Mediated DNA Transfer Research Articles

REVIEW ON TRANSGENIC TECHNOLOGY IN LIVESTOCK: CURRENT STATUS AND FUTURE HORIZONS


 
 
 Transgenic technologies are one of the most promising arenas in the field of biotechnology. These techniques employ genetic engineering techniques to insert foreign genes of interest into genome of an individual. These foreign genes are inherited and expressed by the recipient‟s offspring.
 
 
 
 Two things determine the success of any transgenic technique namely the transport of foreign piece of DNA across the recipient‟s cell membrane and delivery of that DNA across the nuclear membrane gaining access to the chromosomes of the recipient. Many transgenic techniques are employed for genetic manipulation like microinjection, somatic cell nuclear transfer, chemical methods, restriction enzyme mediated gene transfer, retrovirus mediated gene transfer as well as embryonic stem cell and sperm mediated DNA transfer etc. The transgenic animals can be employed in drug discovery, disease research and xenotransplantation etc. This review summarizes various transgenic techniques, applications and future prospects of the transgenic technology.
 
 
 
 
 

The Rhodobacter capsulatus gene transfer agent is induced by nutrient depletion and the RNAP omega subunit.

Small bacteriophage-like particles called gene transfer agents (GTAs) that mediate DNA transfer between cells are produced by a variety of prokaryotes. The model GTA, produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), is controlled by several cellular regulators, and production is induced upon entry into the stationary phase. We report that RcGTA production and gene transfer are stimulated by nutrient depletion. Cells depleted of organic carbon or blocked for amino acid biosynthesis increased RcGTA production and release from cells. Furthermore, cells lacking the sole RelA-SpoT homologue produced decreased levels of RcGTA, and the RNA polymerase omega (ω) subunit was required for appreciable production of RcGTA.

Development of pollen mediated activation tagging system for Phalaenopsis and Doritaenopsis

In the present study, a novel plant transformation system for Doritaenopsis and Phalaenopsis has been developed. The pollen-mediated activation tagging system was established by artificial pollination. The pollens, co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring an activation tagging vector (pTAG-8), were used for pollination. In order to optimize the transformation efficiency, several factors (concentration of A. tumefaciens , concentration of acetosyringone during co-cultivation and the duration of co-cultivation) known to influence Agrobacterium -mediated DNA transfer were examined. A concentration of 0.5-1 x 10 8 CFU/ml for A. tumefaciens, 0.1 mM acetosyringone, and 6 hrs of co-culture period were found to be the optimal condition for high transformation efficiency. Integration of T-DNA into the genome of putative transgenic plants was confirmed by PCR and DNA blot analyses. Single copy of the transgene was observed in all transgenic plants analyzed. Most of the transgenic plants had a morphologically normal phenotype and the overall capsule formation efficiency was similar to control plant. Our results showed a new approach of genetic transformation in orchids and this method can be employed for genetic improvement of the orchids.

Elicitation of Epithelial Cell-Derived Immune Effectors by Outer Membrane Vesicles of Nontypeable Haemophilus influenzae

Outer membrane vesicles (OMVs) are produced by all Gram-negative microorganisms studied to date. The contributions of OMVs to biological processes are diverse and include mediation of bacterial stress responses, selective packaging and secretion of virulence determinants, modulation of the host immune response, and contributions to biofilm formation and stability. First characterized as transformasomes in Haemophilus, these membranous blebs facilitate transfer of DNA among bacteria. Nontypeable Haemophilus influenzae (NTHI), an opportunistic pathogen of the upper and lower respiratory tracts, produces OMVs in vivo, but there is a paucity of information regarding both the composition and role of OMVs during NTHI colonization and pathogenesis. We demonstrated that purified NTHI vesicles are 20 to 200 nm in diameter and contain DNA, adhesin P5, IgA endopeptidase, serine protease, and heme utilization protein, suggesting a multifaceted role in virulence. NTHI OMVs can bind to human pharyngeal epithelial cells, resulting in a time- and temperature-dependent aggregation on the host cell surface, with subsequent internalization. OMVs colocalize with the endocytosis protein caveolin, indicating that internalization is mediated by caveolae, which are cholesterol-rich lipid raft domains. Upon interaction with epithelial cells, NTHI OMVs stimulate significant release of the immunomodulatory cytokine interleukin-8 (IL-8) as well as the antimicrobial peptide LL-37. Thus, we demonstrated that NTHI OMVs contain virulence-associated proteins that dynamically interact with and invade host epithelial cells. Beyond their ability to mediate DNA transfer in Haemophilus, OMV stimulation of host immunomodulatory cytokine and antimicrobial peptide release supports a dynamic role for vesiculation in NTHI pathogenesis and clinically relevant disease progression.

