Site-specific integrase-mediated transgenesis in mice via pronuclear injection
Bosiljka Tasic1, Simon Hippenmeyer1, Charlene Wang2, Hui Zong3, Yanru Chen-Tsai2, Liqun Luo1
1
HHMI, Department of Biology, Stanford University, Stanford, California, 94305
Transgenic Facility, Stanford Cancer Center, Stanford University School of Medicine, Stanford,
California, 94305
3
Institute of Molecular Biology, University of Oregon, Eugene, Oregon, 97403
2
Abstract:
Although microinjection of recombinant DNA into zygotic pronuclei is widely used for
producing transgenic mice, 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 injection, where an intact single-copy transgene can be inserted into
predetermined genetic loci with high efficiency (up to 40%). The site-specific integration of a
marker transgene driven by a ubiquitous promoter leads to global high-level marker
expression, and the transgene is faithfully transmitted across generations. Our analyses also
reveal that a neighboring tissue-specific promoter and bacterial DNA cause profound silencing
and expression variability of this transgene. The site-specific transgenesis method presented
here will enable precise structure-function dissection of gene function and regulation in vivo.
Production of transgenic mice via microinjection of DNA into zygotic pronuclei1-3 has served
mammalian genetics for 30 years. Although still the predominant method used to produce transgenic
mice, this method has several important limitations: the insertion site, integrity and copy number of
the transgene cannot be controlled. The insertion of DNA into different chromosomal loci at random
could disrupt the function of endogenous genes. Moreover, it subjects the transgenes to the local
chromatin environment (position effect) that can lead to transgene silencing or ectopic expression4-7.
An additional concern is that the transgenic DNA concatemerized into a large array is subject to
repeat-induced gene silencing8.
Single-copy transgenesis in mice can be achieved with retroviruses9 and transposons10,11, but these
approaches integrate transgenes throughout the genome. They also subject transgenes to the local
chromatin environment and can cause endogenous gene disruption, although depending on the
purpose of transgenesis, the mutagenic properties of transposons are sometimes desirable10. These
problems can be overcome by targeting the transgene to a specific chromosomal locus via
homologous recombination in embryonic stem (ES) cells12,13. 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 systems14,15 (+cite the fly transgenesis papers if there is space <51 references).
Integrases catalyze irreversible recombination between an appropriate attB and attP sites, one in
circular DNA and the other one in the genome14,16. C31 integrase has previously been used for
transgene integration in transgenic flies17-19. In mice, C31 integrase from a Streptomyces phage and
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its recognition sites (attB and attP) have been used for a number of applications. As an integrase, it
has been used to catalyze integration of circular DNA into pseudo-attP sites in the genome for genetherapy20, and for low-efficiency transgenesis21. Moreover, it has been used as a “recombinase”, for
example, in mouse ES cells for cassette exchange22, and in mice for removal of undesirable
transgene portions or reporter activation23,24.
Here, we describe a new C31 integrase-mediated highly efficient method for site-specific
transgenesis in mice via pronuclear microinjection, with integration efficiencies of up to 40%. The
system depends on a pre-targeted attP site into the mouse genome and injection of a DNA/RNA mix
into mouse zygotes (Fig. 1). In our system, C31 integrase catalyzes recombination between an attB
site from a circular recombinant DNA, and an attP site that we previously inserted into a specific
locus in the mouse genome. We also show that the expression from integrated transgenes is affected
by plasmid’s bacterial backbone (BB) and a nearby transgenic tissue-specific promoter, and that the
removal of these elements results in robust expression of the transgene from a ubiquitous promoter.
RESULTS
Integrase-mediated strategy for site-specific single-copy transgenesis
To generate embryos containing attP sites for C31 integrase-mediated transgenesis, we used
standard homologous recombination-based methods in mouse ES cells12,25. We inserted three
shortened tandem C31 integrase attP sites (attPx3) or a single “full length” attP site14 into two loci:
the Rosa26 (R26) locus on mouse chromosome 626 and an intergenic Hipp11 (H11) locus on mouse
chromosome 1127 (Fig. 1). The R26 locus supports global marker expression of a single copy
knockin transgene driven by a combination of the CMV enhancer and the chicken -actin promoter
(pCA)28,29. Knockin experiments confirmed that H11 supports high-level global marker expression
from the pCA promoter27 (SH, LL, unpublished data). Our knockin cassettes also contained a
mammalian codon-optimized C31 integrase (C31o)23 driven by a fragment of the mouse VASA
promoter sufficient for germline expression30, and a neomycin-resistance gene, flanked by FRT5
sites31 (Supplementary Fig. 1 online). This “VASA cassette” (abbreviated in the genotypes as NV)
was designed to provide the integrase in embryos in situ. 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 knockin cassettes.
We carried out a series of experiments to test integration of transgenes at the knocked-in attP sites.
