BACKGROUND
transgenic  mice have been extensively used for in vivo analysis of gene function and generation of human disease models.
since generation of the first tg mice was reported by gordon et al.
in  <dig>  who performed pronuclear injection  of purified dna into zygotes, pi-mediated transgenesis has been the most common method for more than 30 years  <cit> .
in general, for obtaining several tg mouse lines harboring a specific transgene, it typically requires approximately  <dig> or more eggs.
unfortunately, the pi-mediated transgenesis is often associated with wide variability in the level and pattern of transgene expression, which causes phenotypic variability among individual tg lines, probably due to the random nature of copy number, configuration, and insertion site of the transgene.
due to such variability in transgene expression, researchers need to extensively screen various lines and choose the most suitable tg mouse line showing desired transgene expression, which is a very laborious and time-consuming task  <cit> .
to overcome the pitfalls associated with random transgenesis, single-copy insertion of a transgene into a predetermined genomic locus can be accomplished using embryonic stem  cell-mediated gene targeting, which is the gold-standard for generation of targeted tg mice  <cit> .
however, this approach is laborious, expensive, and time-consuming when compared to pi-based transgenesis.
strategies using recombinase- or integrase-based targeted insertion through pi have been recently described by us and others  <cit> .
we termed this technology as “pronuclear injection-based targeted transgenesis .
the pitt system requires a ‘seed mice strain’ containing a suitable landing pad at a predetermined genomic locus as an embryo donor for pi.
the method requires, in its initial stage, to establish the seed mouse by targeted insertion of the landing pad via conventional es cell-based gene targeting approach.
such an established mouse line serves as a “seed” strain, from which tg mice harboring the dna of interest  are generated by injection of donor vector into zygotes along with dna/mrna for recombinase or integrase.
for performing targeted insertion of doi using a seed strain, we and another group used cre-loxp recombination system  <cit> , while a third group used phic <dig> integrase system  <cit> .
despite the advantages of pitt approach compared to traditional methods, its full potential is not discovered yet, because the available methods are only single-enzyme based pitt versions.
we hypothesized that further improving of the pitt by combining multiple recombinase-integrase systems, the method can be made more efficient, versatile and capable of multiplexing.
specifically, we anticipated that such a strategy should make the pitt method able to rapidly generate multiple different transgenic lines simultaneously, that are targeted precisely to the same genomic location.
this would ultimately facilitate many areas of biomedical research that depend on rapid generation of multiple, reliable transgenic mice since it would address the major constraints such as time, costs and variable efficiencies associated with the use of es cell-based targeted transgenesis approach while taking care of several pitfalls associated with random transgenesis approaches.
therefore, in this report we further improved the technique by creating the next generation seed mouse with a combination of elements including mutant loxp, mutant frt, and attp sites at the rosa <dig> locus.
we termed the new seed mouse as tokmo- <dig>  and the method as improved-pitt .
this system adds many new features and capabilities to the previously described pitt, and to the targeted transgenesis approaches in general.
these features are: 1) the i-pitt method enables the use of any of the three insertion systems, viz.
cre, phic <dig> or flp, or combinations, for inserting the tg cassettes.
2) simultaneous use of two systems, for e.g., cre-loxp-mediated recombination and phic <dig> integration, significantly enhances the targeted insertion efficiency.
3) many tg doi constructs can be co-injected to produce tg lines for each of the constructs injected.
4) the multiplexing capability allows the generation of transgenic lines with  <dig> zygotes per donor tg construct or less .
5) the new seed mouse is under the c57bl/6n genetic background, which is widely used as a standard strain by the scientific community, including the international knockout mouse consortium   <cit> .
recently there have been remarkable advances in knockout technology using crispr/cas <dig> system in which multiple gene knockouts can be made in a single microinjection session.
the i-pitt method described here offers the advantage of creating multiple tg models in a single experimental session and thus serves as an analogous tool for multiplexing in tg technology similar to crispr/cas <dig> tool for ko technology.
RESULTS
i-pitt strategy
the overall scheme of i-pitt is illustrated in figure  <dig>  a new pitt seed mouse  under c57bl/6n genetic background was engineered by inserting a landing pad containing loxp derivatives , attp, and frt derivatives  into the rosa <dig> locus through es cell-based targeting.
a donor vector carrying tdtomato-pa, loxp derivatives , attb, and frt derivatives  was designed to perform site-specific insertion of a transgene .
the transgene flanked by the f <dig> and f <dig> sites in the donor vector was inserted into target locus of fertilized eggs via i-pitt  that offers options for any of the three enzymes cre, phic <dig> or flp to choose for targeted insertion.
the resulting embryos will exhibit “targeted insertion with extra sequence ” allele.
the strategy is designed in such way that the extra sequences will be flanked by f <dig> and f <dig> elements which enable the removal of extra sequences by breeding with flp mouse.
this results in cleanly inserted transgene flanked by only the f <dig> and f <dig> sites .
although the structures of tiex allele differ among the site-specific insertion systems employed , the structure of final tiΔex alleles generated will be identical in all the strategies.
combination of mutant loxps  was same as in our original pitt system  <cit> .
among the multitude of mutant frt sites reported previously, we chose f <dig> and f <dig>  and frt-l/r , since f14/f <dig> pairing was known to be incompatible  <cit>  and thus we anticipated that combination of the spacer variants and the inverted repeat variant would show high recombination efficiency similar to jt15/jtz <dig> and lox <dig> combinations for the cre-loxp system  <cit> .
the targeted allele also contains a 43-bp fragment corresponding to human oct <dig> gene sequence  that can serve as a zinc finger nuclease target to aid in insertion of additional sequences for future needs, but the component is not tested yet.figure  <dig>
schematic of pitt-mediated targeted insertion using the new seed mice, tokom- <dig>  cre-pitt  and phic31-pitt  confer insertion of the donor vector containing.
dna of interest , into a target site in the genome, to generate tiex allele  <dig> or  <dig> respectively.
doi shown in this example is tdtomato-poly cassette in this figure.
introduction of flp removes extra sequence in the tiex allele  <dig> and  <dig>  resulting in generation of tiΔex allele.
see additional file 1: figures s <dig>  s <dig> and s <dig> for other possible outcomes recombination.
gtnop: a cassette containing “egfp-t2a-neomycin resistant gene-hoct4-polya”.
verification of landing pad insertion in es cells
to confirm that three kinds of landing pads work in the genomic context, we first established an es cell clone  that contains mutant loxp, mutant frt and attp sites at the rosa <dig> locus.
the #bdu <dig> was then transfected with a pbdr donor vector carrying a promoter-less tdtomato gene, the recombinase  and/or integrase  expression vectors.
the tdtomato expression will be driven by endogenous rosa <dig> promoter only if a correct insertion of the cassette at the target locus occurs.
the red fluorescence in successfully targeted cells was detected by facs analysis.
two days after transfection, approximately 1% of cells were positive for tdtomato fluorescence when icre- or phic31o-expression vector  was used , but when flpo-expression vector was used, only a few cells showed tdtomato fluorescence .
notably, the percentage of fluorescent cells increased when any two expression vectors  were co-transfected .
