Expression of mRNA electroporated into plant and animal cells

Abstract

A general method to introduce RNA molecules into plant protoplasts and animal cells is described. This technique utilizes the ability of electric pulses of high field strength to form pores in biomembranes. RNA molecules containing the coding region for the bacterial enzyme chloramphenicol acetyltransferase (CAT) were used as a model system. The presence of CAT activity as a result of the in vivo translation of the introduced RNA is entirely dependent on the presence of a 5' cap and greatly increased by the presence of a poly A tail at the 3' end. The introduction of RNA into eukaryotic cells has broad applicability both as an assay for the uptake of nucleic acids into cells independent of transcriptional activity and as a tool to study eukaryotic mRNA translation. Introduction of RNA into plant protoplasts Maize and carrot protoplasts were prepared and the electroporation solutions used were as described previously (22) with the addition of an extra wash prior to electroporation. Protoplasts INIRODUCfION

Transfection of Leishmania enriettii and expression of chloramphenicol acetyltransferase gene.

“…3, transfection with the hybrid plasmid pALT1-1 results in expression of CAT activity. In contrast, no CAT activity was detected in mock-transfected parasites or parasites transfected with pSP65CATA, which contains a CAT gene and upstream signals necessary for expression of CAT in plant cells and mammalian cells (33). This experiment has been repeated several times with similar results.…”

Electroporation: basic principles, practical considerations and applications in molecular biology

“…Foreign DNA has been successfully introduced into the plant cells [6][7][8][9], fungi [10], intact yeast cells [11][12][13] yeast spheroplasts [14], animal cells [15][16][17] and bacteria [18][19][20][21][22][23][24][25][26][27][28][29]. Electroporation can be applied to a number of strains and species reported to be untransformable so far and in some cases it is the only method of choice available for the transformation of intact cells [30,31].…”

Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation.

“…Electroporation, or reversible electrical membrane breakdown, involves exposing cell membranes to electric field pulses of high intensity for short durations (15,22). Electroporation has been used to induce uptake of macromolecules (including DNA, RNA, and proteins) and molecules (including nucleotides, dyes, and ions) into cell types ranging from prokaryotes and lower eukaryotes to higher plant and animal cells (3,12,13,15,19,21). Several factors influence the success of electroporation, including field strength, pulse length, medium composition and temperature, and the membrane characteristics of the particular cell type (22).…”