First, we injected embryos homozygous for attPx3 and the VASA cassette knocked-in at the H11
locus (H11P3NV) with a circular plasmid, pattB-pCA-GFP. This plasmid contains a “full-length”
attB site14 and the sequence for a thermotolerant GFP32,33 driven by the ubiquitous pCA promoter
(Fig. 2a). We analyzed F0 embryos at embryonic day 10 or 11 (E10/E11) by PCR to detect newly
formed junctions due to site-specific insertions (Fig. 2a). We could not obtain any site-specific
integration (0/32 F0s; Table 1) despite occasional random integrations. We suspected that the VASA
promoter does not promote sufficient C31o expression to enable site-specific insertions. We then
co-injected the pattB-pCA-GFP plasmid with in vitro transcribed mRNA for C31o into
homozygous H11P3NV embryos. These experiments yielded site-specific integrations (Fig. 2a,b;
Table 1), indicating that site-specific integrase-mediated integration of transgenes can be achieved
with exogenously supplied C31o mRNA.
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Because the VASA cassette did not provide sufficient integrase activity in situ, we produced the
H11P3 allele, where the VASA cassette from H11P3NV had been excised by FLPo-mediated
recombination (Supplementary Fig. 1 online) (add the part from the methods to the legend). In a
second series of experiments, we co-injected pattB-pCA-GFP and C31o mRNA into homozygous
H11P3 embryos. These experiments also produced site-specific integrants (Fig. 2c, left and 2d, top;
Table 1). All site-specific founder pups derived from injecting pattB-pCA-GFP into H11P3NV or
H11P3 expressed GFP when examined as embryos (data not shown). Moreover, integrated
transgenes were properly transmitted from founders to progeny (Table 2). These data represent the
proof-of-principle for our site-specific integrase-mediated transgenesis in mice (Fig. 1).
Removal of bacterial backbone is necessary for proper transgene expression
Despite proper transmission of the site-specifically integrated transgenes, GFP expression levels in
the progeny of these transgenic founders exhibited a wide range in embryos (Fig. 2e) and adult tails
(data not shown). Moreover, the GFP expression in the progeny was mosaic in several internal
tissues including the heart, brain, and particularly the liver (Fig. 3a and Supplementary Fig. 2
online). We first observed this variable and mosaic expression with H11P3NV as the host. We
suspected that the nearby germline-specific VASA promoter could affect the expression of the pCAGFP introduced into H11 through integrase-mediated transgenesis. Indeed, pCA-GFP transgenes
produced in H11P3 host, where the VASA cassette had been removed, produced more uniform GFP
expression (Fig. 2e, first row vs. second row; Fig. 3a, first column vs. second column;
Supplementary Fig. 2 online). However, considerable variability in pCA-GFP transgene expression
still persisted especially in the livers (Fig. 3a) of F1 or F2 animals derived from a number of
transgenic founders (Supplementary Fig. 2 online).
It has been reported that plasmid bacterial backbone (BB) in extrachromosomal (episomal)
transgenes can silence the rest of the covalently linked DNA34,35 by recruitment of heterochromatic
factors36. To test if the bacterial DNA backbone that is part of the integrated transgene contributes to
the variability of GFP expression, we developed an in vitro system for producing minicircle DNA—
circular DNA containing desired transgene elements, but devoid of the BB (Supplementary Fig. 3
online). We injected the minicircle DNA into H11P3 embryos to produce transgenic animals (Fig.
2c, and 2d, middle; Table 1). For simplicity, hereafter, we designate mice derived from integration
of the entire pCA-GFP plasmid (which contains the BB) as pCA-GFP-BB (Fig. 1C1 left), and mice
derived from integration of the pCA-GFP minicircle as pCA-GFP (Fig. 1C1 right). Transgenic
animals derived from the pCA-GFP minicircle exhibited higher and much more uniform expression
in embryos (Fig. 2e, bottom row) and all tissues examined (Fig. 3a, third column; Supplementary
Fig. 2 online).
These data demonstrate that pCA-GFP at the H11 locus can express GFP ubiquitously in the absence
of the VASA cassette and the BB. Since the greatest variability was observed in the liver, we used it
as a model to determine the relative contributions of the VASA cassette and the BB to GFP
expression variability in these transgenic mice (Fig. 3a; Supplementary Fig. 2 online). In addition,
to test for the possible differences between single attP vs. attPx3, and to probe the effect of genetic
background, we also generated transgenic mice starting from H11P, or H11P3 mice that had been
outcrossed to the FVB strain for 4 generations. We compared total GFP fluorescence of liver
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sections from different transgenic animals under identical conditions. 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 strain for transgenesis (Supplementary Fig. 4
online). Therefore, we segregated all data only according to the presence of the VASA cassette and/or
the BB (Fig. 3b). We found that, in the presence of both the VASA cassette and the BB, GFP
expression was detectable in the liver in a small number of cells and at a very low level (Fig. 3a, top
left), but was statistically indistinguishable from negative controls when total fluorescence was
compared quantitatively (Fig. 3b, 2nd column versus 1st column). However in other organs analyzed
(heart and brain), GFP expression was apparent but mosaic (Fig. 3b; Supplementary Fig. 2 online).