we then tested if inclusion of six copies of tandem repeats of recombination/integration sites in the donor vector would increase the insertion efficiency  and found that it was slightly improved  in the cre-loxp-based system, when 6xjtz <dig> was included in a donor vector.
no obvious improvement was noticed in the phic31-based system .figure  <dig>
targeted insertion in es cells.
introduction of donor vector  harboring promoter-less tdtomato gene into targeted es cells  with each expression vector for icre recombinase, flpo recombinase or phic <dig> integrase.
red fluorescent es cells, as analyzed by facs, are regarded as successfully targeted tdtomato cassette inserted cells.
co-introduction of combinations of recombinases or integrase expression vectors increases transgene insertion efficiency.
these results indicated that 1) the landing pad architecture we designed worked as expected, and 2) the cre-based and phic31-based gene insertion systems showed comparable targeted insertion efficiencies, while overall efficiency of the flp-based system was relatively poor compared to phic31- and cre-based systems, 3) the combinatorial use of two insertion systems  increased the targeted insertion efficiency, and 4) tandem repeats of recombinase recognition sites in the donor vectors enhanced cre-loxp-mediated targeted insertion of doi.
validation of i-pitt
after confirming that desired dna cassettes can be successfully inserted into the landing pads by using recombinases and/or integrase in es cells, a novel seed mouse line was established using c57bl/6n-derived es cell clones harboring the targeted allele.
similar to our previous seed mouse , both homozygotes and heterozygotes of the new seed mouse  were viable with normal reproductive ability.
to test phic31-based pitt on the new seed mouse tokmo- <dig>  we injected various concentrations of phic31o mrna along with 10 ng/μl of pber donor vector  into fertilized eggs that were generated by in vitro fertilization  of c57bl/6n eggs with the epididymal spermatozoa isolated from a male homozygous tokmo- <dig> mouse.
based on the results  presented in additional file 1: table s <dig>  we decided to use  <dig>  or 15 ng/μl of phic31o mrna in subsequent experiments.
these concentrations are relatively higher compared to icre mrna concentrations  that is typically needed for pitt  <cit> .
the pitt efficiency for cre- or phic31-based systems was assessed by injecting zygotes, culturing them until blastocysts, and subsequent analysis for targeted insertion .
targeted insertion was detected in  <dig> % or  <dig> % of developed blastocysts  when icre mrna or phic31o mrna were injected .
interestingly, co-injection of icre and phic31o mrnas resulted in increased insertion efficiency of up to  <dig> % of developed blastocysts  .
notably, insertion rate as high as  <dig> % among developed blastocysts was achieved in a single experiment .
based on these results, we decided to follow a co-injection strategy using icre mrna and phic31o mrna at concentrations  <dig>  ng/μl and  <dig>  ng/μl respectively in subsequent experiments.figure  <dig>
pitt using tokom- <dig> seed mice.
schematic diagram of the pitt injection experiment.
donor vector containing doi is injected into the fertilized eggs harboring landing pad together with icre and/or phic31o mrna.
successful targeted transgenesis in blastocysts derived from zygotes injected with pber + icre mrna + phic31o mrna.
successful targeted transgenesis in day  <dig>  fetuses.
zygotes/fetuses exhibiting red fluorescence indicate successful targeted insertion of doi.
the pitt results in blastocysts  and fetuses/pups .
we next examined whether co-injection of icre and phic31o mrnas into zygotes leads to production of targeted tg fetuses or newborns .
two different donor vectors were included in the injection, pber and pbgo.
the targeted insertion efficiencies were  <dig> %  and  <dig> % , and  <dig> % and  <dig> % for total injected zygotes, respectively .
these results indicate that 1) both phic <dig> and icre-based targeted insertions were effective in using the new seed mouse tokmo- <dig>  and that 2) combinatorial use of the two systems enhanced the in vivo targeted insertion efficiency.
simultaneous production of multiple different targeted tg mouse lines using i-pitt
given the ability of the original pitt system to insert a single copy transgene through specially designed donor vectors, we hypothesized that when a mixture of multiple donor vectors is injected into zygotes, the resulting independent tg founder mouse lines should contain only one type of transgene from the mixture.
if this occurs, more than two types of tg lines can be produced at once in a single injection event, which offers versatility to the system and also would reduce the cost significantly, compared to the traditional tg production method.
as a preliminary test, we first assessed this possibility by injecting a mixture of donor vectors pbgv, pbgw and pbdr that confer expression of mcfp, mcitrine and tdtomato respectively, when they are correctly inserted.
the injection mix containing the three donor vectors along with icre and phic31o mrnas was microinjected into the fertilized eggs harboring hemizygous targeted allele .
the injected eggs were cultured up to blastocyst stage and observed for transgene expression under a fluorescent microscope .
among a total of  <dig> eggs injected in two experimental days ,  <dig> eggs developed to blastocysts and  <dig> of them showed either green  or red fluorescence   and the remaining  <dig> embryos were non-fluorescent.
nested pcr assay revealed successfully targeted insertion of doi in all the  <dig> blastocysts  with targeted insertion efficiency of  <dig> % injected zygotes that developed into blastocysts  .figure  <dig>
production of multiple targeted tg mouse lines using
i-
pitt.
schematic of simultaneous production of multiple tg lines using i-pitt.
multiple donor vectors that harbor different doi are mixed and co-injected with icre and phic31o mrna into the fertilized eggs carrying the i-pitt landing pad in their genome.
appearance of different fluorescent colors indicates successful insertion of doi.
schematic of targeted insertion alleles for each doi.
tiex allele  <dig> is shown as an example.
arrows indicate the pcr primer sets used for genotype identification of the correct insertion.
for detecting targeted transgenesis in blastocyst, 1st pcr was performed using the outer most primer pair sets  and nested pcr using the internal primer pair sets .
for detecting targeted transgenesis in fetuses, pcr with only the purple primer pair is sufficient.
example of simultaneous production of multiple targeted tgs.
blastocysts  and day  <dig>  fetuses  derived from injected zygotes.
zygotes/fetuses exhibiting blue, green or red fluorescence indicate successful insertion of doi from pbgv, pbgw or pbdr vectors respectively.
the results of pcr-based genotyping are shown below the images; arrows indicate positive samples.
the results of i-pitt experiment in blastocyst embryos  and fetuses/pups .
next, we tested whether multiple tg fetuses or mice can be obtained simultaneously by co-injecting two or three different donor vectors along with icre and phic31o mrnas into zygotes and transferring them to pseudopregnant recipients .
the microinjections were repeated on three separate sessions.
as shown in figure 4c and e, it was possible to obtain simultaneously tg lines derived from donor vectors containing two to three separate doi.
targeted insertion efficiency was  <dig> % and  <dig> % or  <dig> % when analyzed at newborn and fetal stages respectively.
the overall targeted tg rate ranged from  <dig>  to  <dig> % for injected zygotes analyzed at both the stages .
taken together, the results showed that i-pitt enables simultaneous production of up to three targeted tg lines with different dna of interest.
recombination outcomes and genotyping
theoretically, co-introduction of both icre and phic31o mrnas can result in one of several possible insertion alleles such as tiex allele  <dig>   <dig>   <dig>   <dig> and  <dig> .
