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Hydrodynamic gene delivery is a common method for gene transfer to the liver of small animals, and its clinical applicability in large animals has been demonstrated. Previous studies focused on functional analyses of therapeutic genes in animals with normal livers and little, however, is known regarding its effectiveness and safety in animals with liver fibrosis. Therefore, this study aimed to examine the effects of liver fibrosis on hydrodynamic gene delivery efficiency using a rat liver fibrosis model. We demonstrated for the first time, using pCMV-Luc plasmid, that this procedure is safe and that the amount of fibrotic tissue in the liver decreases gene delivery efficiency, resulting in decrease in luciferase activity depending on the volume of fibrotic tissue in the liver and the number of hepatocytes that are immunohistochemically stained positive for transgene product. We further demonstrate that antifibrotic gene therapy with matrix metalloproteinase-13 gene reduces liver fibrosis and improves efficiency of hydrodynamic gene delivery. These results demonstrate the negative effects of fibrotic tissue on hydrodynamic gene delivery and its recovery by appropriate antifibrotic therapy.

Liver fibrosis is the final stage of liver diseases that lead to liver failure and cancer. While various diagnostic methods, including the use of serum marker, have been established, no standard therapy has been developed. The objective of this study was to assess the approach of overexpressing matrix metalloproteinase-13 gene (MMP13) in rat liver to prevent liver fibrosis progression. A rat liver fibrosis model was established by ligating the bile duct, followed by liver-targeted hydrodynamic gene delivery of a MMP13 expression vector, containing a CAG promoter-MMP13-IRES-tdTomato-polyA cassette. After 14 days, the serum level of MMP13 peaked at 71.7 pg/ml in MMP13-treated group, whereas the nontreated group only showed a level of ~5 pg/ml (P < 0.001). These levels were sustained for the next 60 days. The statistically lower level of the hyaluronic acids in treated group versus the nontreated group (P < 0.05) reveals the therapeutic effect of MMP13 overexpression. Quantitative analysis of tissue stained with sirius red showed a statistically larger volume of fibrotic tissue in the nontreated group compared to that of MMP13-treated rats (P < 0.05). These results suggest that the liver-targeted hydrodynamic delivery of MMP13 gene could be effective in the prevention of liver fibrosis.

The CRISPR revolution that began in 2013 has been adopted and embraced by many researchers worldwide, including the mouse molecular genetics community. CRISPR represents one of only a few radical and transformative shifts in transgenic technologies over the past 30 years. This chapter discusses the paradigm shift that CRISPR technology has brought about in the field of mouse genome editing.

Transgenic technologies used for creating a desired genomic change in animals involve three critical steps: isolation of fertilized eggs, microinjection of transgenic DNA into them and their subsequent transfer to recipient females. These ex vivo steps have been widely used for over 3 decades and they were also readily adapted for the latest genome editing technologies such as ZFNs, TALENs, and CRISPR/Cas9 systems. We recently developed a method called GONAD (Genome editing via Oviductal Nucleic Acids Delivery) that does not require all the three critical steps of transgenesis and therefore relieves the bottlenecks of widely used animal transgenic technologies. Here we provide protocols for the GONAD system.

Neurons transmit active potentials through axons, which are essential for the brain to function. In this study, the axonal networks of the murine brain were visualized with X-ray tomographic microscopy, also known as X-ray microtomography or micro-CT. Murine brain samples were freeze-dried to reconstitute the intrinsic contrast of tissue constituents and subjected to X-ray visualization. A whole brain hemisphere visualized by absorption contrast illustrated three-dimensional structures including those of the striatum, corpus callosum, and anterior commissure. Axonal tracts observed in the striatum start from the basal surface of the cerebral cortex and end at various positions in the basal ganglia. The distribution of X-ray attenuation coefficients indicated that differences in water and phospholipid content between the myelin sheath and surrounding tissue constituents account for the observed contrast. A rod-shaped cutout of brain tissue was also analyzed with a phase retrieval method, wherein tissue microstructures could be resolved with up to 2.7 {\mu}m resolution. Structures of axonal networks of the striatum were reconstructed by tracing axonal tracts. Such an analysis should be able to delineate the functional relationships of the brain regions involved in the observed network., Comment: 28 pages, 6 figures, 1 table

The aim of this study was to investigate whether direct injection of nonviral DNA into the oviductal lumen and subsequent in vivo electroporation leads to in vivo gene transfer in mouse preimplantation embryos present within an oviduct, as an alternative to the pre-existing pronuclear microinjection-based transgenesis. With this technique, effects of expression of the gene of interest (GOI) on mouse preimplantation development can be monitored with relative ease. Superovulated 4-week-old B6C3F1 female mice (hybrids between C57BL/6N and C3H/HeN) were mated with adult B6C3F1 male mice. Two days later, females that had been identified as pregnant, based on the presence of copulation plugs, were injected with 1 µl of a solution containing an enhanced green fluorescent protein (EGFP) expression plasmid (0.5 µg) and 0.05% trypan blue. The entire oviduct was then electroporated using tweezer-type electrodes with 8 square-wave pulses of 50 V each with 50-ms duration. The next day, the 8-cell stage embryos were collected, and their number, morphology, and EGFP-derived fluorescence were recorded. Of the 12 oviducts (6 females used) examined, 3 contained fluorescent 8-cell stage embryos (33%, 19/58 tested), but the intensity of fluorescence varied among the embryos. In total, 10% (19/192 tested) of the embryos were fluorescent and the fluorescence was maintained in these embryos after 1 day of culture. However, the fluorescence disappeared in the late gestational stage fetuses, and the transgenes could not be detected. Our results indicate that it is possible to transfect in vivo preimplantation embryos, although the success rate appears to be relatively low and gene expression is transient. This technology may provide a new method for manipulating preimplantation embryos in vivo, by using, for example, Cre-mediated conditional DNA recombination.