When the VASA cassette was removed but the BB was still present, average GFP fluorescence
intensity became significantly higher (Fig. 3b, 3rd column versus 2nd column). Finally, when the BB
was removed, GFP fluorescence intensity was even higher than that from transgenes containing only
the BB (Fig. 3b, 4th column versus 3rd column). Thus, both the VASA cassette and the BB
significantly affect transgene expression. The reduction of total fluorescence intensity could be
caused by low level of expression in every cell, absence of expression in a subset of cells, or a
combination of the above. As is evident from Figures 3b and Supplementary Fig. 2 online, both
factors contribute to the reduced level of transgene expression in the presence of the VASA cassette
or the BB. In summary, the minicircle approach was essential for removing the remaining expression
variability.
To test whether C31-mediated integration is applicable to other genomic loci, we injected pattBpCA-GFP into embryos homozygous for R26P3NV (attPx3+NV integrated at the Rosa26 locus)
and obtained integrants (Table 1). We have removed the VASA cassette from R26P3NV using Flpo,
and have recently created homozygous R26P3 mice to provide a second locus for integrase-mediated
transgenesis.
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; see
Supplementary Table 2 for more details). Although on pooled data, 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 indistinguishable (Table 1
and Supplementary Table 3 online, Fisher’s exact test). In contrast, backcrossing the H11P3 mice
to the FVB strain for 4 generations (FVB N4) significantly increased the integration efficiency to
~40% (Table 1 and Supplementary Table 3 online, Fisher’s exact test). This efficiency is
comparable to or better than the efficiency of traditional transgenesis with random integration.
Circular DNAs with sizes from 3-6 kb appeared to have similar efficiencies of integration (Table 1).
Although we used circular DNA for injections, we also observed insertions at locations other than
our intended attP sites (Table 1) at a highly variable frequency (0-20%). In 20 out of 23 founders
that transmitted their site-specific transgene 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 (See Online Methods). In rare cases, when site-specific and random integration
occurred in the same transgenic founder, the two integrations could be readily segregated in the F1
progeny.
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DISCUSSION
Our method is considerably simpler than transgenesis using homologous recombination in ES cells
and offers many technical advantages compared to the most-widely used method of random
integration of transgenes via pronuclear injection1-3. Transgenes produced from our site-specific
integration method are intact, have a defined copy number and chromosomal environment, and do
not disrupt endogenous genes. These properties will increase the reliability of many transgenesisbased experiments and enable new ones. For example, the relationships between amino acid
sequences or domain structures of a protein and its in vivo biological functions can be more reliably
compared if a series of transgenes expressing different variants of a protein are expressed at the same
level. The regulatory elements that control gene expression can also be systematically dissected
when reporter transgenes from the same integration site are compared; subtle differences in levels or
patterns of transgene expression that would be overwhelmed by positional effects and differences in
copy numbers in randomly integrated transgenes can now be deciphered using site-specific
integration of transgenes. In this study, we have already generated mice that allow integration at two
defined loci, a widely used Rosa26 locus26 and a new Hipp11 locus27, which support high-level
ubiquitous expression of integrated transgenes. Future generation and characterization of additional
transgenes and integration sites should enable tissue-specificity and inducibility of transgene
expression.
Very recently, two other reports of site-specific transgenesis in mice using pronuclear injection were
published37,38. One uses zinc-finger nucleases to report initial success in integration in the Rosa26
locus at frequency ~2.5% (2 events out of 80 embryos), but does not report germline transmission of
the transgene37. The other report uses Cre to catalyze cassette-exchange in the Rosa26 locus and a
tissue-specific H2-Tw3 locus at an average frequency of ~4.3% 38. In comparison to our method, our
highest integration frequency is about an order of magnitude higher than the efficiencies in the other
two reports (the fact is we are comparing apples and oranges, their Rosa and our H11 in FVB, and if
we use our Rosa expt to compare, then the difference is not so dramatic; Also, it seems based on the
methods, that the Cre-based study uses heterozygous embryos for injection!). The zinc-finger
method permits its use in any mouse strain (should we say this, as this is really their biggest
advantage?), but requires further improvements in efficiency and proofs of germline transmission
and expression in F1 progeny. The Cre-based method, although it displays somewhat higher
efficiency than the zinc-finger method, cannot be used for the generation of Cre-reporters
(transgenes containing loxP-STOP-loxP, as this sequence would be deleted in the process of
integration). These types of transgenes are very desirable for reporting Cre activity or perturbing
gene function in cells in which Cre is expressed.
Our study also revealed that a neighboring tissue-specific promoter-driven transgene and plasmid’s
bacterial backbone have profound effects on the expression reliability of our GFP transgenes driven
by a ubiquitous promoter. This observation may preclude a practice that is standard in other model
organisms, where a marker driven by a tissue or cell specific promoter is co-integrated with a
transgene of interest together with a full plasmid backbone (standard practice for P-element and
integrase mediated transgenesis in Drosophila)(REF – Liqun, can you suggest a reference for this, if
we want to keep this sentence?). The effect of BB has been reported in episomal transgenes34-36, and
other native bacterial sequences like the lacZ gene have been suspected to cause variegation in
transgenic animals39,40. Very recently, the silencing by non-mammalian transgene elements in the
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Cre-catalyzed transgenesis has also been reported38. Our experiments based on site-specific
integration enabled us to unambiguously establish this phenomenon for single-copy chromosomally
integrated transgenes and to systematically and quantitatively characterize these effects. Our study
reveals intricacies of single-copy transgene expression in vivo, emphasizes the requirements for gene
expression reliability in mammals including gene therapy in humans, and provides an efficient
system for studying gene expression and function in vivo.
