Gene transfer by electroporation

Lurquin, Paul F.

doi:10.1007/bf02821542

Cited by 84 publications

(53 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The exact nature of the molecular sieve through which proteins have to pass in order to leave osmotically shocked cells is unknown. An interesting clue is provided by our finding that electroporation of cells, known to generate transient pores in biological membranes (19,24), causes selective release of the same subset of proteins, presumably passing through the same molecular sieve. Furthermore, solutions containing EDTA and a membrane-permeabilizing agent, such as Triton X-100 or polymyxin B, have been reported to extract from E. coli the same proteins as the osmotic shock procedure (23).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked Escherichia coli

Vázquez‐Laslop

Lee

et al. 2001

J Bacteriol

View full text Add to dashboard Cite

Escherichia coli cells, the outer membrane of which is permeabilized with EDTA, release a specific subset of cytoplasmic proteins upon a sudden drop in osmolarity in the surrounding medium. This subset includes EF-Tu, thioredoxin, and DnaK among other proteins, and comprises ϳ10% of the total bacterial protein content. As we demonstrate here, the same proteins are released from electroporated E. coli cells pretreated with EDTA. Although known for several decades, the phenomenon of selective release of proteins has received no satisfactory explanation. Here we show that the subset of released proteins is almost identical to the subset of proteins that are able to pass through a 100-kDa-cutoff cellulose membrane upon molecular filtration of an E. coli homogenate. This finding indicates that in osmotically shocked or electroporated bacteria, proteins are strained through a molecular sieve formed by the transiently damaged bacterial envelope. As a result, proteins of small native sizes are selectively released, whereas large proteins and large protein complexes are retained by bacterial cells.The procedure of osmotic shock was originally introduced for the purpose of extracting periplasmic enzymes of Escherichia coli (20). In this procedure, bacterial cells are preincubated in a hyperosmotic sucrose solution supplemented with EDTA to permeabilize their outer membrane and then transferred into a solution of low osmolarity. The resulting osmotic pressure within shocked cells was believed to cause extrusion of the contents of the periplasm. Later experiments have shown, however, that osmotically shocked bacteria, while remaining viable, also release a number of cytoplasmic molecules, including ions, metabolites (22), and certain proteins. In fact, the major protein released from the shocked cells is a cytoplasmic constituent, translation elongation factor EF-Tu, at least half of which is extracted by the osmotic shock procedure (12, 13). The subset of released cytoplasmic proteins also includes thioredoxin (1, 17), molecular chaperone DnaK (6,8), and proteins involved in the biosynthesis of enterobactin (9), among others (5). Some foreign proteins expressed in the cytoplasm of E. coli have also been successfully extracted by the osmotic shock procedure (16, 23).It has been unclear what determines the selectivity of protein release from osmotically shocked bacteria, since the released cytoplasmic proteins share no apparent common characteristics distinguishing them from the majority of proteins, which are retained by the cells. How these proteins leave the cytoplasm in the absence of cell lysis has also remained an enigma. The most frequently entertained hypothesis postulates that these proteins normally concentrate in a hypothetical "osmotically sensitive compartment" of the bacterial cytoplasm, which is presumably adjacent to the zones of adhesion between the plasma membrane and the outer membrane and, therefore, is selectively extruded from the shocked cells (4, 8-10, 18, 23).This hypothesis leaves open, however, a s...

show abstract

Section: Discussionmentioning

confidence: 99%

“…It is well established that electroporation causes formation of transient pores in biological membranes (19,24). In view of the similarity in protein release between the two procedures, it is tempting to speculate that osmotic shock also causes transient perforation of the plasma membrane.…”

Section: Vol 183 2001mentioning

confidence: 99%

Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked Escherichia coli

Vázquez‐Laslop

Lee

et al. 2001

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…Electroporation has several advantages. It is technically simple, can be used to treat whole population of cells, has broad application for transfer of any macromolecule, provides greater efficiency of transfection for many cell lines, and can be applied equally successfully to prokaryotic and eukaryotic cells (Lurquin, 1997). Nevertheless, it needs to empirically determine through extensive studies, the conditions for efficient transfection for each cell type (Andreason, 1993).…”

Section: Resultsmentioning

confidence: 99%

Multiple pulses improve electroporation efficiency in Agrobacterium tumefaciens

Mahmood¹,

Zar²,

Naqvi³

2008

Electron. J. Biotechnol.

View full text Add to dashboard Cite

Electroporation entails brief, high intensity pulse to create transient pores in the cell membrane to facilitate the entry of exogenous macromolecules, which may otherwise be excluded. Removal of the external field leads to the resealing of the membrane electropores permitting the survival of the electrically stimulated recipient cells. Using this technique foreign deoxyribonucleic acid (DNA) has been successfully introduced into many cell types both from prokaryotes and eukaryotes. Increase in pulse voltage and length beyond a critical limit has been reported to decrease transformation efficiency, hence in this study we have investigated another strategy i.e. increase in the number of pulses at constant high voltage and pulse duration. Commonly used Agrobacterium strains LBA4404 and EHA101 and binary vector pCAMBIA1301 were used. Transformants were selected on a combination of hygromycin and kanamycin, and confirmed by polymerase chain reaction (PCR) and restriction analysis. Increase in the number of pulses was found to show a significant and linear increase in transformation efficiency.Application of deoxyribonucleic acid (DNA) technology to genetically manipulate cellular functions requires a suitable high efficiency transformation system. There are many approaches available for introducing foreign DNA into eukaryotic and prokaryotic cells, however electroporation is the most commonly used technique and currently of more practical interest. It is a gene transfection technique, which entails brief, high intensity pulse to facilitate the entry of exogenous molecules like DNA, ribonucleic acid (RNA), and protein. Removal of the external field leads to the resealing of the membrane electropores.Applying high intensity electric fields to reversibly permeablize biomembranes to create structural distortions, allowing the uptake of DNA was first demonstrated by Neuman in mouse myeloma cells (Neuman et al. 1982).

show abstract

“…However, microinjection is a time consuming process. Electroporation is an another choice for gene transfer and has been applied successfully in bacteria, yeast, cultured cells, and embryos (Lurquin, 1997;Muramatsu et al, 1998).…”

Section: Discussionmentioning

confidence: 99%