Neomycin Phosphotransferase Research Articles

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

BackgroundClustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.ResultsWe describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.ConclusionsHere we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).

Establishment of neomycin phosphotransferase II (nptII) selection for transformation of soybean somatic embryogenic cultures

Soybean transformation is limited by the lack of multiple efficient selectable marker systems. Biolistic transformation of somatic proliferative embryogenic cultures, one of the commonly used soybean transformation methods, relies largely on hygromycin phosphotransferase II (hptII) selection. The purpose of the present study was to establish another efficient selectable marker system to facilitate multiple gene transformations of soybean. We tested neomycin phosphotransferase II (nptII) that has been used successfully in cotyledonary node transformation, but with limited success in transformation of embryogenic cultures. Transgenic events were obtained using nptII with improved G418 selection without generating escapes. G418 selection required longer recovery and selection periods, and resulted in a lower efficiency of initial transformants compared to hygromycin selection. Six independent fertile transgenic plants were recovered using nptII and G418, a frequency similar to that obtained with hygromycin selection. Soybean embryogenic cultures co-transformed with the hptII and nptII markers showed resistance to both hygromycin B and G418, while regeneration and plant fertility were not adversely affected. The nptII will be useful as a second selectable marker for multiple gene transformations in basic and applied soybean research.

Agrobacterium-mediated transformation of Camelina sativa for production of transgenic plants.

Camelina sativa (C. sativa), an oilseed species rich in poly-unsaturated fatty acids, has gained great importance as an industrial oil platform crop in recent years. Despite the potential benefits of C. sativa for bioenergy applications, limited research has been conducted to improve its agronomic qualities. Hence, a simple and efficient technique for production of transgenic C. sativa plants is warranted. In the present study, shoot apical meristems of two C. sativa cultivars (Pl650159 and Pl650161) were transformed with Agrobacterium strain ‘EHA 105’ harboring the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase II (nptII) genes. After two days of co-cultivation in the dark, explants were transferred to selection medium. Transgenic shoots were identified on the basis of green fluorescence and kanamycin resistance. Shoots were then rooted and transferred to potting mix soil for acclimatization. This protocol describes an efficient method to generate transgenic C. sativa plants in as little as 4 weeks.

Development of a multiplex fluorescence quantitative PCR for detection of genetically modified organisms

The commercially available genetically modified plants authorized worldwide and therefore the target sequences for molecular detection of genetically modified organisms (GMOs) are ever-increasing. The European Union has implemented a set of very strict procedures for approval to grow, import and/or utilize GMOs as food or food ingredients. As a result, GMO laboratories and food production industry currently are forced to apply different methods to test raw material and complex processed food products. Three exogenous genes (the 35 s promoter of the cauliflower mosaic virus (35 s), nos terminator from Agrobacterium tumefaciens (nos), and the neomycin phosphotransferase II (nptII) gene) are commonly used in GMO detection. In this paper, a multiplex quantitative real-time PCR (qPCR) system was developed which allows simultaneously detection of the three exogenous genes in one reaction tube. The determined limits for the multiplex qPCR assays were 4 copies/reaction in maize samples. The specificity of the assays was demonstrated to be 100% according to the detection results of 23 genetically modified (GM) crops and 97 complex processed food products. The validation data show the individual PCR efficiency was accredited with negligible impacts between three detection channels in 7500 fluorescence quantitative PCR machine. These results indicate that this high-throughput multiplex qPCR method which combined with a reference gene is feasible for screening of GMOs, even for the processed food.

Efficient genetic transformation of Momordica charantia L. by microprojectile bombardment.