ACKNOWLEDGMENTS
We thank Yanfeng Li, Jennifer Lin, Hong Zeng, Ying Jiang and Carlota Manalac for technical
support, Michele Calos for plasmids, Russell Fernald for the use of the real-time PCR machine, and
Kazunari Miyamichi and Tim Mosca for comments on the manuscript. This work is supported by an
NIH grant (R01-NS050835). B.T. was a Damon Runyon Fellow supported by the Damon Runyon
Cancer Research Foundation (DRG-1819-04). S.H. was supported by postdoctoral 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 investigator of the Howard Hughes Medical Institute.
AUTHOR CONTRIBUTIONS
B.T., H.Z., Y.C.-T. and L.L. designed the experiments. B.T. carried out all the experiments except
mouse ES cell manipulation (done by Hong Zeng), pronuclear injections (done by C.W., Yanfeng Li
and Jennifer Lin) and immunohistochemistry on brain slices (done by H.S.). H.S. also provided
information and targeting construct for Hipp11 locus. B.T. and L.L. wrote the paper.
FIGURE LEGENDS
Figure 1 Schematic overview for site-specific C31 integrase-mediated transgenesis via pronuclear
injection in mice. Both H11 and R26 loci were modified with attP sites (note the different
orientation of attP sites for the two loci), but for simplicity, the scheme details the process for H11
only. After homologous recombination in mouse ES cells and germline transmission of the
transgene, homozygous lines were made to serve as donors of single-cell embryos. Each embryo was
injected into a single pronucleus with circular DNA and in vitro transcribed mRNA for C31. The
embryos that survived the injection were implanted into foster mothers to obtain transgenic animals.
Figure 2 Site-specific transgenesis – proof of principle. (a) Schematic of the recipient H11P3NV
locus and the modified H11P3-pCA-GFP-BB-NV locus obtained by C31-catalyzed site-specific
insertion of the attB-pCA-GFP plasmid into the first attP site. All three attP sites are suitable
recipients for the transgene and the site used in any particular case can be determined by PCR. (b)
PCR results confirming site-specific integration. The DNA template for each PCR panel was
obtained from the mice of the genotype designated below each gel. The red numbers correspond to
the PCR products designated on the schemes by red brackets and numbers in (a). The primer set #8
amplified a band smaller than expected due to the deletion of two attP sites during integration
(spade, see Online Methods). The wt H11 locus is also amplified by primer set #2 (asterisk, see (c)).
(c) Schematic of the recipient H11P3 locus, obtained by Flpo-mediated removal of the VASA
cassette (NV), and the two products obtained by C31-catalyzed site-specific insertion of the attBpCA-GFP plasmid (left) or the attB-pCA-GFP minicircle (right). The corresponding modified loci
are: H11P3-pCA-GFP-BB (left) and H11P3-pCA-GFP (right), respectively. (d) PCR results
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confirming site-specific integration, as explained in (b). (e) GFP expression in F2 mouse embryos at
the embryonic day 11 (E11). Each row shows representative embryos from a single pregnancy with
genotypes designated above. Images were obtained under identical conditions, except that “5x-exp”
designates five-fold longer exposure time than for the rest of the images. The insets in top right
corners represent the corresponding bright field images of each embryo. pSV40, SV40 promoter;
pVASA, VASA promoter; U, unique sequence; FRT5, a mutant version of FRT that is compatible
with itself but not with wt FRT; pCA, -actin promoter and CMV enhancer; G, GFP; pA, polyA
signal; BB, plasmid’s bacterial backbone; MC, minicircle; attB and attP, C31 attB and attP sites.
Figure 3 GFP expression in adult animals carrying site-specific pCA-GFP transgenes introduced by
C31 integrase-mediated transgenesis. (a) Representative fluorescence microscopy images from
liver, heart and cerebellum of F1 or F2 animals for the genotypes shown on top. The livers and
hearts are represented by epifluorescence images of 10 µm sections 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) and anti-calbindin (red) for Purkinje cells. (b) Average
fluorescence in the GFP channel for liver sections of the genotypes shown below (see Online
Methods). The number 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.
Mouse designations are numbers used to represent each mouse in Fig. S2. Statistical significance
was calculated with ANOVA and post-hoc pairwise Tukey’s test. ns, not significant; *, p<0.05; ***,
p<0.001. (have to figure out how to report exact P values, Nature Genetics asks for those and Prism
is not giving them for this test) Each set of data was represented by a mean  standard deviation. NT,
non-transgenic.