Sperm and testis mediated DNA transfer as a means of gene therapy

Assisted reproductive technologies (ART) have revolutionized the treatment of infertility. However, many types of infertility may still not be addressable by ART. With recent successes in identifying many of the genetic factors responsible for male infertility and the future prospect of whole individual human genome sequencing to identify disease causing genes, the possible use of gene therapy for treating infertility deserves serious consideration. Gene therapy in the sperm and testis offers both opportunities and obstacles. The opportunities stem from the fact that numerous different approaches have been developed for introducing transgenes into the sperm and testis, mainly because of the interest in using sperm mediated gene transfer and testis mediated gene transfer as ways to generate transgenic animals. The obstacles arise from the fact that it may be very difficult to carry out gene therapy of the testis and sperm without also affecting the germline. Here we consider new developments in both sperm and testis mediated gene transfer, including the use of viral vectors, as well as the technical and ethical challenges facing those who would seek to use these approaches for gene therapy as a way to treat male infertility.

Factors affecting the efficiency of producing porcine embryos expressing enhanced green fluorescence protein by ICSI-mediated gene transfer method

This study aims to investigate factors that affect the efficiency of blastocyst development and enhanced green fluorescence protein (EGFP) expression in porcine embryos following intracytoplasmic sperm injection (ICSI)-mediated DNA transfer. Frozen-thawed dead spermatozoa were exposed to different concentrations (0.01 μg/mL, 0.05 μg/mL or 0.1 μg/mL) of EGFP DNA solution, and then microinjected into in vitro matured oocytes. The optimal concentration for EGFP expression of resultant embryos was 0.05 μg/mL. When oocytes were microinjected on a warm stage at 30 °C, the percentage of EGFP-expressing embryos was higher than that at 38.5 °C (40.1% vs. 20.9%, P < 0.01). The efficiency of EGFP expression in embryos following ICSI using linear EGFP DNA-exposed spermatozoa was higher than using circular DNA (40.8% vs. 28.2%, P < 0.05). ICSI oocytes treated with 6-DMAP after electro-activation had a higher percentage of embryos expressing EGFP than those not treated (52.5% vs. 26.3%, P < 0.01). However, neither incubation temperatures of spermatozoa and DNA (4 °C, 24 °C or 39 °C) nor BSA addition to the incubation medium affected the efficiency of producing EGFP-expressing embryos. Furthermore, treatment with DNase I after preincubation of sperm and DNA prevented the embryos from expressing EGFP. The EGFP expression of ICSI oocytes was affected neither by intracytoplasmic injection using sperm heads or whole spermatozoa, nor by washing of the sperm after preincubation. The above-mentioned factors did not affect embryonic developmental competence, apart from 6-DMAP treatment after electro-activation. In conclusion, most exogenous DNA molecules were tightly bound on the membranes of sperm head after incubation of DNA and sperm, and the temperature during ICSI, 6-DMAP treatment, exogenous DNA concentrations and constructs could significantly affect EGFP expression in porcine embryos following ICSI-mediated DNA transfer.

FACTORS AFFECTING PRODUCTION OF TRANSGENIC MICE BY ICSI-MEDIATED DNA TRANSFER

Transgenesis in mice is currently most-efficiently performed using pronuclear injection. In the current study, we sought to evaluate whether optimization of intracytoplasmic sperm injection (ICSI)-mediated DNA transfer could increase the efficiency of transgenesis in mice. Competency of B6D2F1-derived epididymal spermatozoa was achieved by freeze and storage in two kinds of medium (NIM, Tris-EGTA). The sperm heads were then exposed for 1 min to different concentrations (2.5 to 7.5 μg/ml) of an isolated DNA linear fragment containing an enhanced green fluorescent protein (EGFP) gene expression cassette, and then microinjected into oocytes. We found that processing of sperm in Tris-EGTA medium was improved from that of NIM. The optimal concentration of EGFP DNA was 2.5–5.0 μg/ml, as determined by the in vitro developmental competence to blastocysts and the extent of EGFP expression in the resultant embryos. Following adoptive transfer of ICSI embryos, we observed optimal transgenesis efficiencies of 4.9% (N= 101 embryos). EGFP positive founder transgenic mice transmitted the transgene to their progeny demonstrating it was indeed integrated into the mouse germ line. Interesting, one founder contained multiple integration sites giving rise to offspring with different levels of EGFP expression. (poster)