Here, we report the optimized conditions for biolistic particle delivery-mediated genetic transformation of bitter melon using petiole segments. In this study, DNA-coated gold particles of 0.6µm were used for optimizing the parameters of transformation and eventually regeneration of bitter melon putative transgenics. Initially, biolistic parameters namely helium pressure and macrocarrier to target tissue distance, were optimized using binary vector pBI121 carrying both β-glucuronidase gene (GUS) and neomycin phosphotransferase II gene (npt II) as a reporter and as a selectable marker gene, respectively. The effect of optimized physical parameters on the frequency of transient (79.2±1.52%) and stable (41.9%) expressions has been investigated. The optimized biolistic parameters for petiole segments of Momordica charantia L. were determined as follows: 650 psi helium pressure and 6cm target distance. Using the optimized parameters, transformation of bitter melon was carried out for generation of putative transformants from bombarded tissues on SRM-K medium, with a mean number of 50.3 explants surviving at the end of the final selection (50mgl-1 kanamycin) round. Finally, the transformants produced were subjected to GUS histochemical assay, and integration of the transgenes (GUS and npt II) into the nuclear genome was confirmed by PCR analysis. DNA blot analysis confirmed the transgene integration in the transformed plantlet genomes. The present study may be used for developing transplastomic technology in this valuable medicinal plant for enhanced metabolic engineering pathways and production of biopharmaceuticals.

Genetic transformation of the white-rot fungus Dichomitus squalens using a new commercial protoplasting cocktail

D. squalens, a white-rot fungus that efficiently degrades lignocellulose in nature, can be used in various biotechnological applications and has several strains with sequenced and annotated genomes. Here we present a method for the transformation of this basidiomycete fungus, using a recently introduced commercial ascomycete protoplasting enzyme cocktail, Protoplast F. In protoplasting of D. squalens mycelia, Protoplast F outperformed two other cocktails while releasing similar amounts of protoplasts to a third cocktail. The protoplasts released using Protoplast F had a regeneration rate of 12.5% (±6 SE). Using Protoplast F, the D. squalens monokaryon CBS464.89 was conferred with resistance to the antibiotics hygromycin and G418 via polyethylene glycol mediated protoplast transformation with resistance cassettes expressing the hygromycin phosphotransferase (hph) and neomycin phosphotransferase (nptII) genes, respectively. The hph gene was expressed in D. squalens using heterologous promoters from genes encoding β-tubulin or glyceraldehyde 3-phosphate dehydrogenase. A Southern blot confirmed integration of a resistance cassette into the D. squalens genome. An average of six transformants (±2 SE) were obtained when at least several million protoplasts were used (a transformation efficiency of 0.8 (±0.3 SE) transformants per μg DNA). Transformation of D. squalens demonstrates the suitability of the Protoplast F cocktail for basidiomycete transformation and furthermore can facilitate understanding of basidiomycete gene function and development of improved strains for biotechnological applications.

Effective Screening of Transgenic Pigeonpea in Presence of Negative Selection Agents

Selection of putative transformants is an essential step in transgenic plant development program of important pulse crop pigeonpea. The multiple shoot induction of pigeonpea was studied under the influence of three negative selection agents kanamycin, hygromycin and glufosinate ammonium. Minimum inhibitory concentration of these selection agents on shoot regeneration was determined. These observations were then applied to genetic transformation studies where 100 mg/l kanamycin and 2.0 mg/l glufosinate ammonium were used to select the neomycin phosphotransferase II (nptII) and bar expressing T0 pigeonpea transformants, respectively. Hygromycin was found to be detrimental for shoot regeneration in all applied concentrations. T1 seeds obtained from nptII positive events were screened by incubating 5 h in 100 mg/l kanamycin solution prior to germination. T1 events containing bar gene were selected by painting 2.0 mg/l solution of glufosinate ammonium on adaxial surfaces of the leaves. Positive transformants survived in both the selection processes, were further confirmed by polymerase chain reaction analysis. Knowledge generated through this study will facilitate the incorporation of agronomically important traits in transgenic pigeonpea for its betterment.

Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

ABSTRACTAn efficient indirect somatic embryogenesis and Agrobacterium-mediated transformation protocol for Limonium sinense has been established, wherein neomycin phosphotransferase II (npt II) and β-glucuronidase (GUS) genes were used as selectable and screenable markers, respectively. The efficiency of plantlet regeneration from transformed tissue was compared between direct embryogenesis from leaf and indirect embryogenesis from callus. Embryogenic callus (EC) was initiated from leaf explants on MS medium supplemented with 6.7 μM 2,4-D and 2.22 μM BA. The somatic embryos were induced, matured, and germinated when ECs were transferred onto MS medium supplemented with 4.44 μM BA and 1.07 μM NAA. Agrobacterium tumefaciens strain LBA 4404 containing the vector pBI121 was used for the transformation. Transient GUS expression frequency was evaluated and putative transgenic plants were successfully grown on culture medium in presence of kanamycin (80–100 mg L−1). PCR analysis of putative transgenic plants confirmed the presence of GUS and nptII genes. The transformation efficiency obtained through indirect embryogenesis from calluses (4%) was much higher than through direct embryogenesis from leaf explants (0.9%).

An efficient method for transgenic callus induction from Vitis amurensis petiole.

Transformation is the main platform for genetic improvement and gene function studies in plants. However, the established somatic embryo transformation system for grapevines is time-consuming and has low efficiency, which limits its utilization in functional genomics research. Vitis amurensis is a wild Vitis species with remarkable cold tolerance. The lack of an efficient genetic transformation system for it has significantly hindered the functional identification of cold stress related genes in the species. Herein, an efficient method was established to produce transformed calli of V. amurensis. Segments of petioles from micropropagated plantlets of V. amurensis exhibited better capacity to differentiate calli than leaf-discs and stem segments, and thus was chosen as target tissue for Agrobacterium-mediated transformation. Both neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (eGFP) genes were used for simultaneous selection of transgenic calli based on kanamycin resistance and eGFP fluorescence. Several parameters affecting the transformation efficiency were optimized including the concentration of kanamycin, Agrobacterium stains, bacterial densities, infection treatments and co-cultivation time. The transgenic callus lines were verified by checking the integration of NPTII gene into calli genomes, the expression of eGFP gene and the fluorescence of eGFP. Up to 20% of the petiole segments produced transformed calli after 2 months of cultivation. This efficient transformation system will facilitate the functional analysis of agronomic characteristics and related genes not only in V. amurensis but also in other grapevine species.

PREPARATION OF PLANT VECTOR CONSTRUCT CONTAINING DEHYDRIN GENE At2g21490

This work is focused on preparation of a plant transformation vector pDH2 containing the expression unit of the dehydrin gene At2g21490 under a control of the constitutive dCaMV 35S promoter and the selectable marker neomycin phosphotransferase gene controlled by the nos promoter. The gene At2g21490 is classified as a dehydrin of the type K2SY2. The gene was isolated from Arabidopsis thaliana by PCR approach. The plasmid pDH2 was transformed into the strain Agrobacterium tumefaciens LBA 4404. The stability of the plasmid pDH2 in Agrobacterium has been verified by restriction analysis after re-transformation of the pDH2 into Escherichia coli.

5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level.

Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2days.

Transformation of pomegranate (Punica granatum l.) a difficult-to-transform tree

Abstract In this study, an Agrobacterium-mediated transformation method was developed for pomegranate, a difficult-to-transform plant. We first optimized callus induction and shoot regeneration efficiency. Induction of calluses was best achieved from the intermodal stem sections on WPM medium supplemented with 12 μM BA and 8 μM NAA. The highest frequency of shoot regeneration (69.33%) and number of shoots (7.16) per piece of callus were obtained when calluses were incubated on WPM medium supplemented with 12 μM BA and 2 μM NAA. Callus pieces were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBin19 carrying the neomycin phosphotransferase (nptII) gene as a selectable marker and green fluorescent protein (GFP) gene as a reporter. After 4 weeks in WPM selection medium (the best callus induction medium supplemented with 50 mg L−1 kanamycin), putative transgenic calluses were obtained. From such calluses transformed shoots began to appear approximately 3 week after the kanamycin resistance transgenic callus pieces had been placed on the best shoot regeneration medium containing 50 mg L−1 kanamycin. GFP fluorescence was observed in putatively transformed calluses and shoots. Molecular analysis confirmed the integration of the nptII transgene in transformed shoots. The transgenic shoots were rooted and successfully acclimatized.