METHODS
Recombinant DNA. We used standard methods of recombinant DNA to construct all plasmids used
in this study. All PCR for DNA construction was done with Phusion DNA polymerase (Finnzymes,
Finland). All DNA fragments that were amplified by PCR were fully sequenced after cloning.
pBT296 (pBS-U-attP-FRT5-pSV40-Neo-pA-FRT5): Into a modified pBluescript, we subcloned: 1) a
unique sequence “U” from the promoter of yeast his3 gene:
(GGTGATAGGTGGCAAGTGGTATTCCGTAAGGATATC); 2) the single “full-length” attP site
from pTA-attP (gift of Michelle Calos)14. 3) FRT5
(GAAGTTCCTATTCCGAAGTTCCTATTCTTCAAAAGGTATAGGAACTTC) 31,41-flanked
neomycin resistance gene driven by an SV40 promoter.
pBT298 (pBS-U-attPx3-FRT5-pSV40-Neo-pA-FRT5): Same as pBT296, but the single attP site was
replaced by three sequential attP sites (70 bp each, sequence of a single site:
CGGGAGTAGTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGCGTAGGGTC
G) synthesized by Celtek Genes (Nashville, TN).
pBT305 (pBS-U-attPx3-FRT5-pSV40-Neo-pA-PL-FRT5): PL represents a polylinker SbfI-HpaIAatII. The plasmid was generated by inserting annealed oligos: PR402
(ctagCCTGCAGGaattaaGTTAACaattaaGACGTC) and PR403
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(ctagGACGTCttaattGTTAACttaattCCTGCAGG) into the XbaI site of pBT298 and thereby
destroying the XbaI sites on both ends of the PL.
pBT307 (pBS-U-attPx3-FRT5-pSV40-Neo-pA-C31o-pA-FRT5): C31o was amplified by PCR
from pPGKFC31obpA23 and subloned into pBT305.
pBT308b (pTOPO-pVasa): A previously described fragment of the VASA promoter 30 was
amplified by PCR from genomic DNA of the FVB strain and cloned into pTOPO.
pBT309a (pBS-U-attPx3-FRT5-pSV40-Neo-pA-pVasa-C31o-pA-FRT5): pVasa was subcloned from
pBT308b (pTOPO-pVasa) into pBT307.
pBT310 (pBS-U-attP-FRT5-pSV40-Neo-pA-pVasa-C31o-pA-FRT5): NheI/AscI fragment from
pBT309a was subcloned into NheI/AscI digested pBT296.
pBT311 (pH11-U-attPx3-FRT5-pSV40-Neo-pA-pVasa-C31o-pA-FRT5): Generated from
PmeI/AscI-digested pHIPP1127 and SwaI/AscI-digested pBT309a.
pBT312 (pH11-U-attP-FRT5-pSV40-Neo-pA-pVasa-C31o-pA-FRT5): Generated from PmeI/AscIdigested pHIPP1127 and SwaI/AscI-digested pBT310.
pBT313 (pR26-U-attPx3-FRT5-pSV40-Neo-pA-pVasa-C31o-pA-FRT5): SwaI/AscI-digested insert
from pBT309a was subcloned into SwaI/AscI-digested pROSA2642.
pBT314 (pR26-U-attP-FRT5-pSV40-Neo-pA-pVasa-C31o-pA-FRT5): SwaI/AscI-digested insert
from pBT310 was subcloned into SwaI/AscI-digested pROSA2642.
pBT316 (pattB-pCA-GFP): A “full-length” attB site was cloned from pTA-attB14 as SalI fragment
into the SalI site of pBT255 (pCA-GFP4m-pA).
pBT317 (pETC31opA): C31o gene was amplified by PCR from pPGKC31obpA23 using primers
PR437 (AACCAACCttaaCCGCCACCATGGATACCTAC) and PR438
(AATAggatccTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTctcgagTCACACTTTCCGCTTTTTCTT
AGG). The PCR was digested with BamHI and MseI and cloned into BamHI/NdeI-digested
pET11C31pA21.
pBT344 (pattB-pCA-GFP-FRT5): A single FRT5 site was inserted downstream of SV40 polyA
signal.
pBT346 (p-attB-pCA-GFP): I-SceI restriction site and the  attL1 were amplified from pENTRTopoD using PR493
(aaagaGGTACCagttacgctagggataacagggtaatatagCAAATAATGATTTTATTTTGACTGATAG) and
PR494 (aaataCTCGAGagcctGCTTTTTTGTACAAAGTTG). The PCR product was digested with
Acc65I and XhoI and inserted into the Acc65I/XhoI-digested pBT316. Subsequently, the  attR1 site
was amplified from pENTR-TopoD using PR495
(aagaaGCGGCCGCacaagtttgtacaaaaaagcTGAACG) and PR496
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(AAGAAgagctcCATAGTGACTGGATATGTTGTGTTTTA) and cloned into the SacI/NotIdigested construction intermediate to generate pBT346.
Gene targeting in mouse ES cells. We used standard techniques to modify R1 mouse ES cells 43.
