RESOURCES

Recombinant Virus Technology

RNAi (shRNA/miRNA) and ORF (cDNA) expression constructs can be efficiently introduced into most cell types by using recombinant viruses as vector carriers. Recombinant virus transduction technology has a wide range of applications in molecular and cell biology research and in functional genomics. Viruses are used as routine gene construct delivery tools in many laboratories, and there are many applications of viral delivery systems such as:

  • Gene therapy research
  • Genetic or genomic level screening
  • Discovery and validation of druggable genes
  • Pathway identification

Genome Biology Unit libraries has both human and mouse lentiviral genome-wide TRC lentiviral shRNA library and genome-wide CRISPR/Cas9 gRNA knock-out library for loss-of-function studies.

Production of Recombinant Lentiviruses and Retroviruses

Recombinant lentivirus particles are produced in Biomedicum Virus Core by co-transfecting a highly transfectable cell line (HEK293 FT) with a transfer vector, which carries the shRNA, cDNA, gRNA etc. of interest, together with two packaging constructs. The packaging constructs produce the proteins required for viral capsid structure, assembly, and function as well as the proteins that determine the viral tropism. The virus particles are assembled within the cells and then released to the culture supernatant. These recombinant virus particles will only carry the sequences of transfer vector in their genome and therefore, they can transduce the target cells with high efficiency, yet the target cells will not propagate the infection. However, the transduced cell lines or cultures need to be completely virus-free before they can be taken out from a BSL-2 virus lab.

 

Transfer vectors used in production of recombinant viruses

Transfer vectors express shRNA, miRNA adapted shRNA or mRNA from DNA inserts, which have been cloned into a virus vector backbone. These constructs are designed to either silence or ectopically express the gene of interest. A customer can either supply us with their own virus vectors for lentiviral production or ask for a full service from HelVi-BVC.

 

Principles of Recombinant Lentivirus Production

Biomedicum Virus Core uses a standard three-plasmid lentiviral system to produce high-titre VSV-G pseudo type lentiviral particles. The recombinant lentivirus particles are produced by transient transfection of the gene/shRNA transfer vector along with two packaging plasmids to an optimized derivative of highly transfectable HEK293FT cells.

The HEK293FT and derivative cells can be efficiently transfected by many standard methods, including liposomes and calcium phosphate. For example, commercial Lipofectamine (Invitrogen) and jetPEI™ (Polyplus) can be used as transfection reagent. At the time of transfection, the confluency of the cells should be approximately 50-70%. Proceed with transfection according to the manufacturer’s instructions and follow the protocol. After transfection, the cells are incubated at +37°C, and depending on the transfection reagent and the protocol media is then changed or added after 4-24h post transfection. After 72h incubation (post-transduction), the virus-containing supernatant is collected and filtered through 0,45 µm PES filter. Virus supernatant is then aliquoted (or concentrated and then aliquoted) in cryovials and finally stored in -80°C.

Lentiviral cell culture medium can be concentrated by ultracentrifugation or with concentrating regents (e.g. PEG-it (System Biosciences) or Lenti-X concentrator, (Takara)) to increase viral particle titre (approximately 100-fold). Sucrose centrifugation separates viruses in sucrose density gradient from toxic cell debris and proteins, which makes viruses consistently non-toxic for use in animal experiments and prevents possible immunogenic reactions in vivo.

Titre of the lentiviral supernatant is measured by p24 test (e.g. PerkinElmer kit), which is carried out for both concentrated and unconcentrated samples. With every virus batch produced, HelVi-BVC also uses internal controls to ensure the high quality of the process.

Ret­ro­virus pro­duc­tion

Recombinant retroviruses are produced in a similar way as lentiviruses, the main difference in the protocol being specific packaging cells. Retroviral packaging cells contain stably integrated viral packaging genome, either in Ecotropic packaging system (capable of delivering genes to murine cells) or in Amphotropic system (delivery to most mammalian cells including human). Therefore, only the transfer vector needs to be transfected to the packaging cells to produce recombinant retroviruses.

Retroviral particles cannot be concentrated by ultracentrifugation because of instability of envelope proteins and viral internal core (DOI: 10.14348/molcells.2017.0043).

