Transfected viruses

Use of a provirus vector such as RCAS. For prolonged overexpression, a combination of a provirus vector and virus-sensitive egg may be used. One can directly electroporate provirus vectors to avoid preparing virus particles. When a lineage trace is needed, virus-insensitive eggs are useful, since the expression of the gene of interest is restricted to the descendants of the transfected cell in such virus-insensitive embryos (7). Transfected virus-sensitive tissue transplanted to an insensitive host produces a tissue-specific transfection. 

Fetal bovine serum (FBS) (v/p) is added to the medium of SF900 II SFM for Sf9 propagation, transfection, virus amplification and intracellular expression. FBS is omitted from the medium for extracellular expression of secreted protein. The presence of FBS in the medium may mask the expression of the secreted target protein and may interfere with the purification procedure. 

Viruses are infectious particles formed by nucleic acid, proteins, and in some cases lipids. As viruses (for example, retro- and adenoviruses) transfer viral genes into cells with high efficiency, modified forms are sometimes used as vectors for gene transfer. However, procedures using virus-based vectors are often significantly more complicated and time-consuming than other transfection methods. In addition, viral vectors are potentially hazardous, and biological safety issues need to be considered carefully. Therefore, techniques that combine... 

Neural progenitor cells Lawrence et al. (2004) When nestin+ multi-potential cells are differentiated towards a neuronal lineage post-infection, there is a negligible increase in virus rephcation. When these cells were transfected with a pNL4-3 molecular clone and stimulated with TNP-a, there is an upregulation in virus production, suggestive of reactivation from latency... 

Subsequently, similar experiments were done with viral nucleic acids. The pure viral nucleic acid, when added to cells, led to the synthesis of complete virus particles the protein coat was not required. This process is called transfection. More recently, DNA has been used in cell-free extracts to program the synthesis of RNA that functions as the template for the synthesis of proteins characteristic of the DNA... 

Brash, D. E., R. R. Reddel, M. Quanrud, K. Yang, M. P. Farrell, and C. C. Harris. 1987. Strontium phosphate transfection of human cells in primary culture Stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells. Mol Cell Biol 7(5) 2031-4. 

The DNA or cDNA library is then introduced into a preparation of bacterial host cells. Usually, the first host selected is a laboratory strain of E. coli which has been grown and pretreated with inorganic salts to make uptake of DNA easier. The ability to take up foreign DNA is called competence, cells which have been specially prepared for the purpose are called competent cells. Other methods to transfer DNA into cells include electroporation (application of an external electric field to permeabUize the cell wall), transfection (where a recombinant bacterial virus is used to transfer the DNA to the target cell) or ballistic methods (by using DNA-coated particle projectiles). The last method has been used to introduce foreign DNA into plant cells and mammalian cells. 

Genes can be introduced by the application of naked DNA alone however, better efficiency is achieved when the DNA is incorporated into a delivery vector. These delivery vectors consist of viral, those utilizing modified virus particles for DNA delivery, and nonviral, for which various chemicals are used to aid DNA packaging and delivery. Viral vectors confer significantly better transfection efficiency than nonviral vectors however, recently the toxicity and oncogenic side effects of viral vectors have become a major concern (6). Nonviral vectors do not have such serious side effects but lack the efficiency (7). 

Peptides can also help overcome the most significant drawback to using liposome vectors when compared to viral vectors, which is lower transfection efficiency. Additional benefits include promotion of compaction, assisting cellular uptake of the DNA. Even peptides derived from viruses themselves can be used to compensate this deficit (e.g., adenovirus p protein and the HIV TAT). 

Pipette 100 xl of the dilute virus transfection supernatant onto each plate except for the control. 

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