Ovarian follicle development is a complex process mediated by interactions between oocytes and surrounding follicular cells. In an ovary, oocytes are ultimately released from Graafian follicles, which develop from antral follicles localized near the surface of an ovary. To examine the molecular interaction between these 2 cell types, direct gene transfer to follicular cells as well as oocytes appears to be a promising approach, but few studies have applied this technique. The aim of the present study was to develop a technique for gene transfer to antral follicle cells based on their accessibility near the surface of an ovary. B6C3F1 (a hybrid between C57BL6/N and C3H/HeN) female mice aged 4 or 8 w were anesthesized and their ovaries were exposed. About 100 nl of a solution containing reporter plasmid DNA (0.5 µg/μl) and 0.1% trypan blue was injected into a follicle using a glass micropipette attached to the mouthpiece. A total of 6 follicles were injected per ovary. After injection, the ovary was immediately subjected to in vivo electroporation (EP) using an electroporator with 8 square electric pulses of 50 ms and 50 V. After 24 h, the treated ovaries were excised to examine the expression of reporter constructs by histochemistry. All the injected follicles expressed reporter genes to different extents. Inspection of cryostat sections of ovaries injected with the lacZ expression plasmid demonstrated that 50-100% of follicular cells within a follicle were successfully transfected. However, there were no oocytes within the antral follicles that were negative for such staining (15 follicles tested). Similar results were obtained when the enhanced green fluorescent protein expression plasmid was introduced. The present method based on in vivo EP was found to be very effective for transfection of follicular cells. This approach might be useful to explore the roles of genes related to oogenesis/folliculogenesis, and for reproductive manipulation targeted to antral follicles.

Transgenic (Tg) mice have been extensively used as valuable tools for analyses of gene function and have also served as models for many human diseases. Typically, a transgenic mouse is created by microinjection of DNA into pronuclei in which the DNA gets integrated at random locations in the genome. Frequently however, the random integration of multiple copies of a transgene results in transgene silencing, probably because of a positional effect and/or repeat-induced gene silencing. The transgene silencing issue has been overcome by single-copy transgene integration into a predetermined locus through ES cell-mediated transgenesis, despite it being expensive and more time-consuming compared with pronuclear injection (PI)-mediated transgenesis. Recently, several groups have reported novel approaches that employ PI for targeted transgenesis. They are based on site-specific recombination catalyzed by a recombinase or an integrase or homologous recombination enhanced by a zinc-finger nuclease via PI. These next-generation transgenesis methods, which we termed as PI-based Targeted Transgenesis (PITT), are more convenient and faster than ES cell-based transgenesis. Furthermore, the Tg mice generated by these newer methods contain a single-copy transgene and exhibit reliable expression of the transgene. The objective of this review is to present the recent progress in mouse targeted transgenesis.

Porcine embryonic fibroblasts (PEFs) are widely used as donor cells for somatic cell nuclear transfer (SCNT) in pigs. Transfection of PEFs with exogenous DNA is essential for producing genetically modified (GM; transgenic or knockout) pigs via SCNT. In this case, selectable markers are strictly required selecting and enriching stably transfected cells. The most frequently used selective drug for this purpose is a neomycin analogue (G418/geneticin); neo has been widely used as a selectable marker gene in the genomic manipulation of pigs. However, little is known about optimal concentrations of other selection drugs. This often hampers functional analysis of the porcine genome and development of individual GM pigs. This study explores the optimal concentrations of selective drugs, other than neomycin, that can be used for the selection of transfected PEFs. Porcine embryonic fibroblasts were incubated in media containing different concentrations of drugs for up to 10 days, to determine the optimal drug concentrations fatal for PEFs. The following concentrations were found to be optimal selective concentrations for use with PEFs: G418/geneticin, 400 μg/ml; blasticidin S, 8 μg/ml; hygromycin B, 40 μg/ml; puromycin, 2 μg/ml; and zeocin, 800 μg/ml. Repeated transfections with plasmids carrying selectable markers resulted in the generation of multidrug-resistant swine transfectants. Furthermore, these markers were found to be independent. The present information will be useful for the production of SCNT-mediated GM piglets that express multiple transgenes.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is highly expressed in reactive astrocytes. Increased production of GFAP is a hallmark of astrogliosis in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, the physiological and pathological roles of GFAP, particularly in chronic neurodegenerative conditions, remain unclear. To address this issue, we here investigate whether absence of GFAP affects the phenotypic expression of motor neuron disease (MND) using an H46R mutant Cu/Zn superoxide dismutase-expressing mouse model of ALS (SOD1(H46R)). GFAP deficient SOD1(H46R) mice showed a significant shorter lifespan than SOD1(H46R) littermates. Further, at the end stage of disease, loss of GFAP resulted in increased levels of Vim and Aif1 mRNAs, encoding vimentin and allograft inflammatory factor 1 (AIF1), respectively, in the spinal cord, although no discernible differences in the levels and distribution of these proteins between SOD1(H46R) and GFAP-deficient SOD1(H46R) mice were observed. These results suggest that loss of GFAP in SOD1(H46R) mice marginally accelerates the disease progression by moderately enhancing glial cell activation. Our findings in a mouse model of ALS may have implication that GFAP is not necessary for the initiation of disease, but it rather plays some modulatory roles in the progression of ALS/MND.

