Transformation microinjection

Another approach that has been explored for plastid transformation is microinjection. Microinjection into discrete plastids of intact plant cells entails the use of a syringe consisting of a submicron diameter glass capillary driven by the thermal expansion of galinstan, a liquid metal alloy... 

Fig. 7.1. A planarian individual of a line transformed with a Hermes transposon-derived vector 12 months after microinjection and subsequent electroporation. Besides the transposon part, the vector contained the E(enhanced)GFP reporter gene under the control of an artificial promoter that responds to the transcription factor Pax6 (Gonzalez-Estevez et ai, 2003). Binding sites for this transcription factor are found in rhodopsin and other photoreceptor-specific genes. Reporter-gene activity was found in the eyes (arrows) of transformed individuals or in regenerated heads as a mosaic of EGFP-positive photoreceptor cells. Figure courtesy of Dr E. Salo.

For transformation experiments in general, biochemical or physical methods such as microinjection, electroporation or lipofection are available. These methods have been successfully applied for a variety of organisms including protozoan parasites (Clayton, 1999 De Koning-Ward et al., 2000) and were consequently tested for their ability to generate transiently transformed schistosomes by different laboratories. 

Capecchi, M R. (1980) High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell, 22, 479 188. 

Between the methods of Agrobacterium and microprojectile transfer, nearly every plant species can be transformed effectively [35]. However, these methods are covered by patent claims and may result in limited transformation efficiency for some cell types. For this reason, the use of alternative gene-transfer methods is an active area of research for all cell types. Alternative gene-transfer techniques include electroporation, microinjection, liposome fusion, direct transfer into protoplasts, and laser treatment [38]. In electroporation, DNA is transferred into the cell using a high-voltage electrical pulse [39]. Standard... 

Wick, R, Angelova, M. L, Walde, P. et al. (1996). Microinjection into giant vesicles and light microscopy investigation of enzyme-mediated vesicle transformations. 

In some circumstances the introduction of a cloning vector into a host cell is a trivial process. For example, phage vectors are designed so they introduce recombinant DNA in an infective process called transfection, and some bacteria take up plasmids unaided. However, most host cells must be induced to take up foreign DNA. Several methods are used. In some prokaryotic and eukaryotic cells, the addition of Ca2+ to the medium promotes uptake. In others, a process called electroporation, in which cells are treated with an electric current, is used. One of the most effective methods for transforming animal and plant cells is the direct microinjection of genetic material. Transgenic animals, for example, are created by the microinjection of recombinant DNA into fertilized ova. 

Plant transformation techniques allow the delivery of the transforming DNA through the cell wall and plasma and nuclear membranes, without compromising the viability of the cell. Gene delivery can be performed either via a biological vector (plant viruses or bacteria) or by non-biological vector-free procedures (chemical methods, microinjection, particle bombardment, etc.) (Birch 1997). 

Available methods for carrying DNA into an animal cell vary in efficiency and convenience. Some success has been achieved with spontaneous uptake of DNA or electroporation, techniques roughly comparable to the common methods used to transform bacteria. They are inefficient in animal cells, however, transforming only 1 in 100 to 10,000 cells. Microinjection—the injection of DNA directly into a nucleus, using a very fine needle—has a high success rate for skilled practitioners, but the total number of cells that can be treated is small, because each must be injected individually. 

When REFs were exposed to the membrane-permeant PKA activator, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), they underwent the same morphological transformation as described above (disruption of actin stress fibers leading to cell shape changes). In contrast, cells microinjected with the caged peptide (5 pM estimated intracellular concentration) and exposed to UV irradiation were unable to respond to the CPT-cAMP stimulus, demonstrating that the CPT-cAMP activation of the PKA pathway had been efficiently blocked in vivo by the decaged peptide [152],... 

The evidence indicating an anti-apoptotic effect of E2F1 was first provided by the studies of E2F1 overexpression. " Microinjection of the wild type-E2Fl cDNA, but not mutated E2F1 cDNA, into quiescent cells can activate DNA synthesis and induce S-phase entry. " Further studies indicated that ovetexpression of E2F1 can cause transformation of primary cells. In contrast, mutation of E2F1 is sufficient to block cell proliferation completely." ... 

Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 77 7380-7384. 

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