Agrobacterium -induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium -mediated DNA transfer

High necrosis and poor survival rate of target plant tissues are some of the major factors that affect the efficiency of Agrobacterium -mediated T-DNA transfer into plant cells. These factors may be the result of, or linked to, hypersensitive defense reaction in plants to Agrobacterium infection, which may involve the recognition of specific signals from the Agrobacterium that triggers the burst of reactive oxygen species at the infection site. Evidences of Agrobacterium -induced necrosis in target plant tissues and its link to reactive oxygen species are presented. Application of antioxidants, addition of acetosyringone and optimization of pre-culture conditions suppress the Agrobacterium -induced hypersensitive necrotic response in target plant tissues, thereby enhancing stable transformation. Key words: Agrobacterium ; hypersensitive reaction; necrosis; signal transduction; oxidative burst; transformation. African Journal of Biotechnology Vol. 4 (8), pp. 752-757

Agrobacterium -induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium -mediated DNA transfer

High necrosis and poor survival rate of target plant tissues are some of the major factors that affect the efficiency of Agrobacterium -mediated T-DNA transfer into plant cells. These factors may be the result of, or linked to, hypersensitive defense reaction in plants to Agrobacterium infection, which may involve the recognition of specific signals from the Agrobacterium that triggers the burst of reactive oxygen species at the infection site. Evidences of Agrobacterium -induced necrosis in target plant tissues and its link to reactive oxygen species are presented. Application of antioxidants, addition of acetosyringone and optimization of pre-culture conditions suppress the Agrobacterium -induced hypersensitive necrotic response in target plant tissues, thereby enhancing stable transformation. Key words: Agrobacterium ; hypersensitive reaction; necrosis; signal transduction; oxidative burst; transformation. African Journal of Biotechnology Vol. 4 (8), pp. 752-757

Factors affecting production of transgenic rats by ICSI-mediated DNA transfer: Effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA

Factors affecting the efficiency of producing transgenic rats by intracytoplasmic sperm injection (ICSI)-mediated DNA transfer were investigated. Epididymal spermatozoa from Sprague-Dawley (SD) rats were sonicated and/or frozen-thawed for cutting the tail and membrane disruption. The sperm heads were exposed for 1 min to different concentrations (0.02-2.5 microg/ml) of 3.0 kb enhanced green fluorescent protein (EGFP) DNA solution, and then microinjected into the denuded F1 hybrid (Donryu x LEW) rat oocytes. The optimal concentration of EGFP DNA solution was 0.1 microg/ml, as determined by the in vitro developmental competence into morulae/blastocysts of the ICSI oocytes and the EGFP expression of the resultant embryos. The efficiency of producing transgenic rat offspring (per transferred zygote) was 2.8%, 1.6%, and 3.3% in the oocytes injected with sonicated, frozen-thawed, and sonicated + frozen-thawed sperm heads, respectively. The founder transgenic rats carrying the EGFP gene transmitted their transgenes to their progeny according to the Mendelian fashion, suggesting the stable incorporation of the transgenes into the rat genomes. Four rat strains (F344, LEW, Donryu, and SD) were compared for their suitability as sperm/oocyte donors for the production of transgenic rats by ICSI with sonicated, frozen-thawed and solution of EGFP DNA-exposed sperm heads. The efficiency of producing transgenic rats in the SD strain (8.2%) was higher than that in the LEW strain (0.9%), while those in the F344 and Donryu strains (4.3%-4.4%) were intermediate. One plasmid DNA (Fyn, 5.0 kb) and two BAC DNA (BAC/Fyn, 208 kb; Svet1/IRES-Cre, 186 kb) were successfully introduced into the SD rat genomes via ICSI, with the producing efficiencies of 2.8%, 0.9%, and 2.4%, respectively.