tiex allele  <dig> and  <dig> are the result of cre-based recombination or phic31-based integration systems, respectively.
various genotyping assays were employed to detect targeted insertion of doi: primer sets ‘a’, ‘b’, ‘d’ and ‘e’ amplify the 5’ or 3’ junction regions generated by recombination/integration whereas primer set ‘c’ amplifies internal regions of the transgene .
targeted insertion of doi generated by cre-based recombination only  or by phic <dig> integration system only  can be detected with the primer sets ‘a’ and ‘b’  or primer sets ‘a’ and ‘d’ .
since pcr with primer set ‘a’ is applicable to identification of targeted insertion of doi performed by either systems, we used this primer set for the first screening in all i-pitt experiments.figure  <dig>
pcr-based genotyping.
the various possible allele outcomes and primer sets  used for detecting them.
the black and blue arrows indicate universal and doi-specific primers, respectively.
the green arrows indicate semi-universal primers used for identification of targeted insertion for several constructs including pbgx and pbgt.
example of pcr-based genotyping.
pcr was performed using  <dig> samples derived from mixed injection of pbgx and pbgt .
the primer sets used were as follows : “m273/m839”, “m273/m874”, “m274/m376”, “m645/m646”, “m873/m874”, “#235/m026”, “m273/m879”, “m958/m839”, “m958/m874”, “m953/m839”, “m953/m874”, “m953/m026” and “m273/m026”.
see text for more details.
there are other possible scenarios of cre and/or phic <dig> mediated insertions.
1) combined effect of recombination and integration systems can generate another allele  as a result of two alternative sequential events: a) phic <dig> mediated-insertion first followed by cre mediated-recombination via lox <dig> , and b) cre mediated-insertion first via lox <dig> followed by phic <dig> mediated-recombination .
this allele can be distinguished from other alleles by pcr using primer sets ‘b’, ‘g’ and ‘f ’  or by sequencing of the pcr-amplified fragments.
2) another possibility is that the inserted dna may get deleted to generate Δti allele after two sequential events: a) phic <dig> mediated-insertion first followed by cre mediated-recombination via jt15/jtz <dig> , and b) cre mediated-insertion first via jt15/jtz <dig> followed by phic <dig> mediated-recombination .
this allele can be detected by pcr using primer set ‘h’ .
pcr-genotyping of genomic dna from i-pitt derived samples demonstrated that all of the positive f <dig> fetuses/pups harbored either tiex allele  <dig> or  <dig> .
none of positive f <dig> fetuses/pups had tiex allele  <dig> , suggesting that the allele may have been changed to be tiex allele  <dig> immediately after cre-mediated recombination via lox <dig> .
we also detected Δti allele by pcr using primer set ‘h’ .
in addition, we performed pcr with primer set ‘i’ for all the samples showing targeted insertion to detect transgene mosaicism with respect to the presence of targeted allele .
as a result, we could detect the transgene mosaicism in  <dig> out of  <dig>  of f <dig> fetuses/pups .
the results shown in figure 5b indicated that offspring no.
<dig>   <dig> have a targeted fragment derived from pbgt, and offspring no.
<dig> from pbgx.
offspring no  <dig> exhibited transgene mosaicism harboring tiex allele  <dig>  while offspring no.
<dig> and  <dig> were pure tg individuals harboring tiex allele  <dig>
reproducibility of i-pitt technology
we next evaluated if the newly developed seed mouse can be efficiently used in another laboratory to successfully perform i-pitt.
to test this idea, we suspended four cauda epididymides dissected from two homozygous seed mice in a  <dig>  ml cryotube containing mineral oil, and shipped the cryotube to riken bioresource center   overnight under refrigerated temperatures , as described by mochida et al.
<cit> .
at the recipient laboratory, the epididymal spermatozoa were recovered from the transported samples and used for ivf with c57bl/6n-derived oocytes  and the resulting zygotes were subjected to i-pitt.
the injection mix used in the recipient lab was from an experiment conducted at the originating lab  that was shipped under frozen conditions.
among a total of  <dig> eggs injected  <dig> fetuses were recovered and  <dig> of them were identified as targeted tg fetuses with a target insertion efficiency was  <dig> % for fetuses recovered and  <dig> % for total zygotes injected .
this result demonstrates the robustness and reproducibility of the i-pitt method.
notably, because of this capability of i-pitt technology, there is no necessity of 1) transportation of seed mice 2) quarantine housing of the mice at the recipient facility, 3) re-derivation  of mice, as may be necessary in some cases, 4) maintenance of seed mice at the recipient animal facility.
this system also enables that multiple different facilities can obtain the cauda epididymide samples from one central facility.
discussion
in this study, we designed and generated a new pitt seed mouse called tokmo- <dig> to develop an improved pitt  system.
the landing pad in the new seed mouse constitutes mutant loxp, attp and mutant frt sites to enable targeted insertion of transgenes using cre or phic <dig> or flp-based pitt.
the seed mouse line is with the c57bl/6n genetic background and thus the pitt derived tg founders under pure genetic background are immediately available for analysis without the need for backcrossing.
combinatorial use of cre-loxp and phic31-attp/b system resulted in improved targeted insertion efficiency of transgene from the newly designed donor vectors.
the i-pitt strategy also enabled simultaneous production of multiple different tg mouse lines in a single micro-injection session.
the reproducibility of i-pitt was proven by performing it in two different laboratories.
in es cell-based targeted insertion experiments, we confirmed that both cre-loxp and phic <dig> systems worked as expected.
the insertion efficiency in both cre-loxp and phic <dig> systems was comparable or slightly higher in the latter than the former.
to our knowledge, pitt using flp-frt system has not been developed yet.
there are a few reported mutant frt sites tested for recombinase-mediated cassette exchange  in cultured cells  <cit> ; of these, we included the most newly described genuine 48-bp variant frt sites in our system.
the targeted recombination efficiency of flp-frt system using mutant frt sites  was considerably low when compared to cre and phic31-based insertion.
notably, turan et al.
<cit>  who developed f <dig> and f <dig> demonstrated that rmce employing these mutant frt sites worked well when transient assay was performed using nih3t <dig> cells.
this implies that targeted insertion by rmce with this frt combination is possible.
we found in this study that the flp-frt system failed to cause efficient targeted insertion of doi .
this may partly be attributed to the es cells used in this study and/or poor transfection efficiency of vectors in those cells.
it remains unclear whether the flp-frt system can yield good in vivo insertion efficiency of doi.
nevertheless, the advantages of flp-frt system in the i-pitt are, i) it allows removal of extra sequences for generation of the “clean” inserted allele  and ii) combinatorial use of flp-frt and cre-loxp systems would result in increased targeted insertion frequency of doi, as described below .
the combinatorial use of cre-loxp and phic <dig> systems resulted in an increased targeted insertion efficiency in es cells and in zygotes.
while it remains unclear why simultaneous introduction of the two systems leads to increased insertion efficiency of doi, a few possible scenarios and plausible reasons are discussed here.
1) cre-loxp- and phic31-based insertions occur independently and the increased targeted insertion efficiency could be due to a simple additive effect.