Porcine embryonic fibroblasts (PEFs) have been used extensively as donor nuclei for the production of cloned pigs via somatic cell nuclear transfer (SCNT). Somatic cell nuclear transfer of gene-targeted PEFs has been the only way to produce gene-targeted pigs, given the lack of germ-line-competent porcine embryonic stem cells. Unlike other primary-cultured cells, such as murine embryonic fibroblasts, a single porcine PEF is unable to proliferate under normal conditions in which a certain number of PEFs (likely over 100) can grow normally. This limitation greatly hampers re-cloning of gene-modified PEFs, which is required for SCNT. Herein, we demonstrate the cultivation of a single PEF transfectant carrying the pEGFP-N1 plasmid with intact normal PEFs (>100) in a Terasaki microtest plate, which resulted in stimulation of the growth of the former cell (doubling time = 2.6 days). In contrast, when a single cell was cultured, it could typically divide only once and never divided more than twice. When a single transfectant was seeded in a well of a 96-well plate together with 5 × 10(4) untransfected PEFs and was subsequently selected in the presence of G418, we obtained a pure cell population of single-cell origin. Thus, this method should be useful for the purification of target recombinant pig clones from mosaic populations that cannot be cultured as a single cell or for which suitable cell growth-promoting conditions are unclear.

Hydrodynamics-based gene delivery (HGD) is an efficient method for transfecting plasmid DNA into hepatocytes in vivo. However, the resulting gene expression is transient, and occurs in a non-tissue specific manner. The piggyBac (PB) transposon system allows chromosomal integration of a transgene in vitro. This study aimed to achieve long-term in vivo expression of a transgene by performing hepatocyte-specific chromosomal integration of the transgene using PB and HGD. Using this approach, we generated a novel mouse model for a hepatic disorder. A distinct signal from the reporter plasmid DNA was discernible in the murine liver approximately two months after the administration of PB transposons carrying a reporter gene. Then, to induce the hepatic disorder, we first administered mice with a PB transposon carrying a CETD unit (loxP-flanked stop cassette, diphtheria toxin-A chain gene, and poly(A) sites), and then with a plasmid expressing the Cre recombinase under the control of a liver-specific promoter. We showed that this system can be used for in situ manipulation and analysis of hepatocyte function in vivo in non-transgenic (Tg) animals.

Akasaka E, Watanabe S, Himaki T, Ohtsuka M, Yoshida M, Miyoshi K, Sato M. Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate. Xenotransplantation 2010; 17: 81–89. © 2010 John Wiley & Sons A/S. Abstract: Background: The recent availability of α-1,3-galatosyltransferase knockout pigs has eliminated anti-Gal antibodies to the galα1-3gal (αgal epitope) as the major barrier to xenotransplantation. These αgal epitope-negative animals can also be produced by somatic cell nuclear transfer of cells overexpressing endo-β-galactosidase (EndoGalC), an enzyme capable of digesting the αgal epitope. For this, selection of cells with highly reduced synthesis of αgal epitope is a prerequisite. In this study, we developed a novel method of selection using isolectin BS-I-B4-conjugated saporin (IB4-SAP), a targeted cytotoxin, that is specific for the terminal αgal epitope. Methods: A mixture of αgal epitope-expressing and non-expressing pig cells was obtained by transfection with an EndoGalC expression vector. These cells were incubated with a solution containing IB4-SAP for 2 h at 37 °C, and subsequently cultivated for over 2 months under general conditions. Results: Almost all (98%) of surviving cells were completely negative for expression of αgal epitope, as confirmed by cytochemical staining using fluorescence-labeled IB4. FACS analysis also confirmed that the IB4-SAP-treated cells exhibited a staining pattern similar to that of the IB4-negative human cells. Extended cultivation (more than 6 months) of these IB4-SAP-treated cells did not alter the above staining pattern. RT-PCR analysis revealed the presence of EndoGalC mRNA in these cells. Conclusions: This IB4-SAP-mediated method of selection of αgal epitope-negative cells will provide an alternative to the present method of cytotoxicity-based selection using specific antibody and complement.

Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak compared with stronger but constitutively expressing viral promoters. In this study, we validated methods of enhancing the transcriptional activity of weak promoters using a Cre-loxP system in vitro and in vivo. We constructed a tester vector, pCTL, which carries a strong systemic cytomegalovirus enhancer/chicken beta-actin promoter (CAG), loxP-flanked CAT, and firefly luciferase (luc) cDNAs. Herpes simplex virus-thymidine kinase (HSV-tk) promoter was used as a weak and systemic promoter and ligated to Cre for construction of pTC. Luc activity was higher (about 10-fold enhancement) in co-transfected (with pCTL and pTC) than in singly (with HSV-tk promoter-driven luc expression vector pTL) transfected NIH3T3 cells. In vivo electroporation-mediated gene delivery of both pCTL and pTC into murine oviductal epithelium yielded results (about 16-fold enhancement) similar to those obtained with in vitro-transfected NIH3T3 cells. To evaluate tissue-specific enhancement of gene expression, podocyte (glomerular visceral epithelial cell)-specific nephrin promoter was ligated to the Cre gene or luc cDNA to create pNC and pNL, respectively. We achieved 2.4-fold improvement of luc gene expression in the mouse kidney in vivo when pCTL and pNC were co-transfected via the tail vein via the lipoplex method. The combination of a weak tissue-specific promoter with the Cre-loxP system could thus be used to enhance the strength of tissue-specific promoters in vitro and in vivo.

Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

Microinjection is considered the gold standard technique for delivery of nucleic acids (NAs; transgenes or genome editing tools such as CRISPR/Cas9 systems) into embryos, for creating genetically modified organisms. It requires sophisticated equipment as wel as well-trained and highly skilled personnel to perform the micro-injection technique. Here, we describe a novel and simple microinjection-independent technique, called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). Using GONAD, we show that NAs (e.g., eGFP mRNA or Cas9 mRNA/sgRNAs) can be effectively delivered to pre-implantation embryos within the intact mouse oviduct by a simple electroporation method and result in the desired genetic modification in the embryos. Thus GONAD can bypass many complex steps in transgenic technology such as isolation of zygotes, microinjection of NAs into them and their subsequent transfer to pseudo-pregnant animals. Furthermore, this method can potentially be used for genome editing in species other than mice.

Ohtsuka, M., Miura, H., Mochida, K. et al. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT). BMC Genomics 16, 274 (2015). https://doi.org/10.1186/s12864-015-1432-5, Background: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) 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 200 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 (PITT). A similar method using PhiC31-attP/B system was reported subsequently. Results: Here, we developed an improved-PITT (i-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 10 to 30%, with 47 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 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice. Conclusions: The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), 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.

Marker assisted speed congenics technique is commonly used to facilitate backcrossing of mouse strains in nearly half the time it normally takes otherwise. Traditionally, the technique is performed by analyzing PCR amplified regions of simple sequence length polymorphism (SSLP) markers between the recipient and donor strains: offspring with the highest number of markers showing the recipient genome across all chromosomes is chosen for the next generation. Although there are well-defined panels of SSLP makers established between certain pairs of mice strains, they are incomplete for most strains. The availability of well-established marker sets for speed congenic screens would enable the scientific community to transfer mutations across strain backgrounds. In this study, we tested the suitability of over 400 SSLP marker sets among 10 mouse strains commonly used for generating genetically engineered models. The panel of markers presented here can readily identify the specified strains and will be quite useful in marker assisted speed congenic screens. Moreover, unlike newer single nucleotide polymorphism (SNP) array methods which require sophisticated equipment, the SSLP markers panel described here only uses PCR and agarose gel electrophoresis of amplified products; therefore it can be performed in most research laboratories.

Gene targeting is a powerful method of specifically modifying genes of interest. It has been most consistently successful in the 129 mouse strain, because the embryonic stem (ES) cells of 129 mice are relatively easy to culture. In gene-targeting experiments, the use of ES cell-derived genomic clones as a source of homology arms is desirable, because the genetic variation among mouse strains results in a reduced frequency of homologous recombination. In this study, we generated an arrayed mouse 129/Ola BAC library derived from E14.1 ES cells, one of the frequently used ES cell lines. More than 135,000 BAC clones with a mean insert size of 110 kb were isolated. This library is estimated to represent a 5.5-fold mouse genome coverage. The BAC clones can be screened within 2 days by PCR. Considering that all 8 loci so far examined are contained in this BAC library, we believe it will be a useful resource for gene targeting studies using E14 ES cells as well as for genome analysis.

The web software CHOP was developed to visualize the ‘wobbling’ in the third codon position of aligned DNA sequences. The simple features of this tool allow users to easily find regions suspected of containing coding sequences (CDSs). The program also allows visualization of the nucleotide diversity between two genomic or gene sequences by graphically plotting the percentage identity between the two sequences. CHOP can also isolate highly conserved regions within both CDSs and non-CDSs. Highly conserved regions within CDSs include the regions with lower rates of synonymous substitution in which nucleotide sequences are expected to be under strong selective pressure. CHOP is available at http://bunsei2.med.u-tokai.ac.jp:8080/~ohtsuka/cds_finding.html.

Double anal fin (Da) is a spontaneous medaka mutant that exhibits an unique ventralizing phenotype, a mirror-image duplication across the lateral midline in the dorsal trunk-tail region. In the mutant, early D-V specification appears normal but the altered phenotype becomes evident during late embryogenesis. In this study, we genetically specified the mutation to a 174-kb region harboring two zinc-finger type transcription factors, zic1 and zic4, and compared the genomic structures of this region between wild-type and Da mutant fish. No mutation was found in the coding regions of either gene of the mutant, while two fragments, 324 bp and 3-4 kb long, were found inserted downstream of zic1 and zic4, respectively. Probably as a result of this, the expression of both genes is lost in the derivatives of the dorsal (epaxial) somite and the region dorsal to the terminal axis bending. All these tissues are morphologically affected or become ventralized in the mutants. In contrast, the expression in the head region and dorsal spinal cord remained unchanged. Detailed characterization of Da phenotypes revealed a novel defect in the axial skeleton (spina bifida occulta) that was also found in zic1-deficient mice. Finally, zic1-morpholino injection partially phenocopied early Da phenotypes. These findings strongly suggest that zic1 and/or zic4 are required for dorsal identity in the trunk-tail region and that loss of their expression in the epaxial somite derivatives and tail region causes the Da phenotypes.

