The goal of this exercise is to learn lentiviral packaging. For this example we will be using the HEK 293T cell line. We will perform two separate transfection reactions with the goal of producing functional viral particles. First, we will transfect cells with a combination of packaging plasmid mix and TurboGFP control vector. The second transfection reaction will contain packaging plasmid mix and Empty control vector. The transfected HEK 293T cells will be monitored for TurboGFP expression, which is indicative of successful viral production.
The plasmids included in the lentiviral packaging mix encode the key structural viral packaging genes and a heterologous viral envelope gene. When combined with a MISSION shRNA pLKO.1 plasmid, this product yields effective high-titer lentivirus, Table 1. Viruses are uniquely adapted to infect even the most resilient cell lines. Therefore, viral systems have been genetically engineered over the last few decades to safely deliver transgenes of interest to target cells. While each viral system has features that are ideal in certain applications, the lentiviral system is an example of a specific retroviral system that has undergone multiple generations of engineering and modification. Generating viral particles can be challenging. We highly recommend the viral format of MISSION TRC shRNA clones to new and experienced users. We have perfected the generation of small and large scale lentiviral particles.
For enhanced safety, the structural and accessory genes necessary to produce viral particles are separated onto multiple plasmids. All wild-type virulence and replication genes are deleted. All our lentiviral transfer vectors contain a modified, self-inactivating 3' long terminal repeat (SIN/LTR) which renders the resulting lentiviral particles replication incompetent. Lentiviral particles are packaged in producer cell lines such as HEK293T cells. Upon co-transfection of the plasmids, all required sequences are available to produce and package a viral particle containing the transgene of interest. Only the region between the viral LTRs of the transfer vector is packaged within the viral capsid.
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