2) cre-loxp- and phic31-based systems work cooperatively.
first insertion might occur through lox <dig> in cre-loxp-based system that generates transient unstable allele  which can be subsequently converted to either ‘tiex allele 1’ through jt15/jtz <dig> recombination or ‘targeted allele’ through lox <dig> by further cre activity.
alternatively, the unstable tiex allele  <dig> produced by initial cre action may be used as a substrate for phic <dig> action to convert the allele to tiex allele  <dig> , which may add synergistic effect to increase the insertion efficiency.
such a cooperative effect was also observed when cre-loxp and flp-frt systems are employed together as shown in figure 2b.
it should be noted that the tiex allele  <dig> can be generated not only through “cre first-phic <dig> next” recombination/integration event , but also through “phic <dig> first-cre next” event .
the present co-injection strategy results in generation of only tiex allele  <dig> or  <dig> .
this presents another advantage of this system, since nearly “clean” inserted allele lacking the vector can be obtained.
on the other hand, our current donor vector design has a risk that self-elimination of inserted cassette occurs, leading to generation of Δti allele .
in fact, we observed the presence of Δti allele in  <dig> % of f <dig> mice .
such unwanted outcomes can be avoided by reversing the order of attb and jtz <dig> in the donor vector, i.e., from “attb-jtz17” to “jtz17-attb”.
thus, we presume that jtz17-attb architecture in the donor vector and the employment of tandemly arranged six copies of jtz <dig>  can further improve insertion efficiency of doi to make the i-pitt system even more efficient.
although complementary, the i-pitt system provides advantages to the current state of use of newer nuclease-mediated genome editing technologies, such as crispr/cas <dig>  for targeted insertion  <cit> .
for example, 1) compared to the use of the well-tested and reliably-used recombinase systems, additional development and testing of crispr/cas <dig> to generate multiple genetic insertion events will be necessary to ensure widespread use and application.
2) the designer nucleases can also introduce un-wanted alleles such as indels along with targeted insertion allele in a mosaic fashion, which often makes it challenging to identify the desired mutation by genotyping.
on the other hand, i-pitt system generates only the expected target insertion allele that can be easily identified by genotyping.
3) designer nuclease strategies often require re-constructing of targeting vectors with significantly longer homology arms that would occasionally force researchers to compromise on the size of the inserted cassettes.
on the contrary, the pitt donor vectors readily accept cassettes as high as 10 kb size: these vectors do not require re-designing and building of special targeting vectors with homology arms for each project.
simultaneous injection of multiple donors in separate sessions resulted in production of as many as three distinctly targeted tg mice, each of which contained a transgene with a single-copy configuration.
notably, the number of zygotes used was well under  <dig> in each session, while traditional approaches typically use more than  <dig> zygotes to generate tg founders for only one tg construct, i-pitt offers over three fold efficiency in generating tg founders for multiple transgenes.
thus, the i-pitt strategy could serve as a high-throughput and cost-saving method to produce targeted tg mice.
this is in contrast with the traditional production of tg mice which is based on random insertion of multiple copy numbers of transgenes  <cit> .
although traditional transgenesis-based pronuclear injection of dna containing multiple transgenes has been used for production of double or triple tg mice that contain multiple transgenes per animal  <cit> , such a strategy is not commonly followed due to inherent and obvious problems that come along with it.
another major pitfall of such multiple transgene injection strategies using traditional approaches is that several lines need to be screened to obtain reliable tg strains for further work.
the mouse strain c57bl/6n has been considered as one of the most widely and frequently used strains in many fields of research  <cit> .
despite the usefulness of this inbred strain, many random insertion-based tg lines are still being generated using mice with mixed genetic background  or fvb/n inbred strain because the tg rate is more efficient and less difficult with hybrid or fvb/n strains than with c57bl/ <dig>   <cit> .
in this study, using the newly developed i-pitt system in c57bl/ <dig> background, our tg production efficiencies ranged from  <dig>  to  <dig> %.
unlike the tg mice generated using traditional approach, the models generated using tokmo- <dig> do not need to go through extensive and time-consuming steps of backcrossing.
this is particularly useful when the genetic backgrounds of multiple mutants need to be maintained in a pure background.
for example, tissue-specific cre and reporter alleles can be created using tokmo- <dig> lines in the correct genetic background as needed for breeding them with lines produced through global programs such as the international knockout mouse consortium   <cit> .
studies have shown that tissue-specific promoter driven transgenes targeted to specific sites in the genome are reliably expressed  <cit> .
the usefulness of tokmo- <dig> seed mice for generation of tissue-specific promoter driven transgenes is now under investigation and has already been verified for some promoters using tokmo- <dig> model, the first generation seed mouse .
for a newly established technology to become widely usable it should be easily reproducible in other labs and be flexible.
we confirmed the reproducibility and flexibility of i-pitt with the tokmo- <dig> mice by performing it in two different facilities  .
the flexibility of the i-pitt to use sperm isolation and ivf to generate zygotes, instead of maintaining a stud male colony for mating with females, was also assessed.
overnight refrigerated transportation of cauda epididymides dissected from tokmo- <dig> mice  <cit>  were used in an ivf session with c57bl/6n-derived oocytes at the receiving end and found to work well.
this strategy relieves the requirement to maintain tokmo- <dig> mice colony at the receiving facility, and thus is time- and cost-saving and convenient.
this inter-lab./institution transportation of refrigerated cauda epididymides has additional merits over the existing methods of transfer of frozen embryos/sperm or live individuals  <cit> .
for example, the cost of transportation is significantly reduced.
in addition, avoiding transportation of live mice is preferable from an animal welfare point of view  <cit> .
the targeted transgenesis approach using i-pitt enables stable, reliable, predictable, and repeatable transgene expression which cannot be achieved using traditional random-insertion transgenesis approaches.
the i-pitt technology described here can generate multiple targeted tg models in one-step.
the tokmo- <dig> mouse and vectors developed in this study will be made available to the scientific community globally through experimental animal division of riken brc 26sor<tm10maoh>, id: rbrc06517), a part of federation of international mouse resources-  and also through tokai university .
the plasmid vectors developed in this study are deposited to addgene.org.
while the i-pitt can be used for generating any type of transgenic mice, this technology will be particularly suitable for simultaneously generating tg lines for multiple different polymorphic variants or mutant forms of a gene.
considering that individual tg lines generated using traditional approaches invariably differ from each other due to the position effect variation, for the projects that require generation of multiple different transgenes and their comparison with each other, the i-pitt system offers the perfect solution for such high-throughput transgene production projects.
CONCLUSIONS
due to the inherent pitfalls that come with mouse random transgenesis methods, some groups have been developing newer methods to achieve targeted transgenesis particularly through direct injection of dna into zygotes.
the new method we report here, termed improved-pitt , offers many new features compared to the published targeted transgenesis approaches, including the original pitt.
the transgenic efficiency with i-pitt was greatly improved with a typical range of 10-30%, up to 62%.
i-pitt also offers the advantage of creating multiple tg models in a single experimental session and thus serves as an analogous tool for multiplexing in tg technology similar to crispr/cas <dig> tool for ko technology.
in summary, i-pitt offers i) superior targeting efficiency, ii) multiplexing capability to insert different cassettes simultaneously, iii) reduction in the number of animals used; an important animal welfare consideration, iv) uniform genetic background.
moreover, these mice will be made readily available to the scientific community.

