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Tutorials : Jun 1, 2008 ( )
Inducement of Gene Expression or Silencing
Technology Rearranges and Adds Vector Elements to Enhance Transfection!--h2>
A major challenge shared by drug discovery, biomedical, and gene-therapy researchers is how to deliver gene expression or knockdown constructs into the greatest number of cells with reproducibility and maximal activity. Primary and nondividing cells most relevant to research and disease models can often be refractory to transfection. Direct injection of integrating DNA sequences can be laborious, technically demanding, and produce only a small number of cells expressing the construct of interest. Delivery into living animals presents additional challenges.
Oncoretroviral vectors can be used both in vitro and in vivo and circumvent the toxicity and inefficiencies of mechanical- or chemical-driven gene-delivery methods. Their utility, however, is limited by low titers, instability, and their inability to transduce nondividing cells. Of the available viral-delivery solutions, lentivirus is unique in offering a combination of ease of production, efficient delivery, and the ability to stably integrate into the host genome.
Pseudotyped lentivirus can also transduce a wide variety of cell types including nondividing or untransfectable cells. Conveniently, the same lentiviral constructs used and validated in cell-based studies may be used in downstream in vivo applications.
A key advantage of the lentivirus is the simplicity of generating replication-incompetent pseudoviral particles. The numerous safety features of Invitrogen’s (www.invitrogen.com) Virapower™ system make this work possible in ordinary BL-2 facilities. These features include removal of most HIV genes, deletion of the viral LTR promoter and enhancer sequences, and the provision of a minimal complement of packaging genes separate frm the virus.
Generating Viral Particles
To make the virus, expression constructs are transfected into 293FT cells along with plasmids encoding the packaging components. The virus is then assembled and secreted into the culture medium for 48 hours. The supernatant can be stored at 80°C for over one year for later use.
A healthy, high-titer viral stock is key to maximizing the percent of cells that are transduced and expressing the gene of interest. Improvements in the marketplace have focused on increasing apparent titer. Methods that simply boost the signal strength of the on-board reporter, however, give an exaggerated impression of viral titer without actually increasing the number of functional viral particles produced.
Recently, Invitrogen introduced a new line of lentiviral tools designed to provide increased numbers of functional viral particles and higher protein expression or gene-knockdown performance. Virapower HiPerform™ vectors contain the HIV central poly purine tract (cppt) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Placed directly downstream of the cloning site, the WPRE element boosts protein expression several fold over the previous generation of vectors. The cppt element increases nuclear uptake of the viral transcript, enhancing integration into the host genome, especially in nondividing cells.
Several HiPerform vectors were created to support specific applications. The pLenti7.3 FastTiter™ series was engineered with an EmGFP reporter to enable rapid titering of functional virus in 2–3 days instead of two weeks for drug selection. Transduced cells may also be sorted or isolated by flow cytometry, a feature that lends this system to high-throughput screening applications. The pLenti6.3 series contains a blasticidin-selection marker instead of EmGFP, enabling the creation of stable integrant lines.
For RNAi applications, the Block-iT™ HiPerform vector, pLenti6.4, uses Multisite Gateway™ technology to accept a selection of promoters along with an RNAi cassette coupled to EmGFP.
HiPerform expression vectors were compared to vectors not containing the cppt and WPRE but otherwise identical. Vectors were transfected into 293FT cells and packaged into the virus in 10 mL adherent cultures. Culture supernatants were collected at 48 hours, filtered, diluted, and applied to cultures of HT1080 cells to determine the concentration of functional viral particles.
Titer performance of the pLenti6.3/V5-TOPO vector was compared to a similar vector lacking the enhancing elements. Constructs containing two different genes (erythropoietin or EmGFP) were evaluated. Both HiPerform constructs revealed a substantial increase in blasticidin-resistant transductants, effectively yielding up to twofold more functional virus particles per mL of viral supernatant over vectors lacking these elements (Figure 1).
Titer was estimated using erythropoietin constructs with either blasticidin selection in pLent6.3 or the EmGFP marker in pLenti7.3. Similar titers were observed using both reporter systems, as each marker signals successful transduction and expression (Figure 1).
In many systems, the WPRE enhancer is placed downstream of the selection marker, distal to the cloning site, where it provides only minor benefit to the cloned gene. HiPerform vectors were created with the WPRE element placed immediately downstream of the cloning site where it provides maximum expression of the gene of interest. Expression of EmGFP and erythropoietin from pLenti6.3 was greater than 10-fold higher than from the same vector lacking the WPRE element (Figure 2 A, C, and D). The expression benefit of HiPerform’s enhancer configuration was reiterated with EmGFP.
Cells transduced with the HiPerform vector generated at least a 10-fold greater EmGFP fluorescence signal than the cells transduced with a vector lacking the HiPerform elements (Figure 2 C and D). On a per particle basis, HiPerform offers higher expression levels stemming from robust support of the expression cassette. Using equal volumes of viral preparations reveals that HiPerform yields more functional virus per mL of viral supernatant (Figure 2 C, red bars).
To demonstrate the stability of the integrated pseudovirus, cells transduced with and expressing the erythropoietin construct were propagated without blasticidin selection for 30 days. Erythropoietin expression was found to be essentially unchanged after 30 days, reflecting the stability of the integrant and its resistance to silencing (Figure 2B).
In RNAi evaluations using a luciferase reporter, HiPerform viral preps offered two- to threefold more robust gene knockdown over previous generation vectors stemming from an increase in viral titer (Figure 3).
The benefit from the HiPerform vector modifications is an increase in yield of functional virus, higher transduction efficiency, higher gene expression, and stronger knockdown performance. Optimizations to the HiPerform system continue with an eye toward increasing the productivity and utility of lentiviral technologies for both research and therapeutic discovery.
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