Development of an Efficient Micropropagation-based Agrobacterium-Mediated Genetic Transformation Protocol in Commercial Cultivar of Jute (Corchorus capsularis L.)

The present study describes an efficient in vitro culture protocol for direct plantlet regeneration and Agrobacterium- mediated genetic transformation of Corchorus capsularis L. cultivar JRC 517. In vitro morphogenetic capacity of different explants was used as parameter of performance adjudgement. Nodal explants with immature axillary buds when cultured on MS with 0.1 mg l-1 IAA and 0.1 mg l-1 Kin treatment showed maximum in vitro culture response (95%) with plantlets regeneration of 5.67 ± 0.30 %. A. tumefaciens strain LBA4404 harbouring a binary vector pBI121, containing gus A reporter gene under the transcriptional control of Cauliflower Mosaic Virus (CaMV) 35S promoter and NOS terminator was used in addition with neomycin phosphotransferase (npt-II) as plant selection marker gene. Different parameters were tested to accomplish utmost transformation competence, which indicates that O.D600. - of Agrobacterium cell suspension: 0.3; one day preculture; vacuum infiltration for 10 min at 600 mm Hg pressure; three days co-cultivation; 100 μM acetosyringone (AS) and 0.001 ml l-1 concentration of non-ionic surfactant (Tween 20) were found to be optimum treatment to achieve maximum number of stable genetic transformants (~3.6%). The putative transformants were screened on MS medium supplemented with 50 mg l-1 kanamycin (Kan50) and their transient expression was confirmed through GUS histochimical assay and PCR analysis. The survivor plants were grown under Kan 50 selection pressure, and rooted successfully. Regenerated plantlets were acclimatized, hardened and transplanted to green house. Stable integration of the transgene into the recipient genome was confirmed by PCR using compatible primers of gusA and nptII, and Southern hybridization. The transgenic plants showed normal morphology and most of them followed 3:1 ratio of Mendelian inheritance for single dominant locus. Our findings documenting the in vitro direct shoot regeneration protocol from nodal explants with concurrent transgenic development there from deemed to be successfully involving economically important gene/s and trait enrichment in jute in future.

Genetic transformation of gentian Gentiana tibetica (King) leaf explants with Agrobacterium tumefaciens strain C58C1

Experiments were carried out on transformation of leaf explants derived from the axenic culture of Gentiana tibetica (King) using a co-culture of Agrobacterium tumefaciens. A. tumefaciens octopine strain C58C1 carried neomycine phosphotransferase (nptII) and β-glucuronidase (uidA) genes. The influence of plasmid helper pCH32 on transformation efficiency was stressed. After co-cultivation, explant was cultured on three different regeneration media (RM1-3) at the presence of the timentin and callus formation and plants regeneration occurred. The transgenic character of the selected tissue and transformants T0 in the presence of kanamycin has been confirmed by the histo-chemical analysis of reporter enzyme activity (ß-glucuronidase), polymerase chain reaction, and southern hybridization detecting uidA and nptII genes. l-glutamine used during inoculation period had a significant influence on the transformation efficiency in relation to its concentration and time of treatment.

In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane.