 transgenic mice have been extensively used for in vivo analysis of gene function and generation of human disease models. since generation of the first tg mice was reported by gordon et al. in who performed pronuclear injection of purified dna into zygotes, pi-mediated transgenesis has been the most common method for more than 30 years. in general, for obtaining several tg mouse lines harboring a specific transgene, it typically requires approximately or more eggs. unfortunately, the pi-mediated transgenesis is often associated with wide variability in the level and pattern of transgene expression, which causes phenotypic variability among individual tg lines, probably due to the random nature of copy number, configuration, and insertion site of the transgene. due to such variability in transgene expression, researchers need to extensively screen various lines and choose the most suitable tg mouse line showing desired transgene expression, which is a very laborious and time-consuming task. to overcome the pitfalls associated with random transgenesis, single-copy insertion of a transgene into a predetermined genomic locus can be accomplished using embryonic stem cell-mediated gene targeting, which is the gold-standard for generation of targeted tg mice. however, this approach is laborious, expensive, and time-consuming when compared to pi-based transgenesis. strategies using recombinase- or integrase-based targeted insertion through pi have been recently described by us and others. we termed this technology as “pronuclear injection-based targeted transgenesis. the pitt system requires a ‘seed mice strain’ containing a suitable landing pad at a predetermined genomic locus as an embryo donor for pi. the method requires, in its initial stage, to establish the seed mouse by targeted insertion of the landing pad via conventional es cell-based gene targeting approach. such an established mouse line serves as a “seed” strain, from which tg mice harboring the dna of interest are generated by injection of donor vector into zygotes along with dna/mrna for recombinase or integrase. for performing targeted insertion of doi using a seed strain, we and another group used cre-loxp recombination system, while a third group used phic integrase system. despite the advantages of pitt approach compared to traditional methods, its full potential is not discovered yet, because the available methods are only single-enzyme based pitt versions. we hypothesized that further improving of the pitt by combining multiple recombinase-integrase systems, the method can be made more efficient, versatile and capable of multiplexing. specifically, we anticipated that such a strategy should make the pitt method able to rapidly generate multiple different transgenic lines simultaneously, that are targeted precisely to the same genomic location. this would ultimately facilitate many areas of biomedical research that depend on rapid generation of multiple, reliable transgenic mice since it would address the major constraints such as time, costs and variable efficiencies associated with the use of es cell-based targeted transgenesis approach while taking care of several pitfalls associated with random transgenesis approaches. therefore, in this report we further improved the technique by creating the next generation seed mouse with a combination of elements including mutant loxp, mutant frt, and attp sites at the rosa locus. we termed the new seed mouse as tokmo- and the method as improved-pitt. this system adds many new features and capabilities to the previously described pitt, and to the targeted transgenesis approaches in general. these features are: 1) the i-pitt method enables the use of any of the three insertion systems, viz. cre, phic or flp, or combinations, for inserting the tg cassettes. 2) simultaneous use of two systems, for e.g., cre-loxp-mediated recombination and phic integration, significantly enhances the targeted insertion efficiency. 3) many tg doi constructs can be co-injected to produce tg lines for each of the constructs injected. 4) the multiplexing capability allows the generation of transgenic lines with zygotes per donor tg construct or less. 5) the new seed mouse is under the c57bl/6n genetic background, which is widely used as a standard strain by the scientific community, including the international knockout mouse consortium. recently there have been remarkable advances in knockout technology using crispr/cas system in which multiple gene knockouts can be made in a single microinjection session. the i-pitt method described here offers the advantage of creating multiple tg models in a single experimental session and thus serves as an analogous tool for multiplexing in tg technology similar to crispr/cas tool for ko technology.  i-pitt strategy the overall scheme of i-pitt is illustrated in figure  a new pitt seed mouse under c57bl/6n genetic background was engineered by inserting a landing pad containing loxp derivatives, attp, and frt derivatives into the rosa locus through es cell-based targeting. a donor vector carrying tdtomato-pa, loxp derivatives, attb, and frt derivatives was designed to perform site-specific insertion of a transgene. the transgene flanked by the f and f sites in the donor vector was inserted into target locus of fertilized eggs via i-pitt that offers options for any of the three enzymes cre, phic or flp to choose for targeted insertion. the resulting embryos will exhibit “targeted insertion with extra sequence ” allele. the strategy is designed in such way that the extra sequences will be flanked by f and f elements which enable the removal of extra sequences by breeding with flp mouse. this results in cleanly inserted transgene flanked by only the f and f sites. although the structures of tiex allele differ among the site-specific insertion systems employed, the structure of final tiΔex alleles generated will be identical in all the strategies. combination of mutant loxps was same as in our original pitt system. among the multitude of mutant frt sites reported previously, we chose f and f and frt-l/r, since f14/f pairing was known to be incompatible and thus we anticipated that combination of the spacer variants and the inverted repeat variant would show high recombination efficiency similar to jt15/jtz and lox combinations for the cre-loxp system. the targeted allele also contains a 43-bp fragment corresponding to human oct gene sequence that can serve as a zinc finger nuclease target to aid in insertion of additional sequences for future needs, but the component is not tested yet.figure schematic of pitt-mediated targeted insertion using the new seed mice, tokom- cre-pitt and phic31-pitt confer insertion of the donor vector containing. dna of interest, into a target site in the genome, to generate tiex allele or respectively. doi shown in this example is tdtomato-poly cassette in this figure. introduction of flp removes extra sequence in the tiex allele and resulting in generation of tiΔex allele. see additional file 1: figures s s and s for other possible outcomes recombination. gtnop: a cassette containing “egfp-t2a-neomycin resistant gene-hoct4-polya”. verification of landing pad insertion in es cells to confirm that three kinds of landing pads work in the genomic context, we first established an es cell clone that contains mutant loxp, mutant frt and attp sites at the rosa locus. the #bdu was then transfected with a pbdr donor vector carrying a promoter-less tdtomato gene, the recombinase and/or integrase expression vectors. the tdtomato expression will be driven by endogenous rosa promoter only if a correct insertion of the cassette at the target locus occurs. the red fluorescence in successfully targeted cells was detected by facs analysis. two days after transfection, approximately 1% of cells were positive for tdtomato fluorescence when icre- or phic31o-expression vector was used, but when flpo-expression vector was used, only a few cells showed tdtomato fluorescence. notably, the percentage of fluorescent cells increased when any two expression vectors were co-transfected. we then tested if inclusion of six copies of tandem repeats of recombination/integration sites in the donor vector would increase the insertion efficiency and found that it was slightly improved in the cre-loxp-based system, when 6xjtz was included in a donor vector. no obvious improvement was noticed in the phic31-based system.figure targeted insertion in es cells. introduction of donor vector harboring promoter-less tdtomato gene into targeted es cells with each expression vector for icre recombinase, flpo recombinase or phic integrase. red fluorescent es cells, as analyzed by facs, are regarded as successfully targeted tdtomato cassette inserted cells. co-introduction of combinations of recombinases or integrase expression vectors increases transgene insertion efficiency. these results