Generation of transgenic mice expressing Cre recombinase under the control of the Dll1 mesoderm enhancer element

To study paraxial mesoderm formation in the mouse, transgenic lines that can be used to either selectively delete or express genes of interest in the paraxial mesoderm are required. We have generated a transgenic mouse line that expresses Cre recombinase in the paraxial mesoderm (PAM) beginning at e7.5. A lacZ Cre recombinase reporter line showed that in addition to PAM and its derivatives, lateral plate and intermediate mesoderm derivatives were also exposed to Cre activity, while the node, notochord, and cardiac mesoderm were not. We further demonstrate that 70–75% of the fibroblasts generated from Dll1‐msd Cre, ROSA26‐rtTA embryos possess Cre recombinase activity. These mice can therefore be used in combination with tet‐responsive transgenic lines to generate mesoderm‐derived embryonic fibroblasts that inducibly express a gene of interest. genesis 47:309–313, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of the Arabidopsis vacuolar H+‐pyrophosphatase gene (AVP1) improves the shoot biomass of transgenic barley and increases grain yield in a saline field

Cereal varieties with improved salinity tolerance are needed to achieve profitable grain yields in saline soils. The expression of AVP1, an Arabidopsis gene encoding a vacuolar proton pumping pyrophosphatase (H⁺‐PPase), has been shown to improve the salinity tolerance of transgenic plants in greenhouse conditions. However, the potential for this gene to improve the grain yield of cereal crops in a saline field has yet to be evaluated. Recent advances in high‐throughput nondestructive phenotyping technologies also offer an opportunity to quantitatively evaluate the growth of transgenic plants under abiotic stress through time. In this study, the growth of transgenic barley expressing AVP1 was evaluated under saline conditions in a pot experiment using nondestructive plant imaging and in a saline field trial. Greenhouse‐grown transgenic barley expressing AVP1 produced a larger shoot biomass compared to null segregants, as determined by an increase in projected shoot area, when grown in soil with 150 mm NaCl. This increase in shoot biomass of transgenic AVP1 barley occurred from an early growth stage and also in nonsaline conditions. In a saline field, the transgenic barley expressing AVP1 also showed an increase in shoot biomass and, importantly, produced a greater grain yield per plant compared to wild‐type plants. Interestingly, the expression of AVP1 did not alter barley leaf sodium concentrations in either greenhouse‐ or field‐grown plants. This study validates our greenhouse‐based experiments and indicates that transgenic barley expressing AVP1 is a promising option for increasing cereal crop productivity in saline fields.     

Utility of HoxB2 enhancer‐mediated Cre activity for functional studies in the developing inner ear

The rhombomere 4(r4)‐restricted expression of the mouse Hoxb2 gene is regulated by a 1.4‐kb enhancer‐containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2‐r4‐Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5–E12.5, in the cochleovestibular and facio‐acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2‐r4‐Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected. genesis 47:361–365, 2009. © 2009 Wiley‐Liss, Inc.     

Construction and characterization of a doxycycline‐inducible transgenic system in Msx2 expressing cells

Homeobox gene Msx2 is widely expressed during both embryogenesis and postnatal development and plays important roles during organogenesis. We developed an Msx2‐rtTA BAC transgenic line which can activate TetO‐Cre expression in Msx2‐expressing cells upon doxycycline (Dox) treatment. Using the Rosa26‐LacZ (R26R) reporter line, we show that rtTA is activated in Msx2‐expressing organs including the limb, heart, external genitalia, urogenital system, hair follicles and craniofacial regions. Moreover, we show that in body appendages, the transgene can be activated in different domains depending on the timing of Dox treatment. In addition, the transgene can also be effectively activated in adult tissues such as the hair follicle and the urogenital system. Taken together, this Msx2‐rtTA;TetO‐Cre system is a valuable tool for studying gene function in the development of the aforementioned organs in a temporal and spatially‐restricted manner, as well as for tissue lineage tracing of Msx2‐expressing cells. When induced postnatally, this system can also be used to study gene function in adult tissues without compromising normal development and patterning. genesis 47:352–359, 2009. © 2009 Wiley‐Liss, Inc.     

