Factors influencing stable transformation of maize protoplasts by electroporation

Two chimaeric genes, containing the promoter of the 35S gene of cauliflower mosaic virus coupled to neomycin phosphotransferase (35S-NPT-NOS) or to hygromycin phosphotransferase (35S-HPT-NOS) have been stably transferred to maize (Zea mays cv. Black Mexican sweet) cells by electroporation. Transformation frequencies of 7.6×10^-4 and 8×10^-4, respectively, (based on the number of surviving cells that divided) were obtained with four pulses of 1 ms duration using 400 V capacitive discharge. Cells transformed to kanamycin-resistance and hygromycin-resistance subsequently multiplied to form callus. Southern blot analysis demonstrated the integration of the selectable marker genes, neomycin or hygromycin phosphotransferase, with single or multiple copy numbers. The blots from DNA of hygromycin-resistant calli also suggested the formation of plasmid concatemers.     

Identification and prevention of heterotopias in mouse neocortical neural cell migration incurred by surgical damages during utero electroporation procedures

ABSTRACT

In utero electroporation (IUE) is a useful technique for gene delivery in embryonic mouse brain. IUE technique is used to investigate the mammalian brain development in vivo. However, according to recent studies, IUE methodology has some limitations like the formation of artificial ectopias and heterotopias at the micro-injection site. Thus far, the artificial heterotopias generated by physical trauma during IUE are rarely reported. Here, we reported the artificial heterotopias and ectopias generated from surgical damages of micropipette in detail, and moreover, we described the protocol to avoid these phenotypes. For the experimental purpose, we transferred empty plasmids (pCAGIG-GFP) with green fluorescent-labelled protein into the cortical cortex by IUE and then compared the structure of the cortex region between the injected and un-injected cerebral hemispheres. The coronary section showed that ectopias and heterotopias were appeared on imperfect-injected brains, and layer maker staining, which including Ctip2 and TBR1 and laminin, can differentiate the physical damage, revealing the neurons in artificial ectopic and heterotopic area were not properly arranged. Moreover, premature differentiation of neurons in ectopias and heterotopias were observed. To avoid heterotopias and ectopias, we carefully manipulated the method of IUE application. Thus, this study might be helpful for the in utero electroporator to distinguish the artificial ectopias and heterotopias that caused by the physical injury by microneedle and the ways to avoid those undesirable circumstances.     

Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids

Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl₂) and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO₄) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5±0.2 × 10⁴ transformants ng⁻¹ plasmid DNA) than with the CaCl₂ (8.1±6.1 × 10⁻¹ transformants ng⁻¹ plasmid) and the 'one-step transformation' protocol (2.5±2.7 transformants ng⁻¹ plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl₂ and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.     

Improved conditions for the transformation by electroporation of the extracellular polysaccharide-producing methylotroph Methylobacillus sp.

The transformation efficiency of Methylobacillus sp. strain 12S, using electroporation, was unaffected by the growth phase of the cells but competent cells grown at 21 °C had a 1.9 × 10³ times higher transformation efficiency than those grown at 30 °C. Heat shock treatment further increased the transformation efficiency up to 7 times.     

Effect of exogenous application of sugars on the salt tolerance of perennial ryegrass protoplasts

Various sugars were introduced by electroporation into perennial ryegrass protoplasts, and the involvement of intracellular functional groups of the sugars in salt tolerance was investigated. The protoplasts were prepared from the young leaves of perennial ryegrass, and those into which sugars were introduced were treated with NaCl solution (250 mM, pH 7.0) for 6 h at 10°C. The survival rate of the protoplasts increased when xylitol, cellobiose, 1-kestose, maltose, maltotriose, raffinose and trehalose were introduced, while no changes occurred when fructose, fucose, galactose, glucose, inositol, mannitol, mannose, rhamnose, sorbitol, sorbose, fructobiose, lactose and sucrose were introduced. Cellobiose, 1-kestose, maltose, maltotriose, raffinose and trehalose possess a number of equatorial OH (e-OH) groups that promote the structuration of H₂O. Xylitol, however, structures H₂O even though it does not possess the e-OH groups. Hence, it is suggested that under conditions of NaCl stress, structured H₂O protects the structure of cell membranes and the activity of enzymes, and that e-OH groups are involved in enhancing salt tolerance.