used for the measurements, but exposed
four times longer for easier visualization.
Animal and Reagent Availability. Plasmids (containing attB sites or integrase
cDNA) and H11P3 and R26P3 homozygous frozen embryos and mice will be
distributed through Applied StemCell, Inc. (www.appliedstemcell.com), for
prices comparable to those of other distributors (e.g., Addgene for plasmids,
Jackson Labs for mice). Applied StemCell will also provide services for
making customized, integrase-mediated site-specific transgenic mice.
ACKNOWLEDGMENTS. We thank Yanfeng Li, Jennifer Lin, Hong Zeng, Ying
Jiang, and Carlota Manalac for technical support; Michele Calos for plasmids;
Russell Fernald for real-time PCR machine; Silvia Arber for anti-Hb9 anti-
body; Dritan Agalliu for help with embryo dissections; and Kazunari
Miyamichi and Tim Mosca for comments on the manuscript. This work is
supported by National Institutes of Health Grant R01-NS050835. B.T. was
a Damon Runyon Fellow and was supported by Damon Runyon Cancer
Research Foundation Grant DRG-1819-04. S.H. was supported by postdoc-
toral fellowships from the European Molecular Biology Organization (ALTF
851-2005), Human Frontier Science Program Organization (LT00805/2006-L),
and Swiss National Science Foundation (PA00P3_124160). L.L. is an in-
vestigator of The Howard Hughes Medical Institute.
1. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH (1980) Genetic
transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad
Sci USA 77:7380–7384.
2. Gordon JW, Ruddle FH (1981) Integration and stable germ line transmission of genes
injected into mouse pronuclei. Science 214:1244–1246.
3. Brinster RL, et al. (1981) Somatic expression of herpes thymidine kinase in mice
following injection of a fusion gene into eggs. Cell 27:223–231.
4. Milot E, et al. (1996) Heterochromatin effects on the frequency and duration of LCR-
mediated gene transcription. Cell 87:105–114.
5. Pedram M, et al. (2006) Telomere position effect and silencing of transgenes near
telomeres in the mouse. Mol Cell Biol 26:1865–1878.
6. Gao Q, et al. (2007) Telomeric transgenes are silenced in adult mouse tissues and embryo
fibroblasts but are expressed in embryonic stem cells. Stem Cells 25:3085–3092.
7. Williams A, et al. (2008) Position effect variegation and imprinting of transgenes in
lymphocytes. Nucleic Acids Res 36:2320–2329.
8. Garrick D, Fiering S, Martin DI, Whitelaw E (1998) Repeat-induced gene silencing in
mammals. Nat Genet 18:56–59.
9. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D (2002) Germline transmission and tissue-
specific expression of transgenes delivered by lentiviral vectors. Science 295:868–872.
10. Ding S, et al. (2005) Efficient transposition of the piggyBac (PB) transposon in
mammalian cells and mice. Cell 122:473–483.
11. Mátés L, et al. (2009) Molecular evolution of a novel hyperactive Sleeping Beauty
transposase enables robust stable gene transfer in vertebrates. Nat Genet 41:753–761.
12. Doetschman T, et al. (1987) Targetted correction of a mutant HPRT gene in mouse
embryonic stem cells. Nature 330:576–578.
13. Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in
mouse embryo-derived stem cells. Cell 51:503–512.
14. Groth AC, Olivares EC, Thyagarajan B, Calos MP (2000) A phage integrase directs
efficient site-specific integration in human cells. Proc Natl Acad Sci USA 97:5995–6000.
15. Keravala A, et al. (2006) A diversity of serine phage integrases mediate site-specific
recombination in mammalian cells. Mol Genet Genomics 276:135–146.
16. Thorpe HM, Smith MC (1998) In vitro site-specific integration of bacteriophage DNA
catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci USA
17. Groth AC, Fish M, Nusse R, Calos MP (2004) Construction of transgenic Drosophila by
using the site-specific integrase from phage phiC31. Genetics 166:1775–1782.
18. Venken KJ, He Y, Hoskins RA, Bellen HJ (2006) P[acman]: A BAC transgenic platform for
targeted insertion of large DNA fragments in D. melanogaster. Science 314:1747–1751.
19. Bischof J, Maeda RK, Hediger M, Karch F, Basler K (2007) An optimized transgenesis
system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci
20. Olivares EC, et al. (2002) Site-specific genomic integration produces therapeutic
Factor IX levels in mice. Nat Biotechnol 20:1124–1128.