Medaka is one of the prominent model animals, which also include other fishes such as Fugu and zebrafish. Its genome is relatively compact but has not been well characterized. Here we have sequenced a 229-kb region of medaka, containing the Double anal fin (Da) locus, and compared its structure to those in Fugu, human, and mouse. This region, representing a gene-poor region, contains no major rearrangements and can be readily compared among different species. Comparison of G+C contents and repeats suggested that medaka and Fugu are highly related as expected and that medaka is more similar to mammals than Fugu is. Sequence comparisons of developmental genes zic1 and zic4, identified within this region, revealed that zic1, but not zic4, is highly conserved among vertebrates. The 5' coding region of zic4 is, however, extremely homologous among fishes with little synonymous substitutions, implying its distinct function in fish.

Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100 - 200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50 - 300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.

Medaka (Oryzias latipes) has many advantages for genetic and developmental studies. With recent advances in the genome analyses of other species, rapid accumulation of resources for medaka genomics is expected. In this study, we generated an arrayed medaka cosmid library from the HNI inbred strain, carrying a 40-kb insert on average. The library consists of approximately 120,000 clones with a 6-fold genomic coverage. Cosmid clones can be screened within 2 days using standard polymerase chain reaction. Considering the advantage of the cosmid insert size and the compact genome size of the medaka, this library provides a powerful tool for future genome analyses.

The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much more quickly than the previously used techniques, and, more importantly, multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one-cell mouse embryos to create knockout or knock-in mouse models.

Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice. However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. In addition, random integration sometimes disrupts an endogenous gene that can affect the phenotypes of Tg mice. Our recently developed pronuclear injection-based targeted transgenesis (PITT) method enables the integration of a single-copy transgene into a predetermined genomic locus through Cre-loxP site-specific recombination. The PITT method enables stable and reliable transgene expression in Tg mice and is also applicable for generating knockdown mice. Therefore, the PITT method could represent next-generation transgenesis that overcomes the pitfalls of conventional transgenesis.

Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice. However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. In addition, random integration sometimes disrupts an endogenous gene that can affect the phenotypes of Tg mice. Our recently developed pronuclear injection-based targeted transgenesis (PITT) method enables the integration of a single-copy transgene into a predetermined genomic locus through Cre-loxP site-specific recombination. The PITT method enables stable and reliable transgene expression in Tg mice and is also applicable for generating knockdown mice. Therefore, the PITT method could represent next-generation transgenesis that overcomes the pitfalls of conventional transgenesis.

Double anal fin (Da) is a medaka with an autosomal semidominant mutation that causes mirror image duplication of the ventral region concentrating on the caudal region. The chromosomal location of the Da gene and its sequence have remained unknown. We constructed a medaka linkage map as a first step to approach positional cloning of the gene. The segregation analysis was performed on the basis of genetic recombination during female meiosis using 134 random amplified polymorphic DNA (RAPD) markers, 13 sequence-tagged sites (STSs), 15 polymorphic sequences from known genes, and the Da gene. One hundred forty-six markers from the above markers segregated into 26 linkage groups. The size of the genome was estimated to be 1776 cM in length. We identified four syntenic regions between medaka and zebrafish (and human) by mapping the known genes and found one of them to be located in close proximity to theDa gene. By mapping the region surrounding the Dagene in high resolution, two markers were detected flanking theDa gene at 0.32 and 0.80 cM. The detected markers providing a vital clue to initiate chromosome walking will lead us to the definite location of the Da gene.

Recombineering [1] or Red/ET recombination [2], a recently developed technique based on homologous recombination in Escherichia coli, is useful for the efficient production of a gene-targeting vector (TV) by modification of bacterial artificial chromosome (BAC) clones. This method requires the preparation of a linear DNA recombineering fragment, containing a selectable gene flanked by homologous regions (HRs) for recombination. The PCR method using primers containing HR sequences is the simplest way to produce the recombineering fragment with HRs placed at both ends. This procedure is not advantageous, however, when complex components such as reporter genes are included in the amplified region due to the high degree of mutations possibly generated by PCR. To avoid this problem, Liu and coworkers [3] and Valenzuela and coworkers [4] employed a ligation-based cloning method to introduce HRs into a vector used for recombineering. They used HRs provided as PCR products and oligonucleotides, respectively. In this strategy, however, several restriction enzymes (REs), including six-base cutters, are employed for cloning of HRs. Suitable enzyme sites or HRs, therefore, should be chosen according to the construct introduced. These properties sometimes necessitate screening to identify and select the correct clones. In addition, background clones derived from uncut vector could appear after recombination [3], although most of them can be eliminated by DpnI digestion when PCR-amplified recombineering fragments are used [2].

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ.