Sugarcane (Saccharum sp. hybrids) is one of the most efficient and sustainable feedstocks for commercial production of fuel ethanol. Recent efforts focus on the integration of first and second generation bioethanol conversion technologies for sugarcane to increase biofuel yields. This integrated process will utilize both the cell wall bound sugars of the abundant lignocellulosic sugarcane residues in addition to the sucrose from stem internodes. Enzymatic hydrolysis of lignocellulosic biomass into its component sugars requires significant amounts of cell wall degrading enzymes. In planta production of xylanases has the potential to reduce costs associated with enzymatic hydrolysis but has been reported to compromise plant growth and development. To address this problem, we expressed a hyperthermostable GH10 xylanase, xyl10B in transgenic sugarcane which displays optimal catalytic activity at 105 °C and only residual catalytic activity at temperatures below 70 °C. Transgene integration and expression in sugarcane were confirmed by Southern blot, RT-PCR, ELISA and western blot following biolistic co-transfer of minimal expression cassettes of xyl10B and the selectable neomycin phosphotransferase II. Xylanase activity was detected in 17 transgenic lines with a fluorogenic xylanase activity assay. Up to 1.2% of the total soluble protein fraction of vegetative progenies with integration of chloroplast targeted expression represented the recombinant Xyl10B protein. Xyl10B activity was stable in vegetative progenies. Tissues retained 75% of the xylanase activity after drying of leaves at 35 °C and a 2 month storage period. Transgenic sugarcane plants producing Xyl10B did not differ from non-transgenic sugarcane in growth and development under greenhouse conditions. Sugarcane xylan and bagasse were used as substrate for enzymatic hydrolysis with the in planta produced Xyl10B. TLC and HPLC analysis of hydrolysis products confirmed the superior catalytic activity and stability of the in planta produced Xyl10B with xylobiose as a prominent degradation product. These findings will contribute to advancing consolidated processing of lignocellulosic sugarcane biomass.

Establishment of an efficient Agrobacterium-mediated genetic transformation system in Pelargonium graveolens: an important aromatic plant

Rose-scented geranium is an important aromatic herb, have eminent for oil. The oil of geranium commercially utilized in the perfumery, cosmetic and aromatherapy industries all over the world. It is also helpful to cure many of the diseases, since it possess antibacterial, antifungal, antioxidant, anti-inflammatory and anticancer activities. However rose scented geranium suffer from several biotic and abiotic stresses, which reduced the yield of oil. So there is need to genetically improve the geranium using biotechnological approaches. The present study demonstrates the establishment of direct regeneration and Agrobacterium tumefaciens (LBA4404) mediated transformation protocol in Pelargonium graveolens (cv. CIM-BIO 171). Different media combinations such as benzyl amino purine (BAP), kinetin, naphthalene acetic acid (NAA), and adenine di-sulphate (ADS) were standardised to induce direct regeneration in P. graveolens. The maximum regeneration frequency i.e. 90.56 ± 1.2% per explant was achieved from petiolar segments in medium containing 2.5 mg/l BAP, 0.1 mg/l NAA, 1 mg/l ADS. However, with the leaf explants only 45.94 ± 2.91% frequency was achieved. In the present study, A. tumefaciens strain LBA4404 was used carrying binary vector pBI121 with the gusA as a reporter gene and neomycin phosphotransferase II (nptII) gene as a plant selectable marker. Parameters like bacterial optical density, infection time, acetosyringone concentration and kanamycin concentration were optimised to achieve maximum transformation frequency (69.5 ± 2.3%).The putative transgenic shoots were subsequently rooted on half strength MS medium and successfully transferred to the greenhouse. The transgenic plants were characterised by gus histochemical assay, PCR analysis (nptII-786 bp and gus A- 1707 bp) and Southern hybridization tests using gusA gene probe. The regeneration as well as transformation protocol will no doubt provide the basis to decipher the insights of metabolic pathways in geranium. Also could be useful for genetic improvement, to make it more tolerant/resistant against biotic and abiotic stresses and ultimately fruitful for Indian farmers in agronomic traits like high biomass, oil content, yield and better quality.