indicated that 1) the landing pad architecture we designed worked as expected, and 2) the cre-based and phic31-based gene insertion systems showed comparable targeted insertion efficiencies, while overall efficiency of the flp-based system was relatively poor compared to phic31- and cre-based systems, 3) the combinatorial use of two insertion systems increased the targeted insertion efficiency, and 4) tandem repeats of recombinase recognition sites in the donor vectors enhanced cre-loxp-mediated targeted insertion of doi. validation of i-pitt after confirming that desired dna cassettes can be successfully inserted into the landing pads by using recombinases and/or integrase in es cells, a novel seed mouse line was established using c57bl/6n-derived es cell clones harboring the targeted allele. similar to our previous seed mouse, both homozygotes and heterozygotes of the new seed mouse were viable with normal reproductive ability. to test phic31-based pitt on the new seed mouse tokmo- we injected various concentrations of phic31o mrna along with 10 ng/μl of pber donor vector into fertilized eggs that were generated by in vitro fertilization of c57bl/6n eggs with the epididymal spermatozoa isolated from a male homozygous tokmo- mouse. based on the results presented in additional file 1: table s we decided to use or 15 ng/μl of phic31o mrna in subsequent experiments. these concentrations are relatively higher compared to icre mrna concentrations that is typically needed for pitt. the pitt efficiency for cre- or phic31-based systems was assessed by injecting zygotes, culturing them until blastocysts, and subsequent analysis for targeted insertion. targeted insertion was detected in % or % of developed blastocysts when icre mrna or phic31o mrna were injected. interestingly, co-injection of icre and phic31o mrnas resulted in increased insertion efficiency of up to % of developed blastocysts. notably, insertion rate as high as % among developed blastocysts was achieved in a single experiment. based on these results, we decided to follow a co-injection strategy using icre mrna and phic31o mrna at concentrations  ng/μl and  ng/μl respectively in subsequent experiments.figure pitt using tokom- seed mice. schematic diagram of the pitt injection experiment. donor vector containing doi is injected into the fertilized eggs harboring landing pad together with icre and/or phic31o mrna. successful targeted transgenesis in blastocysts derived from zygotes injected with pber + icre mrna + phic31o mrna. successful targeted transgenesis in day fetuses. zygotes/fetuses exhibiting red fluorescence indicate successful targeted insertion of doi. the pitt results in blastocysts and fetuses/pups. we next examined whether co-injection of icre and phic31o mrnas into zygotes leads to production of targeted tg fetuses or newborns. two different donor vectors were included in the injection, pber and pbgo. the targeted insertion efficiencies were % and %, and % and % for total injected zygotes, respectively. these results indicate that 1) both phic and icre-based targeted insertions were effective in using the new seed mouse tokmo- and that 2) combinatorial use of the two systems enhanced the in vivo targeted insertion efficiency. simultaneous production of multiple different targeted tg mouse lines using i-pitt given the ability of the original pitt system to insert a single copy transgene through specially designed donor vectors, we hypothesized that when a mixture of multiple donor vectors is injected into zygotes, the resulting independent tg founder mouse lines should contain only one type of transgene from the mixture. if this occurs, more than two types of tg lines can be produced at once in a single injection event, which offers versatility to the system and also would reduce the cost significantly, compared to the traditional tg production method. as a preliminary test, we first assessed this possibility by injecting a mixture of donor vectors pbgv, pbgw and pbdr that confer expression of mcfp, mcitrine and tdtomato respectively, when they are correctly inserted. the injection mix containing the three donor vectors along with icre and phic31o mrnas was microinjected into the fertilized eggs harboring hemizygous targeted allele. the injected eggs were cultured up to blastocyst stage and observed for transgene expression under a fluorescent microscope. among a total of eggs injected in two experimental days, eggs developed to blastocysts and of them showed either green or red fluorescence and the remaining embryos were non-fluorescent. nested pcr assay revealed successfully targeted insertion of doi in all the blastocysts with targeted insertion efficiency of % injected zygotes that developed into blastocysts.figure production of multiple targeted tg mouse lines using i- pitt. schematic of simultaneous production of multiple tg lines using i-pitt. multiple donor vectors that harbor different doi are mixed and co-injected with icre and phic31o mrna into the fertilized eggs carrying the i-pitt landing pad in their genome. appearance of different fluorescent colors indicates successful insertion of doi. schematic of targeted insertion alleles for each doi. tiex allele is shown as an example. arrows indicate the pcr primer sets used for genotype identification of the correct insertion. for detecting targeted transgenesis in blastocyst, 1st pcr was performed using the outer most primer pair sets and nested pcr using the internal primer pair sets. for detecting targeted transgenesis in fetuses, pcr with only the purple primer pair is sufficient. example of simultaneous production of multiple targeted tgs. blastocysts and day fetuses derived from injected zygotes. zygotes/fetuses exhibiting blue, green or red fluorescence indicate successful insertion of doi from pbgv, pbgw or pbdr vectors respectively. the results of pcr-based genotyping are shown below the images; arrows indicate positive samples. the results of i-pitt experiment in blastocyst embryos and fetuses/pups. next, we tested whether multiple tg fetuses or mice can be obtained simultaneously by co-injecting two or three different donor vectors along with icre and phic31o mrnas into zygotes and transferring them to pseudopregnant recipients. the microinjections were repeated on three separate sessions. as shown in figure 4c and e, it was possible to obtain simultaneously tg lines derived from donor vectors containing two to three separate doi. targeted insertion efficiency was % and % or % when analyzed at newborn and fetal stages respectively. the overall targeted tg rate ranged from to % for injected zygotes analyzed at both the stages. taken together, the results showed that i-pitt enables simultaneous production of up to three targeted tg lines with different dna of interest. recombination outcomes and genotyping theoretically, co-introduction of both icre and phic31o mrnas can result in one of several possible insertion alleles such as tiex allele and. tiex allele and are the result of cre-based recombination or phic31-based integration systems, respectively. various genotyping assays were employed to detect targeted insertion of doi: primer sets ‘a’, ‘b’, ‘d’ and ‘e’ amplify the 5’ or 3’ junction regions generated by recombination/integration whereas primer set ‘c’ amplifies internal regions of the transgene. targeted insertion of doi generated by cre-based recombination only or by phic integration system only can be detected with the primer sets ‘a’ and ‘b’ or primer sets ‘a’ and ‘d’. since pcr with primer set ‘a’ is applicable to identification of targeted insertion of doi performed by either systems, we used this primer set for the first screening in all i-pitt experiments.figure pcr-based genotyping. the various possible allele outcomes and primer sets used for detecting them. the black and blue arrows indicate universal and doi-specific primers, respectively. the green arrows indicate semi-universal primers used for identification of targeted insertion for several constructs including pbgx and pbgt. example of pcr-based genotyping. pcr was performed using samples derived from mixed injection of pbgx and pbgt. the primer sets used were as follows: “m273/m839”, “m273/m874”, “m274/m376”, “m645/m646”, “m873/m874”, “#235/m026”, “m273/m879”, “m958/m839”, “m958/m874”, “m953/m839”, “m953/m874”, “m953/m026” and “m273/m026”. see text for more details. there are other possible scenarios of cre and/or phic mediated insertions. 1) combined effect of recombination and integration systems can generate another allele as a result of two alternative sequential events: a) phic mediated-insertion first followed by cre mediated-recombination via lox, and b) cre mediated-insertion first via lox followed by phic mediated-recombination. this allele can be distinguished from other alleles by pcr using primer sets ‘b’, ‘g’ and ‘f ’ or by sequencing of the pcr-amplified fragments. 