Transthyretin mouse transgenes direct RFP expression or Cre‐mediated recombination throughout the visceral endoderm

Transthyretin (Ttr) is a thyroid hormone transport protein secreted by cells of the visceral yolk sac and fetal liver in developing embryos, and by hepatocytes and the choroid plexus epithelium of the brain in adult mice. Spatiotemporal localization of Ttr mRNA during embryogenesis suggested that Ttr regulatory elements might drive transgene expression throughout the visceral endoderm of early mouse embryos. We use Ttr cis‐regulatory elements to generate Ttr::RFP and Ttr::Cre strains of mice, driving red fluorescent protein (RFP) and a nuclear‐localized Cre recombinase, respectively. Visualization of RFP fluorescence in Ttr::RFP transgenics confirms reporter localization throughout the visceral endoderm in early embryos and in the visceral yolk sac and fetal liver of later stage embryos. Using both GFP‐based and LacZ‐based Cre reporter strains, we demonstrate that in Ttr::Cre transgenics, Cre‐mediated recombination occurs throughout the visceral endoderm. The Ttr::Cre strain can therefore be used as a tool for genetic modifications within the visceral endoderm lineage. genesis 47:447–455, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of a myrosinase cDNA from Brassica parachinensis and its defense role against Plutella xylostella after suppression

Abstract In Brassicaceae, myrosinase catalyzes the hydrolysis of glucosinolate and plays an important role in anti‐herbivore defense. We have cloned and characterized the full‐length complementary DNA of myrosinase gene from Brassica parachinensis that exhibits high sequence identity with myrosinase genes from other Brassica species. To investigate the role of this myrosinase in defense against the diamondback moth (Plutella xylostella), we constructed an RNA‐interference (RNAi) cassette expressing a double‐stranded RNA that targeted myrosinase and transfected it into B. parachinensis. Myrosinase was suppressed in the resulting transgenic plants. Diamondback moth larvae feeding on transgenic plants had lower larval and pupal weights, longer pupal duration, and lower fecundity than those feeding on non‐transgenic plants, suggesting that the diamondback moth has adapted to the glucosinolate‐myrosinase defensive system. Therefore, the suppression of myrosinase is a potential approach for controlling the diamondback moth.     

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests

The adoption of pest‐resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.     

The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.     

CRISPR/Cas9‐mediated genome modification in the mollusc,Crepidula fornicata

The discovery and application of the CRISPR/Cas9 genome editing method has greatly enhanced the ease with which transgenic manipulation can occur. We applied this technology to the mollusc, Crepidula fornicata, and have successfully created transgenic embryos expressing mCherry fused to endogenous β‐catenin. Specific integration of the fluorescent reporter was achieved by homologous recombination with a β‐catenin‐specific donor DNA containing the mCherry coding sequence. This fluorescent gene knock‐in strategy permits in vivo observations of β‐catenin expression during embryonic development and represents the first demonstration of CRISPR/Cas9‐mediated transgenesis in the Lophotrochozoa superphylum. The CRISPR/Cas9 method is a powerful and economical tool for genome modification and presents an option for analysis of gene expression in not only major model systems, but also in those more diverse species that may not have been amenable to the classic methods of transgenesis. This approach will allow one to generate transgenic lines of snails for future studies. genesis 53:237–244, 2015. © 2014 Wiley Periodicals, Inc.     

Generation of cortactin floxed mice and cellular analysis of motility in fibroblasts

Cortactin is an F‐actin binding protein that has been suggested to play key roles in various cellular functions. Here, we generated mice carrying floxed alleles of the cortactin (Cttn) gene (Cttn^flox/flox mice). Expression of Cre recombinase in mouse embryonic fibroblasts (MEFs) isolated from Cttn^flox/flox embryos depleted cortactin within days, without disturbing F‐actin distribution and localization of multiple actin‐binding proteins. Cre‐mediated deletion of Cttn also did not affect cell migration. To obtain mice with a Cttn null allele, we next crossed Cttn^flox/flox mice with transgenic mice that express Cre recombinase ubiquitously. Western blot and immunocytochemical analysis confirmed complete elimination of cortactin expression in MEFs carrying homozygously Cttn null alleles. However, we found no marked alteration of F‐actin organization and cell migration in Cttn null‐MEFs. Thus, our results indicate that depletion of cortactin in MEFs does not profoundly influence actin‐dependent cell motility. genesis 47:638–646, 2009. © 2009 Wiley‐Liss, Inc.     