Almost all transfection protocols for mammalian cells use a drug resistance gene for the selection of transfected cells. However, it always requires the characterization of each isolated clone regarding transgene expression, which is time-consuming and labor-intensive. In the current study, we developed a novel method to selectively isolate clones with high transgene expression without drug selection. Porcine embryonic fibroblasts were transfected with pCEIEnd, an expression vector that simultaneously expresses enhanced green fluorescent protein (EGFP) and endo-b-galactosidase C(EndoGalC, an enzyme capable of digesting cell surface a-Gal epitope) upon transfection. After transfection, the surviving cells were briefly treated with IB4SAP (a-Gal epitope-specific BS-I-B4 lectin conjugated with a toxin saporin). The treated cells were then allowed to grow in normal medium, during which only cells strongly expressing EndoGalC and EGFP would survive because of the absence of a-Gal epitopes on their cell surface. Almost all the surviving colonies after IB4SAP treatment were in fact negative for BS-I-B4 staining, and also strongly expressed EGFP. This system would be particularly valuable for researchers who wish to perform large-scale production of therapeutically important recombinant proteins.

We recently reported a novel transgenic (Tg) technique named pronuclear injection-based targeted transgenesis (PITT) (Ohtsuka et al. 2010; Ohtsuka et al. 2012a). The PITT technique enables targeted integration of a single-copy transgene into a specified locus by Cre-loxP mediated recombination and it guarantees faithful and reproducible transgene expression. As described in the first report, the PITT technique requires co-injection of a Cre expression plasmid and a donor vector containing loxP sequences into pronuclei of zygotes obtained from the seed mice that carry loxP sequences at the Rosa26 or H2-Tw3 loci. This method has a (targeted) integration efficiency of 26/585 (4.4 %; 95 % CI 2.9–6.4 %) (Ohtsuka et al. 2012b). Although PITT has a lower transgenic rate (4.4 %) when compared to traditional transgenesis (*10–20 % (Fielder and Montoliu 2011)), it is superior to the latter method since just obtaining one transgenic founder is sufficient in case of PITT. With an aim to improve the PITT technique further we attempted to co-inject iCre mRNA [codon improved Cre recombinase; (Shimshek et al. 2002)] into zygotes with the expectation that the Cre protein would be available more readily in the zygotes from injected iCre mRNA compared to when the Creexpression plasmid DNA was co-injected. We coinjected 10 ng/ll of pAOM donor vector (Ohtsuka et al. 2010) along with various concentrations of iCre mRNA (1–45 ng/ll) into zygotes obtained from the Electronic supplementary material The online version of this article (doi:10.1007/s11248-013-9703-x) contains supplementary material, which is available to authorized users.

The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes.

We recently reported a novel method of mouse transgenesis called Pronuclear Injection-based Targeted Transgenisis (PITT) using which a series of fluorescent transgenic (Tg) mice lines were generated. These lines, unlike those generated using conventional random integration methods, express the transgenes faithfully and reproducibly generation after generation. Because of this superior nature, these lines are ideal for the generation of multi-colored aggregation chimeras that can be used to study cell–cell interactions and lineage analyses in living embryos/organs, where the transgenes can be detected and the clonal origin of a given cell population easily traced by its distinct fluorescence. In this study, to verify if Tg fluorescent mice generated through PITT were suitable for such applications, we sought to generate chimeric blastocysts and chimeric-Tg mice by aggregating two- or three-colored 8-cell embryos. Our analyses using these models led to the following observations. First, we noticed that cell mixing was infrequent during the stages of morula to early blastocyst. Second, chimeric fetuses obtained after aggregation of the two-colored 8-cell embryos exhibited uniform cell mixing. And third, in the organs of adult chimeric mice, the mode of cell distribution could be either clonal or polyclonal, as previously pointed out by others. Implications of our novel and improved Tg-chimeric mice approach for clonal cell lineage and developmental studies are discussed.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is highly expressed in reactive astrocytes. Increased production of GFAP is a hallmark of astrogliosis in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, the physiological and pathological roles of GFAP, particularly in chronic neurodegenerative conditions, remain unclear. To address this issue, we here investigate whether absence of GFAP affects the phenotypic expression of motor neuron disease (MND) using an H46R mutant Cu/Zn superoxide dismutase-expressing mouse model of ALS (SOD1(H46R)). GFAP deficient SOD1(H46R) mice showed a significant shorter lifespan than SOD1(H46R) littermates. Further, at the end stage of disease, loss of GFAP resulted in increased levels of Vim and Aif1 mRNAs, encoding vimentin and allograft inflammatory factor 1 (AIF1), respectively, in the spinal cord, although no discernible differences in the levels and distribution of these proteins between SOD1(H46R) and GFAP-deficient SOD1(H46R) mice were observed. These results suggest that loss of GFAP in SOD1(H46R) mice marginally accelerates the disease progression by moderately enhancing glial cell activation. Our findings in a mouse model of ALS may have implication that GFAP is not necessary for the initiation of disease, but it rather plays some modulatory roles in the progression of ALS/MND.

A medaka Double anal fin (Da) mutant, spontaneously isolated from the wild-type, exhibits a unique ventralizing phenotype consisting of a mirror-image duplication across the lateral midline in the dorsal trunk-tail region. No similar mutation has been reported in other model organisms. Hence, the Da mutant provides a unique model system for investigation of the mechanisms controlling the overall dorsal–ventral patterning of the vertebrate trunk-tail region. In this chapter, we introduce (1) a brief history of the Da mutant; (2) details of the morphological phenotype of Da; (3) positional cloning of the Da mutation; (4) the Da mutant in relation to fish evolution; and (5) Da as a potential model for human disease.