2) another possibility is that the inserted dna may get deleted to generate Δti allele after two sequential events: a) phic mediated-insertion first followed by cre mediated-recombination via jt15/jtz, and b) cre mediated-insertion first via jt15/jtz followed by phic mediated-recombination. this allele can be detected by pcr using primer set ‘h’. pcr-genotyping of genomic dna from i-pitt derived samples demonstrated that all of the positive f fetuses/pups harbored either tiex allele or. none of positive f fetuses/pups had tiex allele, suggesting that the allele may have been changed to be tiex allele immediately after cre-mediated recombination via lox. we also detected Δti allele by pcr using primer set ‘h’. in addition, we performed pcr with primer set ‘i’ for all the samples showing targeted insertion to detect transgene mosaicism with respect to the presence of targeted allele. as a result, we could detect the transgene mosaicism in out of of f fetuses/pups. the results shown in figure 5b indicated that offspring no. have a targeted fragment derived from pbgt, and offspring no. from pbgx. offspring no exhibited transgene mosaicism harboring tiex allele while offspring no. and were pure tg individuals harboring tiex allele reproducibility of i-pitt technology we next evaluated if the newly developed seed mouse can be efficiently used in another laboratory to successfully perform i-pitt. to test this idea, we suspended four cauda epididymides dissected from two homozygous seed mice in a  ml cryotube containing mineral oil, and shipped the cryotube to riken bioresource center overnight under refrigerated temperatures, as described by mochida et al.. at the recipient laboratory, the epididymal spermatozoa were recovered from the transported samples and used for ivf with c57bl/6n-derived oocytes and the resulting zygotes were subjected to i-pitt. the injection mix used in the recipient lab was from an experiment conducted at the originating lab that was shipped under frozen conditions. among a total of eggs injected fetuses were recovered and of them were identified as targeted tg fetuses with a target insertion efficiency was % for fetuses recovered and % for total zygotes injected. this result demonstrates the robustness and reproducibility of the i-pitt method. notably, because of this capability of i-pitt technology, there is no necessity of 1) transportation of seed mice 2) quarantine housing of the mice at the recipient facility, 3) re-derivation of mice, as may be necessary in some cases, 4) maintenance of seed mice at the recipient animal facility. this system also enables that multiple different facilities can obtain the cauda epididymide samples from one central facility. discussion in this study, we designed and generated a new pitt seed mouse called tokmo- to develop an improved pitt system. the landing pad in the new seed mouse constitutes mutant loxp, attp and mutant frt sites to enable targeted insertion of transgenes using cre or phic or flp-based pitt. the seed mouse line is with the c57bl/6n genetic background and thus the pitt derived tg founders under pure genetic background are immediately available for analysis without the need for backcrossing. combinatorial use of cre-loxp and phic31-attp/b system resulted in improved targeted insertion efficiency of transgene from the newly designed donor vectors. the i-pitt strategy also enabled simultaneous production of multiple different tg mouse lines in a single micro-injection session. the reproducibility of i-pitt was proven by performing it in two different laboratories. in es cell-based targeted insertion experiments, we confirmed that both cre-loxp and phic systems worked as expected. the insertion efficiency in both cre-loxp and phic systems was comparable or slightly higher in the latter than the former. to our knowledge, pitt using flp-frt system has not been developed yet. there are a few reported mutant frt sites tested for recombinase-mediated cassette exchange in cultured cells; of these, we included the most newly described genuine 48-bp variant frt sites in our system. the targeted recombination efficiency of flp-frt system using mutant frt sites was considerably low when compared to cre and phic31-based insertion. notably, turan et al. who developed f and f demonstrated that rmce employing these mutant frt sites worked well when transient assay was performed using nih3t cells. this implies that targeted insertion by rmce with this frt combination is possible. we found in this study that the flp-frt system failed to cause efficient targeted insertion of doi. this may partly be attributed to the es cells used in this study and/or poor transfection efficiency of vectors in those cells. it remains unclear whether the flp-frt system can yield good in vivo insertion efficiency of doi. nevertheless, the advantages of flp-frt system in the i-pitt are, i) it allows removal of extra sequences for generation of the “clean” inserted allele and ii) combinatorial use of flp-frt and cre-loxp systems would result in increased targeted insertion frequency of doi, as described below. the combinatorial use of cre-loxp and phic systems resulted in an increased targeted insertion efficiency in es cells and in zygotes. while it remains unclear why simultaneous introduction of the two systems leads to increased insertion efficiency of doi, a few possible scenarios and plausible reasons are discussed here. 1) cre-loxp- and phic31-based insertions occur independently and the increased targeted insertion efficiency could be due to a simple additive effect. 2) cre-loxp- and phic31-based systems work cooperatively. first insertion might occur through lox in cre-loxp-based system that generates transient unstable allele which can be subsequently converted to either ‘tiex allele 1’ through jt15/jtz recombination or ‘targeted allele’ through lox by further cre activity. alternatively, the unstable tiex allele produced by initial cre action may be used as a substrate for phic action to convert the allele to tiex allele, which may add synergistic effect to increase the insertion efficiency. such a cooperative effect was also observed when cre-loxp and flp-frt systems are employed together as shown in figure 2b. it should be noted that the tiex allele can be generated not only through “cre first-phic next” recombination/integration event, but also through “phic first-cre next” event. the present co-injection strategy results in generation of only tiex allele or. this presents another advantage of this system, since nearly “clean” inserted allele lacking the vector can be obtained. on the other hand, our current donor vector design has a risk that self-elimination of inserted cassette occurs, leading to generation of Δti allele. in fact, we observed the presence of Δti allele in % of f mice. such unwanted outcomes can be avoided by reversing the order of attb and jtz in the donor vector, i.e., from “attb-jtz17” to “jtz17-attb”. thus, we presume that jtz17-attb architecture in the donor vector and the employment of tandemly arranged six copies of jtz can further improve insertion efficiency of doi to make the i-pitt system even more efficient. although complementary, the i-pitt system provides advantages to the current state of use of newer nuclease-mediated genome editing technologies, such as crispr/cas for targeted insertion. for example, 1) compared to the use of the well-tested and reliably-used recombinase systems, additional development and testing of crispr/cas to generate multiple genetic insertion events will be necessary to ensure widespread use and application. 2) the designer nucleases can also introduce un-wanted alleles such as indels along with targeted insertion allele in a mosaic fashion, which often makes it challenging to identify the desired mutation by genotyping. on the other hand, i-pitt system generates only the expected target insertion allele that can be easily identified by genotyping. 