RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

We have developed an efficient and reproducible method for RNA transfection, using a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propylj-N,N,N-trimethylammonium chloride (DOTMA), incorporated into a liposome (lipofectin). Transfection of 10 ng to 5 jug of Photinus pyrais luciferase mRNA synthesized in vitro into NIH 3T3 mouse cells yields a linear response of luciferase activity. The procedure can be used to efficiently transfect RNA into human, rat, mouse, Xenopus, and Drosophila cells. Using the RNA/ lipofectin transfection procedure, we have analyzed the role of capping and (3-globin 5' and 3' untranslated sequences on the translation efficiency of luciferase RNA synthesized in vitro. Following transfection of NIH 3T3 cells, capped mRNAs with B8-globin untranslated sequences produced at least 1000-fold more luciferase protein than mRNAs lacking these elements.The wide variety of methods to introduce genetic material into cells includes relatively simple manipulations like mixing high molecular weight DNA with calcium phosphate, DEAEdextran, polylysine, or polyornithine. Other methods involve electroporation, protoplast fusion, liposomes, reconstituted viral envelopes, viral vectors, or microinjection. In nearly all cases DNA has been introduced into cells because of its inherent stability and eventual integration in the host genome. By comparison, progress in introducing RNA molecules into cells has been very slow and restricted to a few cases (1-4). Inability to obtain sufficient amounts of intact RNA and its rapid degradation have been a major hindrance in the past. The limitation of obtaining sufficient quantities of RNA can now be alleviated by synthesizing large amounts of RNA in vitro, using bacteriophage RNA polymerases (5).Since we were interested in studying the cis-and transacting factors influencing both the translational efficiency and the stability of eukaryotic mRNAs, we undertook the development of a reliable method to efficiently introduce RNAs into cells. We report the use of RNA transfection mediated by lipofectin (a liposome containing a cationic lipid)

Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.

IntroductionReports of high rates of primary microcephaly and Guillain–Barré syndrome associated with Zika virus infection in French Polynesia and Brazil have raised concerns that the virus circulating in these regions is a rapidly developing neuropathic, teratogenic, emerging infectious public health threat. There are no licensed medical countermeasures (vaccines, therapies or preventive drugs) available for Zika virus infection and disease. The Pan American Health Organization (PAHO) predicts that Zika virus will continue to spread and eventually reach all countries and territories in the Americas with endemic Aedes mosquitoes. This paper reviews the status of the Zika virus outbreak, including medical countermeasure options, with a focus on how the epidemiology, insect vectors, neuropathology, virology and immunology inform options and strategies available for medical countermeasure development and deployment.MethodsMultiple information sources were employed to support the review. These included publically available literature, patents, official communications, English and Lusophone lay press. Online surveys were distributed to physicians in the US, Mexico and Argentina and responses analyzed. Computational epitope analysis as well as infectious disease outbreak modeling and forecasting were implemented. Field observations in Brazil were compiled and interviews conducted with public health officials.