Individual G418-resistant clones were evaluated for homologous recombination by long-range PCR,
using LA Taq (Takara Bio) and the following primers for H11 5’ arm: PR374
(atgtgaggcaggagatgagagaggaatgactggtcac) and PR432
(GATATCCTTACGGAATACCACTTGCCACCTATCACC); H11 3’ arm: PR351
(aataaGCTAGCctcgagGATATCctgtgccttctagttgccag) and PR422 (ccattttttagtacccctctacactcctcc);
R26 5’ arm: Rosa3 (ccactgaccgcacggggattc) and PR432 (see above), and R26 3’ arm: PR351 (see
above) and PR395 (gttgagggcaatctgggaaggt). The clones containing correctly recombined targeting
vectors were used to generate mouse chimeras by injection into C57BL/6J blastocysts. The chimeras
were crossed to B6D2 F1 females (F1 females from a cross between C57BL/6J and DBA2/J mice;
Stock# 100006, Jackson Lab). Agouti progeny were genotyped for the presence of the knockin allele
using the following primers for H11: (PCR1+2 in Fig. 1): SH176 (tggaggaggacaaactggtcac), SH178
(ttccctttctgcttcatcttgc) and PR432 (see above). The expected sizes are: 147 bp for the knockin and
321 bp for wt. For R26 the primers used were: Rosa10 (CTCTGCTGCCTCCTGGCTTCT), Rosa11
(cgaggcggatcacaagcaata), and PR432 (see above). Expected sizes are: 168 bp for the knockin and
330 bp for wt.
Mouse maintenance and breeding. All experimental procedures were carried out in accordance
with the APLAC (Administrative Panel on Laboratory Animal Care) protocol and the institutional
guidelines by the Veterinary Service Center (VSC) at Stanford University.
The F1 attP-knockin animals obtained from the cross of chimeras to B6D2 F1 females were
crossed to each other to establish homozygous knockin mouse lines. These lines were maintained by
intercrosses between homozygous animals. To outcross the mice to FVB (Charles River), we started
from a homozygous male and crossed him and his transgenic male progeny to FVB females, for a
total of 4 generations. During the outcrossing, we preferentially selected transgenic mice of white
coat color. The 4th generation outcrossed mice were crossed to each other to make homozygous
males and females that will be subsequently used to produce zygotes for microinjection. The FVB
N4 homozygous line was subsequently maintained by homozygous crosses. For testing transgenic
founders we crossed F0 animals to wild-type CD1 mice (Charles River). For F2 and F3 generation,
we continued crossing to CD1.
Preparation of DNA and mRNA for microinjection. Plasmid DNA was prepared using Qiagen
mini-preps and was subsequently extracted twice with a phenol:chloroform (50:50) mix and twice
with chloroform only. The DNA was precipitated with 1/10 volume of 3M sodium-acetate pH 5.2
and 2.7 volumes of ethanol, and subsequently dissolved in microinjection TE buffer (miTE; 0.1 mM
EDTA, 10 mM Tris pH 7.5). The DNA was filtered through a sterile 0.2 µm filter and the
concentration was determined using Nanodrop spectrophotometer (Thermo Scientific). The DNA
was diluted to 6 ng/µl by sterile miTE and was kept at -80ºC until the injection. The DNA was tested
to be RNase-free by incubation with an in vitro transcribed RNA at 37ºC for 1h and then by running
the mix on a 1% agarose gel. Prior to loading on the gel, the RNA was denatured as described below
for the analysis of in vitro transcribed RNA.
Capped mRNA for PhiC31o and Flpo was generated using mMESSAGEmMACHINE in
vitro transcription kit from Ambion according to the manufacturer’s instructions from pBT317 and
Tasic et al.
10
pFlpo23, respectively. The integrity of the RNA was assessed by electrophoresis on a 1% agarose gel.
Prior to loading on the gel, the RNA was denatured using the loading buffer provided in the Ambion
kit according to the manufacturer’s instructions.
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 crossed to corresponding males to generate homozygous attP-containing zygotes. A
DNA/mRNA mix of interest was microinjected into a single pronucleus and cytoplasm of each
zygote using a continuous flow injection mode. The surviving zygotes were implanted into oviducts
of pseudo-pregnant CD1 (Charles River) recipient mothers. All injection mixes contained 3 ng/µl
DNA and 48 ng/µl of in vitro transcribed C31o mRNA in microinjection TE buffer (miTE; 0.1 mM
EDTA, 10 mM Tris pH 7.5). The injection mixes were prepared fresh before each injection by
mixing the equal volumes of 6 ng/µl DNA solution and 96 ng/µl mRNA solution.
Microinjection of Flpo mRNA for generation of Flp-out alleles. To remove the VASA cassette,
which is flanked by FRT5 sites31,41, we injected the capped in vitro transcribed Flpo mRNA obtained
from pFlpo23 into the cytoplasm of attP-homozygous embryos. Microinjection was performed as
decribed in the section above. The average efficiency of Flp-out was 25.6% (32 out of 125) for
H11P(3)NV and 7.4% (6 out of 81) for R26P(3)NV. None of the animals obtained were
homozygous Flp-outs (n=206) based on the PCR that can detect both the Flp-out and non-Flp-out
alleles (Figure 1). We mated the animals containing the same Flp-out allele to each other to create
homozygous Flp-out mouse lines.