3) designer nuclease strategies often require re-constructing of targeting vectors with significantly longer homology arms that would occasionally force researchers to compromise on the size of the inserted cassettes. on the contrary, the pitt donor vectors readily accept cassettes as high as 10 kb size: these vectors do not require re-designing and building of special targeting vectors with homology arms for each project. simultaneous injection of multiple donors in separate sessions resulted in production of as many as three distinctly targeted tg mice, each of which contained a transgene with a single-copy configuration. notably, the number of zygotes used was well under in each session, while traditional approaches typically use more than zygotes to generate tg founders for only one tg construct, i-pitt offers over three fold efficiency in generating tg founders for multiple transgenes. thus, the i-pitt strategy could serve as a high-throughput and cost-saving method to produce targeted tg mice. this is in contrast with the traditional production of tg mice which is based on random insertion of multiple copy numbers of transgenes. although traditional transgenesis-based pronuclear injection of dna containing multiple transgenes has been used for production of double or triple tg mice that contain multiple transgenes per animal, such a strategy is not commonly followed due to inherent and obvious problems that come along with it. another major pitfall of such multiple transgene injection strategies using traditional approaches is that several lines need to be screened to obtain reliable tg strains for further work. the mouse strain c57bl/6n has been considered as one of the most widely and frequently used strains in many fields of research. despite the usefulness of this inbred strain, many random insertion-based tg lines are still being generated using mice with mixed genetic background or fvb/n inbred strain because the tg rate is more efficient and less difficult with hybrid or fvb/n strains than with c57bl/. in this study, using the newly developed i-pitt system in c57bl/ background, our tg production efficiencies ranged from to %. unlike the tg mice generated using traditional approach, the models generated using tokmo- do not need to go through extensive and time-consuming steps of backcrossing. this is particularly useful when the genetic backgrounds of multiple mutants need to be maintained in a pure background. for example, tissue-specific cre and reporter alleles can be created using tokmo- lines in the correct genetic background as needed for breeding them with lines produced through global programs such as the international knockout mouse consortium. studies have shown that tissue-specific promoter driven transgenes targeted to specific sites in the genome are reliably expressed. the usefulness of tokmo- seed mice for generation of tissue-specific promoter driven transgenes is now under investigation and has already been verified for some promoters using tokmo- model, the first generation seed mouse. for a newly established technology to become widely usable it should be easily reproducible in other labs and be flexible. we confirmed the reproducibility and flexibility of i-pitt with the tokmo- mice by performing it in two different facilities. the flexibility of the i-pitt to use sperm isolation and ivf to generate zygotes, instead of maintaining a stud male colony for mating with females, was also assessed. overnight refrigerated transportation of cauda epididymides dissected from tokmo- mice were used in an ivf session with c57bl/6n-derived oocytes at the receiving end and found to work well. this strategy relieves the requirement to maintain tokmo- mice colony at the receiving facility, and thus is time- and cost-saving and convenient. this inter-lab./institution transportation of refrigerated cauda epididymides has additional merits over the existing methods of transfer of frozen embryos/sperm or live individuals. for example, the cost of transportation is significantly reduced. in addition, avoiding transportation of live mice is preferable from an animal welfare point of view. the targeted transgenesis approach using i-pitt enables stable, reliable, predictable, and repeatable transgene expression which cannot be achieved using traditional random-insertion transgenesis approaches. the i-pitt technology described here can generate multiple targeted tg models in one-step. the tokmo- mouse and vectors developed in this study will be made available to the scientific community globally through experimental animal division of riken brc 26sor<tm10maoh>, id: rbrc06517), a part of federation of international mouse resources- and also through tokai university. the plasmid vectors developed in this study are deposited to addgene.org. while the i-pitt can be used for generating any type of transgenic mice, this technology will be particularly suitable for simultaneously generating tg lines for multiple different polymorphic variants or mutant forms of a gene. considering that individual tg lines generated using traditional approaches invariably differ from each other due to the position effect variation, for the projects that require generation of multiple different transgenes and their comparison with each other, the i-pitt system offers the perfect solution for such high-throughput transgene production projects.  due to the inherent pitfalls that come with mouse random transgenesis methods, some groups have been developing newer methods to achieve targeted transgenesis particularly through direct injection of dna into zygotes. the new method we report here, termed improved-pitt, offers many new features compared to the published targeted transgenesis approaches, including the original pitt. the transgenic efficiency with i-pitt was greatly improved with a typical range of 10-30%, up to 62%. i-pitt also offers the advantage of creating multiple tg models in a single experimental session and thus serves as an analogous tool for multiplexing in tg technology similar to crispr/cas tool for ko technology. in summary, i-pitt offers i) superior targeting efficiency, ii) multiplexing capability to insert different cassettes simultaneously, iii) reduction in the number of animals used; an important animal welfare consideration, iv) uniform genetic background. moreover, these mice will be made readily available to the scientific community. 
thus, typically at least three or more tg lines are produced by injecting over zygotes and the best line/s among them are selected through laborious screening steps. a similar method using phic31-attp/b system was reported subsequently. the pronuclear injection is the simplest and widely used method to generate transgenic mice. transgenic mousepronuclear injection-based targeted transgenesiscre-loxpphic31-attp/bflp-frtrosa26transportation of the cauda epididymidesissue-copyright-statement© the author 2015 unfortunately, pi-based tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. furthermore, the system could generate multiple tg mice simultaneously. we demonstrate that injection of up to three different tg cassettes in a single injection session into as less as zygotes resulted in production of all three separate tg dna containing targeted tg mice. the i-pitt system offers several advantages compared to previous methods: multiplexing capability, very high efficiency of targeted-transgenesis, significantly reduces animal numbers in mouse-transgenesis and the system is developed under c57bl/6n strain, the most commonly used pure genetic background. further, the i-pitt system is freely accessible to scientific community. the targeted tg efficiency in the i-pitt typically ranged from to 30%, with and 62% in two of the sessions, which is by-far the best tg rate reported. here, we developed an improved-pitt method by combining cre-loxp, phic31-attp/b and flp-frt systems directly under c57bl/6n inbred strain, unlike the mixed strain used in previous reports.
 the pronuclear injection is the simplest and widely used method to generate transgenic mice. unfortunately, pi-based tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. thus, typically at least three or more tg lines are produced by injecting over zygotes and the best line/s among them are selected through laborious screening steps. recently, we developed technologies using cre-loxp system that allow targeted insertion of single-copy transgene into a predetermined locus through pi. we termed the method as pi-based targeted transgenesis. a similar method using phic31-attp/b system was reported subsequently.  here, we developed an improved-pitt method by combining cre-loxp, phic31-attp/b and flp-frt systems directly under c57bl/6n inbred strain, unlike the mixed strain used in previous reports. the targeted tg efficiency in the i-pitt typically ranged from to 30%, with and 62% in two of the sessions, which is by-far the best tg rate reported. furthermore, the system could generate multiple tg mice simultaneously. we demonstrate that injection of up to three different tg cassettes in a single injection session into as less as zygotes resulted in production of all three separate tg dna containing targeted tg mice.  the i-pitt system offers several advantages compared to previous methods: multiplexing capability, very high efficiency of targeted-transgenesis, significantly reduces animal numbers in mouse-transgenesis and the system is developed under c57bl/6n strain, the most commonly used pure genetic background. further, the i-pitt system is freely accessible to scientific community. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords transgenic mousepronuclear injection-based targeted transgenesiscre-loxpphic31-attp/bflp-frtrosa26transportation of the cauda epididymidesissue-copyright-statement© the author 2015 