Preparation of an Endosperm Protoplasts from a Dwarf Rice Variety and Transient Expression of Green-Fluorescent Protein

Vital protoplasts were isolated from developing dwarf rice endosperms about 10 days post anthesis. A yield of 0.98x105±0.12 protoplasts was obtained from 10 developing grains. These protoplasts were viable for at least three days in the culture medium. Transient expression of the green-fluorescent protein (GFP) was detected after four hours of introduction of the GFP gene by the polyethylene glycol (PEG)-mediated DNA transfer. As expected, the fluorescence of GFP without the targeting sequence was observed diffusely in the cytoplasm. On the other hand, the fluorescence of the fusion protein for rice prolamin followed by the GFP was observed in the small particles and the ER-network. These endosperm protoplasts had no autofluorescence upon excitation with 490 nm (blue) light. The vital protoplasts obtained from this dwarf rice variety are very useful for the physiological, biochemical and molecular biology studies of rice endosperm.

Genetic transformation of bean callus via Agrobacterium- mediated DNA transfer

Callus cultures were induced from hypocotyl of young bean seedlings. Callus developed and maintenaned on B5 medium supplemented with 2mg/1 2,4-D and 1 mg/1 kinetin. The results demonstrate that A. tumefacins-mediated transformation is a convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus co-cultivated with A. tumefaciens can be transformed to get heibicide Finale (glufosinate-ammonium) resistant GUS positive tissues. Southern blot analysis of transformed calli showed integration of gusA marker gene carried by a binary vector. Transformed calli were selected on herbicide containing media. Data of molecular analysis (Southern blotting) confirmed the insertion of gusA gene in the genome of herbicide resistant calli with bar gene. There are three evidences that calli are stable transformants: (1) herbicide resistance, (2) GUS activity which is indicative since the coding region containing an intron, (3) the results of Southern hybridization technique.

Challenges and prospects for targeted transgenesis in livestock. Practical applications of gene targeting.

Until recently the only ways of making transgenic livestock were by pro-nuclear injection or, more controversially, by sperm mediated DNA transfer. Both techniques usually result in multiple copies of the transgene at a random site within the genome. This is associated with unpredictable transgene expression often due to gene silencing. In the mouse, this problem has been addressed by directing single copy transgenes to particular sites in the genome (gene targeting). This has been possible because of the availability, in that species, of embryo-derived stem cells (ES cells) which can be modifiedin vitroand then used to create transgenic mice. Despite considerable research effort, however, ES cells are not available for any livestock species. ES-like cells have been derived from sheep, pigs and cattle that can contribute to the formation of chimaeras but they do not contribute to the germline (reviewed by McWhir).

Co-transformation of bean callus using high-velocity microprojectiles- mediated DNA transfer

We have found that 50 mg/I kanamycin and 0.8 Mo1/1 mannitol concentration was sufficient to kill the control callus of bean (Phaseolus vulgaris L.) and differentiate transgenic from the non-transgenic cells. The GeneBooster particle delivery system was used for the bombardment of bean callus. The kanamycin resistance gene was used as a selectable marker. The test was made by transferring the healthy white callus, subcultured for three months on selective and non-selective medium. After selection on kanamycin containing media, several kanamycin resistant calli had been obtained, survived and grew. After selection on mannitol containing media no drought resistant calli had been obtained. Resistance of the selected calli were verified by their ability to grow repeatedly on selective medium containing 150 mg/I kanamycin. Selective pressure was maintained over a period of 8 months.

A comparison of shock wave and sinusoidal-focused ultrasound-induced localized transfection of HeLa cells

Both shock waves and sinusoidal continuous wave ultrasound can mediate DNA transfer into cells. The relative transfection efficiencies of different ultrasound modalities are unclear. The purpose of this paper is to compare the transfection efficiency of lithotripter shock waves and focused sinusoidal ultrasound in vitro. HeLa cells were transfected with β-galactosidase and luciferase plasmid DNA reporter. Shock waves were generated by an electromagnetic sound source. Sixty to 360 pulses at 1 Hz pulse frequency were administered at 13, 16 or 19 kV capacitor voltage. Sinusoidal focused ultrasound was generated by a single focus piezoceramic air-backed disk transducer at a carrier frequency of 1.18 MHz operated in a pulsed mode. Compared to cells mixed with DNA only, shock waves induced up to eightfold more transfected cells at a cell viability of 5%, while sinusoidal-focused ultrasound induced up to 80-fold more transfected cells at a cell viability of 45%. The corresponding transfection efficiencies of the HeLa cells were 0.08% for shock waves and 3% for focused ultrasound. These results may contribute to the selection of the ultrasound modality as a localized, noninvasive and safe tool to mediate gene transfer.