PCR. To test if F1 animals from any particular founder contained both a site-specific insertion and a
random insertion, we performed three PCRs on the progeny:
(PCR3 in Fig. 1): PR425 (ggtgataggtggcaagtggtattc) and PR436 (atcaactaccgccacctcgac). Expected
sizes are: 147 bp, 217 bp, 287 bp, and 244 bp, for insertion into the first attP; second attP, third attP,
or full-length attP, respectively.
(PCR4 in Fig. 1, for H11P(3) alleles): PR522 (CGATGTAGGTCACGGTCTCG) and PR387
(gtgggactgctttttccaga). Expected sizes are: 371 bp, 301 bp, 231 bp, and 313 bp, for insertion into the
first attP; second attP, third attP, or full-length attP, respectively.
(PCR8 in Fig. 1, for H11P3NV alleles): PR522 (see above) and PR428 (ccgaaaagtgccacctgaataat).
Expected sizes are: 178 bp, 248 bp, 318 bp, for insertion into the first attP; second attP, third attP,
respectively.
(PCR7 in Fig. 1): FACS G5' (CTTCAAGTCCGCCATGCCCGA) and GFP2-Hermie
(TCCAGCAGGACCATGTGATCGC). Expected size: 420 bp.
100% correlation between GFP-specific and site-specific integration PCR on F1 animals
suggested that the corresponding F0 founder most likely contained only a single site-specific
insertion. This conclusion was reinforced by quantitative PCR (see below) for GFP to show that a
selected number of F1 animals indeed had a single-copy transgene. In most of the cases (20 out of 28
founders), C31 catalyzed precise recombination between attP and attB. In 6 cases, integration
appeared to occur at two different attP sites, or it caused the deletion of one or more attP sites (for
example, see Fig. B2). These imprecise events occurred only when transgenesis was performed in
animals with three tandem attP sites.
To show that a particular insertion into H11P(3) originated from either a plasmid or a
minicircle we performed the following PCRs:
Tasic et al.
11
(PCR5 in Fig. 1): PR21 (ctgcaaggcgattaagttgg) and PR387 (gtgggactgctttttccaga). Expected sizes
are 498 bp, 428 bp, 358 bp, and 440 bp, for insertion into the first attP; second attP, third attP, or
full-length attP, respectively.
(PCR6 in Fig. 1): PR487 (TCCCCCTGAACCTGAAACAT) and PR387 (see above). Expected
sizes are 612 bp, 542 bp, 472 bp, and 554 bp, for insertion into the first attP; second attP, third attP,
or full-length attP, respectively.
Immunohistochemistry. Tissues were obtained from postnatal day 21 ( 2 days) mice that were
transcardially perfused with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). The
tissues were post-fixed overnight in 4% PFA, washed once with PBS and then cryoprotected in 30%
sucrose overnight. The tissues were embedded in OCT and stored at -80ºC before cryosectioning.
All tissue sectioning was performed using a Leica cryostat. The livers and hearts were sectioned
coronally to obtain 10 µm-thick sections, washed in PBS, stained with DAPI and mounted with
Fluorogel. The livers and hearts were imaged with a fluorescence microscope (Nikon). The brains
were sectioned sagittally to obtain 30 µm-thick sections, the sections were washed 3 times in PBS
and incubated overnight at 4ºC with chicken anti-GFP antibody (Aves Labs) and monoclonal mouse
anti-calbindin antibody (Sigma). Following incubation with fluorophore-conjugated secondary
antibodies (Jackson ImmunoResearch), the slides were mounted in Fluorogel and imaged with a
Zeiss confocal microscope.
Quantification of GFP fluorescence in liver sections. At least 3 individual images were taken from
randomly chosen 10 µm-sections for each liver, by a camera connected to a fluorescence microscope
(Nikon) with a 20x objective. 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 (8) and during two continuous
days of imaging. At this condition, even the samples with brightest fluorescence had no saturated
pixels. Total fluorescence for each image was calculated using ImageJ. We averaged total
fluorescence from all images of the same liver and plotted it on the graph in Fig. S2.
Quantitative PCR. Quantitative PCR was performed as previously described 44,45. Each sample was
tested in triplicate both for GFP and for the internal control. The primers used for GFP were: LL84
(AAGTCGTGCTGCTTCATGTG), and LL85 (ACGTAAACGGCCACAAGTT), and they generate
a product of 187 bp. The internal control primers were: IMR0015 (CAAATGTTGCTTGTCTGGTG)
and IMR0016 (GTCAGTCGAGTGCACAGTTT), and they generate a product of 200 bp.
Tasic et al.