Genetic transformation of pigeonpea (Cajanus cajan L.) and screening transgenic progenies based on lateral root inhibition

Production of transgenic pigeonpea is becoming increasingly important, but the methods currently employed in production and subsequent screening still requires improvement. Here, we describe Agrobacterium-mediated genetic transformation of pigeonpea with reporter uidA (gus) gene and selectable marker, neomycin phospho-transferase (nptII) gene. Histochemical assay demonstrate localization of gus activity in cells and transformed plants. Overall, a transformation frequency of 0.33% was achieved using the protocol. Grafting of in vitro-regenerated healthy shoots indicates higher survival percent (72.6%), when stock and scion are of the same variety. Seeds harvested from primary transgenic plants can be screened based on lateral root inhibition strategy. Approximately 87% of the screened T1 plants were found to be PCR positive. In conclusion, in vitro grafting of transgenic pigeonpea shoots leads to better plant establishment and screening based on lateral root inhibition leads to quick identification of positive segregants.

Distribution of corticotropin-releasing factor neurons in the mouse brain: a study using corticotropin-releasing factor-modified yellow fluorescent protein knock-in mouse.

We examined the morphological features of corticotropin-releasing factor (CRF) neurons in a mouse line in which modified yellow fluorescent protein (Venus) was expressed under the CRF promoter. We previously generated the CRF-Venus knock-in mouse, in which Venus is inserted into the CRF gene locus by homologous recombination. In the present study, the neomycin phosphotransferase gene (Neo), driven by the pgk-1 promoter, was deleted from the CRF-Venus mouse genome, and a CRF-Venus∆Neo mouse was generated. Venus expression is much more prominent in the CRF-Venus∆Neo mouse when compared to the CRF-Venus mouse. In addition, most Venus-expressing neurons co-express CRF mRNA. Venus-expressing neurons constitute a discrete population of neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) that project to the median eminence. Venus-expressing neurons were also found in brain regions outside the neuroendocrine PVH, including the olfactory bulb, the piriform cortex (Pir), the extended amygdala, the hippocampus, the neocortices, Barrington's nucleus, the midbrain/pontine dorsal tegmentum, the periaqueductal gray, and the inferior olivary nucleus (IO). Venus-expressing perikarya co-expressing CRF mRNA could be observed clearly even in regions where CRF-immunoreactive perikarya could hardly be identified. We demonstrated that the CRF neurons contain glutamate in the Pir and IO, while they contain gamma-aminobutyric acid in the neocortex, the bed nucleus of the stria terminalis, the hippocampus, and the amygdala. A population of CRF neurons was demonstrated to be cholinergic in the midbrain tegmentum. The CRF-Venus∆Neo mouse may be useful for studying the structural and functional properties of CRF neurons in the mouse brain.

35S Promoter Methylation in Kanamycin-Resistant Kalanchoe (Kalanchoe pinnata L.) Plants Expressing the Antimicrobial Peptide Cecropin P1 Transgene.

Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.

Modification of plant regeneration medium decreases the time for recovery of Solanum lycopersicum cultivar M82 stable transgenic lines

Tomato (Solanum lycopersicum) has rapidly become a valuable model species for a variety of studies including functional genomics. A high-throughput method to obtain transgenic lines sooner than standard methods would greatly advance gene function studies. The goal of this study was to optimize our current transformation method by investigating medium components that would result in a decreased time for recovery of transgenics. For this study, 6-day-old cotyledon explants from Solanum lycopersicum cultivar M82 in vitro-grown seedlings were infected with the Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBI121. This vector contains the β-glucuronidase reporter gene and the neomycin phosphotransferase II selectable marker gene that confers resistance to kanamycin. Modification of our standard plant regeneration medium with indole-3-acetic acid (IAA) at concentrations of either 0.05 or 0.1 mg/l decreased the recovery time for transgenic lines by 6 weeks as compared to our standard medium that contains zeatin as the only plant growth regulator. We observed 50 and 54 % transformation efficiency on plant regeneration medium containing 0.05 and 0.1 mg/l IAA, respectively. Moreover, addition of 1 mg/l IAA to the root induction medium resulted in earlier root development than medium that did not contain IAA. Addition of IAA to the plant regeneration and rooting media did not have any negative effects on plant development. Recovery of transgenic lines in a shorter time results in higher throughput for the introduction of gene constructs and has the potential to decrease the time and resources needed to complete investigations of gene function.