We developed Cre-loxP-based systems, termed CRTEIL and CETRIZ, which allow gene switching in a noninvasive manner. Single transfection with pCRTEIL resulted in predominant expression of red fluorescence. Cotransfection with pCRTEIL and Cre-expression plasmid (pCAG/NCre) caused switching from red to green fluorescence. Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence. These noninvasive systems will be useful in cell lineage analysis, since descendants of cells exhibiting newly activated gene expression can be continuously monitored in noninvasive fashion.

A large cis-regulatory landscape is a common feature of vertebrate genomes, particularly at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying large genomic regions of interest spanning over hundreds of kilobases are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping trait of the Sleeping Beauty transposon in the mouse genome. We generated embryonic stem cells with a targeted integration of the transposon vector, carrying an enhancer-detecting lacZ reporter and loxP cassette, into the developmentally critical Pax1 gene locus, followed by efficient local transpositions, nested deletion formation and derivation of embryos by tetraploid complementation. Comparative reporter expression analysis among different insertion/deletion embryos substantially facilitated long-range cis-regulatory element mapping in the genomic neighborhood and demonstrated the potential of the transposon-based approach as a versatile tool for exploration of defined genomic intervals of functional or clinical relevance, such as disease-associated microdeletions.

RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (-MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the target gene (eGFP) in +MG hemizygous mice, obtaining -MG amiRNA expression mice, even hemizygotes, was difficult and the animals died perinatally. We obtained only mosaic mice having both "-MG amiRNA" cells and "amiRNA low-expression" cells but they exhibited growth retardation and cataracts, and they could not transmit the -MG amiRNA allele to the next generation. Furthermore, +MG amiRNA homozygotes could not be obtained. These results suggested that excessive amiRNAs transcribed by -MG expression cassettes cause deleterious effects in mice, and the amiRNA expression level in hemizygous +MG amiRNA mice is near the upper limit, where mice can develop normally. In conclusion, the PITT-(+MG amiRNA) system demonstrated here can generate knockdown mouse models that reliably express highest and tolerable levels of amiRNAs.

Hydrodynamic gene delivery has emerged as an effective method for gene transfer in vivo especially to the liver, and the liver-targeted hydrodynamic procedure has been evaluated in various animal models including rats, pigs, and dogs. In an effort to use hydrodynamic gene delivery for treatment of liver diseases that are commonly fibrotic, we have systematically examined the effect of liver fibrosis on efficiency of hydrodynamic gene delivery. Rat liver fibrosis model was developed by the bile duct ligation (BDL) procedure and hyaluronic acid (HA), a serum marker for liver fibrosis, was used to determine the degree of liver fibrosis. Gene delivery efficiency was assessed using luciferase reporter gene plasmid (pCMV-Luc) in normal rats (n=20) and BDL performed rats (liver fibrosis group, n=20) 10 weeks after the BDL, when liver fibrosis group showed significantly higher serum HA concentration of 231±167 ng/ml than that of 28±17 ng/ml in normal rats (p

Liver fibrosis is final stage of chronic liver diseases which may cause liver failure and liver cancer. While various diagnostic methods, including serum marker have been established, no standard therapy has developed. The objective of this study is to assess the approach of overexpressing matrix metalloproteinase-13 gene (MMP13) in rat liver to block liver fibrosis. The rat liver fibrosis model was developed by the bile duct ligation (BDL). And the liver-targeted hydrodynamic gene delivery procedure involves catheter insertion via the inferior vena cava (IVC) to the junction of the IVC and the hepatic veins followed by the hydrodynamic injection of MMP13 expression vector, containing CAG promoter-MMP13-IRES-tdTomato-polyA cassette with temporal blood flow occlusions at the supra- and infra-hepatic IVC. Three groups (n=5 in each group) of animals, BDL alone (non-treated), BDL simultaneously followed by hydrodynamic delivery of pBGI-MMP13 (treated group), and hydrodynamic delivery of pBGI-MMP13 (control) were analyzed for: 1) MMP13 expression in serum and cells in the liver by ELISA, western blotting, immunohistochemical staining, and fluorescent detection; and 2) therapeutic effect on liver fibrosis by analysis of serum fibrotic markers of hyaluronic acid and collagen type IV, and histological analysis including silver staining. Serum concentration of MMP13 in pBGI-MMP13 treated group reached 71.±5.1 pg/ml (p

The GenomiPhi DNA Amplification Kit (Amersham Biosciences Co., Piscataway, NJ, USA) uses a novel, isothermal DNA amplification technique for production of microgram quantities of DNA from picograms of starting material. In this study, we tested whether repeated reactions using the kit would enhance high-fidelity amplification of a small amount (10 ng) of plasmid DNA containing enhanced green fluorescent protein (EGFP) cDNA. Approximately 4.7 x 10(9)-fold amplification of plasmid DNA was achieved after 3 repeated reactions, and the RCA products were sufficient for EGFP expression in cells. Based on the preliminary results, repeated reactions using the kit appears feasible for amplification of the entire genome of organisms or cells (such a sperms and oocytes) carrying very small amounts of DNA.

Type IIS or interrupted palindrome restriction endonuclease digestion produces unique cohesive ends. Utilization of this feature of BglI, SfiI and BstXI has led to direct subcloning of 28.5-kb and 22.9-kb regions, which contains candidate genes for medaka Double anal fin (Da) mutant, from a 229-kb BAC insert. This method does not contain gel-fractionation of the fragments and time-consuming screening process using hybridization. Non-recombinant backgrounds were distinguished from recombinant clones by introducing GFPuv gene into the subcloning vector. This strategy was successfully applicable even when the target region was cut into three fragments by these enzymes.

