Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1975.tb02238.x

Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography.

A simple method is described for converting a standard rabbit reticulocyte cell-free extract (lysate) into an mRNA-dependent protein synthesis system. The lysate is preincubated with CaCl2 and micrococcal nuclease, and then excess ethyleneglycol-bis(2-aminoethylether)-N,N′-tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have negligible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full-sized products of the expected molecular weight up to about 200 000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10656.x

An efficient mRNA-dependent translation system from reticulocyte lysates.

We describe a method for the synthesis of microgram quantities of eucaryotic messenger RNAs. Injection into the cytoplasm of frog oocytes and addition to wheat germ extracts show that these synthetic RNAs function efficiently as messenger RNAs. We confirm that a 5' cap on the mRNA is essential for translation in injected oocytes and show that most of the 3' flanking region, including the poly A tail, can be deleted without the abolition of protein synthesis. The method of mRNA synthesis involves in vitro transcription of cDNAs which have been cloned into SP6 vectors (described in the accompanying paper). This method enables one to produce large amounts of mRNA and consequently protein from any cDNA clone.

Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs

In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.

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mRNA-based therapeutics — developing a new class of drugs

A cDNA coding for human interleukin-2 (IL-2) has been cloned from a cDNA library prepared from partially purified IL-2 mRNA. The DNA sequence codes for a polypeptide which consists of 153 amino acids including a putative signal sequence. A biologically active polypeptide, characteristic of human IL-2, was produced when the cDNA was fused to a simian virus 40 promoter sequence and used to transfect cultured monkey COS cells.

Structure and expression of a cloned cDNA for human interleukin-2

Injected frog eggs and oocytes provide a very sensitive assay system for the identification of messenger RNA and permit the study of translational control in living cells. The translation of each haemoglobin messenger RNA molecule once every 5–10 minutes for at least 24 hours makes it possible to recognize less than 1 ng of this messenger RNA.

Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells

Rabbit haemoglobin synthesis in frog cells: the translation of reticulocyte 9 s RNA in frog oocytes☆

Translational capacity of living frog eggs and oocytes, as judged by messenger RNA injection

Insect protein synthesis in frog cells: the translation of honey bee promelittin messenger RNA in Xenopus oocytes.

Publisher Summary This chapter discusses the use of frog oocytes for the assay of mRNA and the study of the control of gene expression To identify mRNA, it should be demonstrated that it codes for a characteristic protein This means showing that synthesis of the particular protein is caused by adding the putative messenger to a system derived from cells that neither express nor even contain the genetic information for the protein in question In practical terms, this is most simply achieved by adding mRNA from one species to a translational system derived from another species The disadvantage of this approach is that translational systems from one species may be restricted in their ability to handle foreign messengers Restrictions are less likely to exist in a translational system derived from embryonic cells, for these, unlike differentiated cells, give rise to an enormous variety of cell types The sensitivity of such an assay for a particular messenger depends upon the ease of detecting one particular translation product among a wide variety of others and against a background of protein synthesis by the oocyte or egg

C.D. Lane

Papers

Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells

Rabbit haemoglobin synthesis in frog cells: the translation of reticulocyte 9 s RNA in frog oocytes☆

Translational capacity of living frog eggs and oocytes, as judged by messenger RNA injection

Insect protein synthesis in frog cells: the translation of honey bee promelittin messenger RNA in Xenopus oocytes.

THE INJECTION OF RNA INTO LIVING CELLS: THE USE OF FROG OOCYTES FOR THE ASSAY OF mRNA AND THE STUDY OF THE CONTROL OF GENE EXPRESSION