12
SS
F0
(n)
SS%
(from
F0)
R
F0
(n)
R%
(from
F0)
DNA
DNA type
DNA
size
(kb)
Strain
Background
F0
(n)
pattB-pCA-GFP, no RNA
plasmid
~6
H11P3NV
mix
32b
0
0.0
5
15.6
pattB-pCA-GFP
plasmid
~6
H11PNV
mix
30b
2
6.7
0
0.0
pattB-pCA-GFP
plasmid
~6
H11P3NV
mix
64c
10
15.6
4
6.3
pattB-pCA-GFP
plasmid
~6
R26P3NV
mix
22b
2
9.1
2
9.1
pattB-pCA-GFP-FRT5
plasmid
~6
H11P
mix
51
5f
9.8
3
5.9
pattB-pCA-GFP-(FRT5)a
plasmid
~6
H11P3
mix
61
4f
6.6
9
14.8
37.5
1
10.3
4.8
plasmid
~6
H11P3
FVB N4
8
3f
attB-pCA-GFP (MC)
minicircle
~3
H11P
mix
21
1
attB-pCA-GFP (MC)
minicircle
~3
H11P3
mix
39
4
attB-pCA-GFP (MC)
minicircle
~3
H11P3
FVB N4
15
6
pattB-pCA-GFP-FRT5
Significant?d
} ns
} ns
}*
} ns
}*
4.8
1
10.3
1
2.6
40.0
3
20.0
Abbreviations: SS, site-specific integration; R, random integration; mix, mixed background of 129, C57BL/6 and DBA2;
FVB N4, mice of the mixed background were backcrossed for 4 generations to the FVB strain and then intercrossed.
a
Both FRT and non-FRT versons of pattB-pCA-GFP were used.
b
F0s were analyzed only as E10 or E11 embryos.
c
F0s were analyzed either as E10 or E11 embryos or as live pups.
d
Statistical significance was evaluated using Fisher’s exact test. ns, not-siginificant; *, p<0.05.
Table 1. Efficiency of site-specific integration.
Tasic et al.
Transgene
H11P3pCA-GFPBB-NV
H11P3pCA-GFPBB
H11PpCA-GFPBB
H11P3pCA-GFP
13
Founder
Background
Sex
Insertion
into attP
1, 2 or 3
A1
A2
A3
A4
B1
B2
B3
B4
B5
B6
B7
C1
C2
C3
C4
C5
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
mix
mix
mix
mix
mix
mix
mix
mix
FVB N4
FVB N4
FVB N4
mix
mix
mix
mix
mix
mix
mix
mix
mix
FVB N4
FVB N4
FVB N4
FVB N4
FVB N4
FVB N4
M
M
M
M
M
M
Fem
Fem
M
Fem
Fem
Fem
M
Fem
Fem
M
Fem
M
Fem
M
Fem
Fem
M
Fem
M
Fem
1
3
1
3
3
2/3b
2
2
2
2
2/3b
n/a
n/a
n/a
n/a
n/a
1
3
3
3
3
3
3
3
1
2
GT of
SS
(Y/N)a
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
N
Y
Y
Y
Y
N
Y
Y
N
Y
N
Y
F1
(n)
F1
SS
(n)
F1 SS
(%)c
F2/3
(n)
F2/3
SS
(n)
F2/3d
SS
(%)
55
62
36
63
143
29
44
20
24
36
12
10
29
19
11
9
4
9
65.5
19.4***
27.8*
46.0
13.3***
37.9
20.5***
20.0*
37.5
25
16
15
10
60.0
62.5
14
48
85
76
4
20
36
34
28.6
41.7
42.4
44.7
8
15
44
9
1
67
20
33
23
59
32
41
3
11
9
4
0
14
8
10
5
0
6
10
37.5
73.3
20.5***
44.4
0
20.9***
40.0
30.3
21.7*
0***
18.8**
24.4**
14
2
14.3*
R
(Y)
Y
Y
Y
62
25
12
29
15
4
46.8
60.0
33.3
F1
R
(n)
F1
R
(%)
0
0
0
0
11
0
0
0
0
0
0
0
0
7.7
0
0
0
0
3
0
0
2
0
0
0
0
0
0
0
0
37.5
0
0
22.2
0
0
0
0
0
0
0
0
0
0
27
8
29.6
20
10
50.0
0
0
H11PpCA-GFP
E1
mix
M
n/a
Y
39
11 28.2*
0
0
Abbreviations: SS, site-specific integration; R, random integration; GT, germline transmission; mix, mixed background of 129,
C57BL/6 and DBA2; FVB N4, the mice of the mixed background were for backcrossed for 4 generations to the FVB strain and then
intercrossed.
a
The founders that did not transmit were ether sterile (D7 and D9), died upon delivery (C4) or cannibalized the pups (B6).
b
Based on site-specific PCR, both insertions into the second and the third site occurred.
c
Frequency of transmission for site-specific integrations for some founders is sub-Mendelian, suggesting mosaicism in the founders
(Fisher’s exact test, *, p<0.05; **, p<0.01; ***, p<0.001)
d
Frequency of transmission for site-specific integrations for subsequent generations (F2 and/or F3) is statistically indistinguishable
from Mendelian transmission (Fisher’s exact test).
Table 2. A complete list of transgenic founders (n=28) and their germline transmission efficiency.
Tasic et al.
14
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