The GenomiPhi DNA Amplification Kit employs rolling circle amplification (RCA) using phi29 polymerase, dNTPs, and random hexamers. We demonstrated that repeated RCA (at least three times) is useful for high-fidelity amplification of large amounts of plasmid DNA.

Syntenic chromosomal areas share paralogous genes which are believed to have been generated by repeated duplication of an ancestral gene. The human RING3 gene is known to have paralogous relationships with the ORFX, BRDT, and HUNK1 genes. In addition to the mouse Ring3 cDNA clones previously reported, we isolated mouse Orfx, Brdt, and Hunk1 cDNA clones using mouse testis RNA. Among these four paralogous genes, structure and expression profiles were compared. The proteins encoded by these genes exhibited similar amino acid sequences including two conserved bromodomains. While the Ring3, Orfx, and Hunk1 genes were ubiquitously expressed in various tissues of adult mouse, the Ring3, Orfx, and Brdt genes produced testis-specific transcripts and the Hunk1 gene produced a striated muscle-specific transcript. The diversification of expression patterns of Ring3-related genes during evolution may reflect nucleotide variations in regulatory elements associated with ubiquitous or tissue-specific gene expression.

Our aim is to develop a simple gene transfer method into egg cylinder and mid-gestational murine embryos. We examined whether plasmid/lipid complexes injected into the tail veins of pregnant transgenic mice can be transferred to fetuses at E 4.5-13.5. When pregnant CETZ-17 mice carrying a transgene consisting of a ubiquitous promoter, floxed EGFP/CAT and the LacZ gene, were injected with a Cre expression vector DNA/lipid complex, Cre-mediated excision of the transgenes, as evaluated by X-gal staining, occurred in 10-50% of fetuses treated at E 11.5-13.5. Although younger embryos remained unstained, PCR analysis revealed low levels of the Cre vector DNA and recombined transgene. To examine the fate of a solution given intravenously, we injected trypan blue or fluorescence-labeled plasmid DNA/lipid complexes into females at E 5.5-11.5 and E 6.5, respectively. Both collected in the visceral endoderm (VE) lineage, but were undetectable in the embryo proper. These findings suggest that substances in maternal blood are delivered to post-implantation embryos via cells of the VE lineage and placenta, but that most are trapped in the VE. If significantly improved, gene transfer to fetuses by injection into the maternal circulation may become a promising tool in fetal gene therapy and embryological studies.

We recently described a new transgenic technique termed pronuclear injection-based targeted transgenesis (PITT) (Ohtsuka et al. 2010), which enables targeted integration of transgenes into predetermined loci (e.g. Rosa26) by Cre-loxP-based recombination in fertilized eggs, leading to stable and reproducible transgene expression. In this study, two steps of PITT were modified. The donor vector was improved by introducing an FLPe expression cassette (Fig. 1a). Therefore, selfremoval of the extra sequence flanked by FRT sequences occurs without additional FLPe administration. In addition, the plasmid backbone of the donor vector was modified from pUC119-to pBR322based to reduce plasmid instability, which is sometimes associated with genomic DNA fragment cloning into a high-copy plasmid. Moreover, in vitro fertilized eggs were used in this study; in the previous study, fertilized eggs obtained by natural mating were used. In this study, fertilized eggs ([100) were obtained simultaneously using epididymal spermatozoa isolated from a single male carrying the targeted allele. Results are listed in Fig. 1b. Three independent donor vectors, pAWK, pAWV and pAXV, were microinjected with a Cre expression vector into in vitro fertilized eggs. The resulting PITT efficiency was 5/93 (5.4%; 95% CI, 1.8–12.1%), which is similar to that reported previously (4.3%; 95% CI, 2.7–6.5%), indicating the feasibility of this modified method and reproducibility of PITT. Regarding removal of the extra sequence, the recombinasemediated cassette exchange (RMCE) allele was successfully created in 60% of founder mice (3/5; indicated by *). Although the other two founder mice did not possess the RMCE allele, the extra sequences of these founders can be successfully removed by crossing them with an FLPe deleter mouse (Ohtsuka et al. 2010). Notably, all tested lines (5/5) exhibited successful germline transmission Electronic supplementary material The online version of this article (doi:10.1007/s11248-011-9505-y) contains supplementary material, which is available to authorized users.

The Cre-loxP system has been used for genetic engineering in many organisms ranging from E. coli to mouse. It is known that intermolecular recombination between two loxP sites is substantially less frequent than intramolecular recombination between the same sites. Therefore, for the efficient isolation of intermolecular loxP recombination products, it is essential to develop a powerful screening method. Here, we describe the construction of an EYFP variant (EYFP157) that could become a vital tool in such a screening method. EYFP157 contains, between Gln157 and Lys158, the JT15:JTZ17 mutant loxP sequence that is not cleaved by the Cre protein. When EYFP157 was expressed at 37 degrees C in E. coli and mouse cells, fluorescence of the protein was readily detectable under a standard microscope. EYFP157 possessed 1-3% of the fluorescence from the wild-type EYFP. Interestingly, at room temperature EYFP157 fluoresced more efficiently in E. coli cells and thus the difference in fluorescence intensity between EYFP157 and the wild-type EYFP became negligible. The potential application of EYFP157 as a recombination indicator in both E. coli and mouse cells is further discussed.