A Transgenic Mouse Model with Inducible Tyrosinase Gene Expression Using the Tetracycline (Tet‐on) System Allows Regulated Rescue of Abnormal Chiasmatic Projections Found in Albinism

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET‐ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.     

Transgenic zebrafish line with over-expression of Hedgehog on the skin: a useful tool to screen Hedgehog-inhibiting compounds

We generated a transgenic line Tg(k18:shh:RFP) with overexpression of Sonic hedgehog in the skin epidermis. By 5 day-post-fertilization (dpf), many epidermal lesions were clearly observed, including a swollen yolk sac, epidermis growth malformation around the eyes and at the basement of the pectoral fins. Skin histology revealed embryos derived from Tg(k18:shh:RFP) displayed an elevated Nuclear/Cytoplasmic ratio and pleomorphic nuclei compared to their wild type littermates, suggesting the abnormal growth pattern on the epidermis of Tg(k18:shh:RFP) embryos were dysplasia. Later (by 7 dpf), Tg(k18:shh:RFP) embryos displayed broader pectoral fins which are similar to the polydactyly phenotypes of Nevoid basal cell carcinoma syndrome (NBCCS)/Gorlin patients and polydactylous mice. In addition, treatment with cyclopamine is able to enhance and prolong the survival rates and survival durations of Tg(k18:shh:RFP) embryos. In conclusion, this unique Tg(k18:shh:RFP) fish line, should be an excellent experimental animal for screening for a lower toxicity level of the new Hh-inhibitor and can even be used as a new anti-cancer drug-screening platform.     

A novel reporter rat strain that expresses LacZ upon Cre‐mediated recombination

The recent widespread application of Cre/loxP technology has resulted in a new generation of conditional animal models that can better recapitulate many salient features of human disease. These models benefit from the ability to monitor the expression and functionality of Cre protein. We have generated a conditional (Cre/loxP dependent) LacZ reporter rat (termed the LacZ541 rat) to monitor Cre in transgenic rats. When LacZ541 rats were bred with another transgenic rat line expressing Cre recombinase under the control of the CAG promoter, LacZ/Cre double transgenic embryos displayed ubiquitous expression of LacZ, and when LacZ541 rats were bred with transgenic rats expressing Cre/loxP‐dependent oncogenic H‐ or K‐ras, LacZ was expressed in the lesions resulting from the activation of the oncogene. The LacZ541 rat enables evaluation of the performance of Cre‐expressing systems which are based upon transgenic rats or somatic gene transfer vectors and provides efficient and simple lineage marking. genesis 51:268–274. © 2013 Wiley Periodicals, Inc.     

Expression of Indica rice <Emphasis Type="Italic">OsBADH1</Emphasis> gene under salinity stress in transgenic tobacco

Glycine betaine has been reported as an osmoprotectant compound conferring tolerance to salinity and osmotic stresses in plants. We previously found that the expression of betaine aldehyde dehydrogenase 1 gene (OsBADH1), encoding a key enzyme for glycine betaine biosynthesis pathway, showed close correlation with salt tolerance of rice. In this study, the expression of the OsBADH1 gene in transgenic tobacco was investigated in response to salt stress using a transgenic approach. Transgenic tobacco plants expressing the OsBADH1 gene were generated under the control of a promoter from the maize ubiquitin gene. Three homozygous lines of T₂ progenies with single transgene insert were chosen for gene expression analysis. RT-PCR and western blot analysis results indicated that the OsBADH1 gene was effectively expressed in transgenic tobacco leading to the accumulation of glycine betaine. Transgenic lines demonstrated normal seed germination and morphology, and normal growth rates of seedlings under salt stress conditions. These results suggest that the OsBADH1 gene could be an excellent candidate for producing plants with osmotic stress tolerance.     

Expression of Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato confers stress tolerance without affecting yield

Modern‐day plants are subjected to various biotic and abiotic stresses thereby limiting plant productivity and quality. It has previously been reported that the use of a strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive the expression of Arabidopsis CBF1 in tomato improved tolerance to cold, drought and salt loading, at the expense of growth and yield under normal growth conditions. Hence in the present study, the suitability of expressing the Arabidopsis CBF1 driven by three copies of an ABA‐responsive complex (ABRC1) from the barley HAV22 gene in order to improve the agronomic performance of the transgenic tomato plants was investigated. Northern blot analysis indicated that CBF1 gene expression was induced by chilling, water‐deficit and salt treatment in the transgenic tomato plants. Under these tested stress conditions, transgenic tomato plants exhibited enhanced tolerance to chilling, water‐deficit, and salt stress in comparison with untransformed plants. Under normal growing conditions the ABRC1‐CBF1 tomato plants maintained normal growth and yield similar to the untransformed plants. The results demonstrate the promise of using ABRC1‐CBF1 tomato plants in highly stressed conditions which will in turn benefit agriculture.     