21. Hollis RP, et al. (2003) Phage integrases for the construction and manipulation of
transgenic mammals. Reprod Biol Endocrinol 1:79.
22. Belteki G, Gertsenstein M, Ow DW, Nagy A (2003) Site-specific cassette exchange and
germline transmission with mouse ES cells expressing phiC31 integrase. Nat
23. Raymond CS, Soriano P (2007) High-efficiency FLP and PhiC31 site-specific recombination
in mammalian cells. PLoS ONE 2:e162.
24. Sangiorgi E, Shuhua Z, Capecchi MR (2008) In vivo evaluation of PhiC31 recombinase
activity using a self-excision cassette. Nucleic Acids Res 36:e134.
25. Capecchi MR (1989) Altering the genome by homologous recombination. Science 244:
26. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat
27. Hippenmeyer S, et al. (2010) Genetic mosaic dissection of Lis1 and Ndel1 in neuronal
migration. Neuron 68:695–709.
28. Zong H, Espinosa JS, Su HH, Muzumdar MD, Luo L (2005) Mosaic analysis with double
markers in mice. Cell 121:479–492.
29. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescent
Cre reporter mouse. Genesis 45:593–605.
30. Siemering KR, Golbik R, Sever R, Haseloff J (1996) Mutations that suppress the
thermosensitivity of green fluorescent protein. Curr Biol 6:1653–1663.
31. Okada A, Lansford R, Weimann JM, Fraser SE, McConnell SK (1999) Imaging cells in
the developing nervous system with retrovirus expressing modified green fluorescent
protein. Exp Neurol 156:394–406.
32. Gallardo T, Shirley L, John GB, Castrillon DH (2007) Generation of a germ cell-specific
mouse transgenic Cre line, Vasa-Cre. Genesis 45:413–417.
33. Seibler J, Bode J (1997) Double-reciprocal crossover mediated by FLP-recombinase:
A concept and an assay. Biochemistry 36:1740–1747.
34. Townes TM, Lingrel JB, Chen HY, Brinster RL, Palmiter RD (1985) Erythroid-specific
expression of human beta-globin genes in transgenic mice. EMBO J 4:1715–1723.
35. Chen ZY, He CY, Ehrhardt A, Kay MA (2003) Minicircle DNA vectors devoid of bacterial
DNA result in persistent and high-level transgene expression in vivo. Mol Ther 8:495–500.
36. Chen ZY, He CY, Meuse L, Kay MA (2004) Silencing of episomal transgene expression
by plasmid bacterial DNA elements in vivo. Gene Ther 11:856–864.
37. Suzuki M, Kasai K, Saeki Y (2006) Plasmid DNA sequences present in conventional
herpes simplex virus amplicon vectors cause rapid transgene silencing by forming
inactive chromatin. J Virol 80:3293–3300.
38. Arber S, et al. (1999) Requirement for the homeobox gene Hb9 in the consolidation of
motor neuron identity. Neuron 23:659–674.
39. Meyer M, de Angelis MH, Wurst W, Kühn R (2010) Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad
Sci USA 107:15022–15026.
40. Ohtsuka M, et al. (2010) Pronuclear injection-based mouse targeted transgenesis for
reproducible and highly efficient transgene expression. Nucleic Acids Res 38:e198.
41. Cui X, et al. (2011) Targeted integration in rat and mouse embryos with zinc-finger
nucleases. Nat Biotechnol 29:64–67.
42. Montoliu L, Chávez S, Vidal M (2000) Variegation associated with lacZ in transgenic
animals: a warning note. Transgenic Res 9:237–239.
43. Cohen-Tannoudji M, Babinet C, Morello D (2000) lacZ and ubiquitously expressed
genes: Should divorce be pronounced? Transgenic Res 9:233–235.
44. Fiering S, et al. (1995) Targeted deletion of 5’HS2 of the murine beta-globin LCR
reveals that it is not essential for proper regulation of the beta-globin locus. Genes
45. Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC (1993) Derivation of
completely cell culture-derived mice from early-passage embryonic stem cells. Proc
Natl Acad Sci USA 90:8424–8428.
46. Zhao S, Fernald RD (2005) Comprehensive algorithm for quantitative real-time
polymerase chain reaction. J Comput Biol 12:1047–1064.
47. Li L, et al. (2010) Visualizing the distribution of synapses from individual neurons in
the mouse brain. PLoS ONE 5:e11503.
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