In vitro construction of pseudovirions of human papillomavirus type 16: incorporation of plasmid DNA into reassembled L1/L2 capsids.

Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids, which had been self-assembled in insect cells (Sf9) expressing virion proteins L1 and L2, were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a beta-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced beta-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Construction in vitro of HPV pseudovirions containing marker plasmids would be potentially useful in developing methods to assay virus-neutralizing antibodies and to transfer exogenous genes to HPV-susceptible cells.

Lipitoids--novel cationic lipids for cellular delivery of plasmid DNA in vitro.

Although synthetic nonviral vectors hold promise for the delivery of plasmid DNA, their gene-transfer efficiencies are far from matching those of viruses. To systematically investigate the structure-activity relationship of cationic lipids, a small library of cationic lipid-peptoid conjugates (lipitoids) was synthesized. The compounds were evaluated for their ability to form complexes with plasmid DNA and to mediate DNA transfer in vitro. Lipid-peptoid conjugates were conveniently prepared in high yield using solid-phase synthesis. Several lipitoids condensed plasmid DNA into 100 nm spherical particles and protected the DNA and DNase digestion. A subset of lipitoids with a repeated (aminoethyl, neutral, neutral) sidechain trimer motif conjugated with dimyristoyl phosphatidyl-ethanolamine (DMPE) mediated DNA transfer with high efficiency. Automated solid-phase synthesis of cationic lipids allowed the rapid synthesis of a diverse set of transfection reagents. The most active compound DMPE-(Nae-Nmpe-Nmpe)3 (Nae, N-aminoethyl glycine; Nmpe, N-p-methoxyphenethyl-glycine) is more efficient than lipofectin or DMRIE-C (two commercial cationic lipid transfection reagents) and is active in the presence and absence of serum. The activity in the presence of serum suggests potential for applications in vivo.

PEG-mediated protoplast transformation with naked DNA.

Direct introduction of DNA into plant protoplasts facilitates a rapid analysis of transient gene expression, as well as the generation of stably transformed transgenic plants. Transient gene expression assays performed after DNA transformation permit a comparative analysis of cis–actmg regulatory sequences and then function m transcriptional control of plant genes by signaling pathways mediating cellular responses to different environmental and hormonal stimuli (). There are a number of methods for introducing DNA into plant protoplasts, but the most commonly used technique 1s the polyethylene glycol (PEG)–mediated DNA uptake. The PEG–mediated transformation is simple and efficient, allowing a simultaneous processing of many samples, and yields a transformed cell population with high survival and division rates (). The method utihzes inexpensive supplies and equipments, and helps to overcome a hurdle of host range limitations of Agrobactenum–mediated transformation The PEG–mediated DNA transfer can be readily adapted to a wide range of plant species and tissue sources.

Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Transfection and protein expression in mammalian cells.

This is the first report of transient transfection of suspended cells with purified plasmid DNA in bioreactors or spinner flasks. DNA/calcium phosphate complexes were pumped or injected directly into stirred cultures of the immortalized human embryo kidney cell line 293 (HEK-293) which had been adapted to growth in suspension. We identified culture conditions suitable for this approach and modified the protocol for the generation of precipitate complexes, based on our earlier work. In order to stabilize the 'DNA-vehicle'-complex in the culture medium, we identified pH ranges and ion-concentrations which prevent dissolution or aggregation of the precipitate particles. Such conditions maintained suspended fine particles in spinners or bioreactors for up to 6 hr. During that period, cells and precipitate complexes interacted sufficiently to allow DNA transfer and subsequent expression of recombinant protein. In a simple 5 day batch process, with a starting density of 0.3 × 10(6) cells mL(-1), about 0.5 mg L(-1) of a recombinant tissue plasminogen activator variant was observed.

Transient expression of goat growth hormone gene in poplar (Populus alba L.) protoplasts: a quick method for detection of foreign gene expression in mRNA level.

We developed a sensitive, accurate, and fast method to detect foreign gene expression using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) in poplar tree (Populus alba L.) protoplasts. Template total RNA was purified by removing the transfected foreign gene vector completely with DNase I treatment before the RT reaction. Expression of cDNA that encodes goat growth hormone was confirmed at the mRNA level 24 h after electroporation mediated DNA transfer.