The wheat Lr34 multipathogen resistance gene confers resistance to anthracnose and rust in sorghum

The ability of the wheat Lr34 multipathogen resistance gene (Lr34res) to function across a wide taxonomic boundary was investigated in transgenic Sorghum bicolor. Increased resistance to sorghum rust and anthracnose disease symptoms following infection with the biotrophic pathogen Puccinia purpurea and the hemibiotroph Colletotrichum sublineolum, respectively, occurred in transgenic plants expressing the Lr34res ABC transporter. Transgenic sorghum lines that highly expressed the wheat Lr34res gene exhibited immunity to sorghum rust compared to the low‐expressing single copy Lr34res genotype that conferred partial resistance. Pathogen‐induced pigmentation mediated by flavonoid phytoalexins was evident on transgenic sorghum leaves following P. purpurea infection within 24–72 h, which paralleled Lr34res gene expression. Elevated expression of flavone synthase II, flavanone 4‐reductase and dihydroflavonol reductase genes which control the biosynthesis of flavonoid phytoalexins characterized the highly expressing Lr34res transgenic lines 24‐h post‐inoculation with P. purpurea. Metabolite analysis of mesocotyls infected with C. sublineolum showed increased levels of 3‐deoxyanthocyanidin metabolites were associated with Lr34res expression, concomitant with reduced symptoms of anthracnose.     

Hairpin RNA derived from viral NIa gene confers immunity to wheat streak mosaic virus infection in transgenic wheat plants

Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importance in the Great Plains of the United States and Canada. Recently, the virus has been identified in Australia, where it has spread quickly to all major wheat growing areas. The difficulties in finding adequate natural resistance in wheat prompted us to develop transgenic resistance based on RNA interference (RNAi). An RNAi construct was designed to target the nuclear inclusion protein ‘a’ (NIa) gene of WSMV. Wheat was stably cotransformed with two plasmids: pStargate‐NIa expressing hairpin RNA (hpRNA) including WSMV sequence and pCMneoSTLS2 with the nptII selectable marker. When T₁ progeny were assayed against WSMV, ten of sixteen families showed complete resistance in transgenic segregants. The resistance was classified as immunity by four criteria: no disease symptoms were produced; ELISA readings were as in uninoculated plants; viral sequences could not be detected by RT‐PCR from leaf extracts; and leaf extracts failed to give infections in susceptible plants when used in test‐inoculation experiments. Southern blot hybridization analysis indicated hpRNA transgene integrated into the wheat genome. Moreover, accumulation of small RNAs derived from the hpRNA transgene sequence positively correlated with immunity. We also showed that the selectable marker gene nptII segregated independently of the hpRNA transgene in some transgenics, and therefore demonstrated that it is possible using these techniques, to produce marker‐free WSMV immune transgenic plants. This is the first report of immunity in wheat to WSMV using a spliceable intron hpRNA strategy.     

Transgenic Tobacco Expressing Zephyranthes candida Agglutinin Showing Enhanced Resistance to Aphids

Aphid is one of the most serious, sap‐sucking insect pests which cause significant losses of crop yields. The aim of this study is to investigate whether transgenic plants expressing Zephyranthes candida agglutinin (ZCA) could confer enhanced resistance to aphids. Tobacco was transformed with a plasmid, pCAMBIAZCA, containing the marker genes nptII and gusA and the Zephyranthes candida agglutinin gene (zca) via Agrobacterium tumefaciens‐mediated transformation. Twenty‐six independent transgenic plants were regenerated. Western blot analysis revealed ZCA expression at various levels in transgenic plants. Insect bioassay tests showed that transgenic plants expressing a high level of ZCA significantly inhibited the growth of the population of peach potato aphids (Myzus persicae Sulzer). This is the first report in which transgenic plants expressing ZCA conferred enhanced resistance to aphids. Our study suggests the zca gene could be a useful candidate for genetic engineering strategies in plants to provide aphid resistance.