 

Viral tropism and pseudotyping

The envelope proteins of retro- and lentiviruses determine the host cell range (viral tropism). There are several options for envelope proteins since they are encoded by separate vector in the lentiviral systems. In the retroviral systems the envelope is selected by choosing a desired packaging cell line. The selection of an envelope protein to determine the viral tropism is called pseudotyping. HelVi-BVC pseudotypes recombinant lentiviruses with Vesicular stomatitis virus G (VSV-G), which allows infection of broad range of mammalian and non-mammalian host cells including human, primate, mouse, rat, hamster, and fish.

In addition to lentiviral particles, HelVi-BVC unit also produces Moloney mouse leukemia virus (MMLV) and mouse stem cell virus (MSCV) vector based retroviruses pseudotyped with ecotropic or amphotropic envelope. Ecotropic retroviruses infect mouse and rat cells but not human cells. Amphotropic viruses have broader host range infecting for example human, primate, mouse, rat, and rabbit cells.

 

Biosafety

The safety practises and procedures followed in our core unit and in our BSL-2 facility:

Recombinant virus particles are considered as BSL-2 material, and need to be maintained at BSL-2 premises and facilities. Infected cells can be transferred into “normal” BSL-1 laboratory conditions only after the replication incompetence of the virus products in the target cells has been proved, for example with and RCV test.

BSL-2 safety practices should be followed when preparing and handling lentiviral particles. Personal protective clothing should be always worn when working in a BSL-2 facility. Use disposable plastic pipettes, and avoid using sharps, such as glass pasteurs or needles. HelVi-BVC facilities are sharp-safe and we don’t allow scalpels, needles or glass pipettes in our labs. All liquid waste should be decontaminated with viral inactivator reagent, such as Virkon. Laboratory materials that encounter viral particles should be treated as biohazardous waste and autoclaved. Therefore use of disposal items is recommended.

Please follow all safety guidelines from your institution for work in a BSL-2 facility. If you have any questions about what safety practice to follow, please contact your institution’s safety office. HelVi-BVC customer support can also assist with biosafety issues!

Use of Lentiviral Products

The recombinant viral particles are biohazardous material and must be handled appropriately at BSL-2 space. Once the transduced adherent cells have been passaged at least three times and a negative RCV test result has been received, the cells can be transferred to normal laboratory spaces (BSL-1). Alternatively, cell lysates or fixed cells can be brought to BSL-1 laboratories without RCV testing. HelVi-BVC performs the RCV test as a service every second Wednesday from unfiltered cell culture media.

The following transduction protocol is suitable for HelVi-BVC-produced MINI and MIDI scale lentiviral as well as ecotropic and amphotropic retroviral particles. We generally use two methods for transduction (i.e. infection): classic transduction and centrifugation-based spin transduction.

Classic transduction procedure is gentler for the cells than spin transduction. However, spin transduction maximizes the infection efficiency by low-speed centrifugation. The following protocol examples are for 6-well plates. Scale up or down according to your needs.

 

Protocols

 

Lentivirus infection protocol with optimal MOIs in common cell lines

 

Additional resources:

Addgene lentiviral guide:

Addgene has put together a webinar ( Lentivirus 101: Plasmids and Viral Production ) with Bitesize Bio focused on understanding the components of lentiviruses and how they are produced in the lab. The webinar covers:

  1. Plasmids required to generate lentivirus (both 2nd and 3rd generation systems)
  2. Safety Concerns

Lentiviral-based applications: https://www.addgene.org/guides/lentivirus/

Addgene FAQ: https://www.addgene.org/guides/lentivirus/#faq

Genome-wide CRISPR/Cas9 gRNA Knock-Out library

The formed FuGU Virus’ CRISPR gRNA libraries are maintained and overseen by Genome Biology Unit (GBU). For more information about the libraries please visit GBU web page or contact their customer support.

Basics of CRISPR/Cas9 technology

The clustered regularly interspaced short palindromic repeats (CRISPR) type II system, originally an adaptive immune system widely distributed in prokaryotes, is currently commonly used for RNA-guided, endonuclease-mediated genome engineering. The system has two components: codon optimised Cas9 endonuclease and a single guide RNA (gRNA, fusion of bacterial crRNA and tracrRNA). The gRNAs base pair with complementary DNA sequences and direct Cas9 nuclease to its target site in DNA. In addition, a protospacer adjacent motif (PAM) sequence must be located immediately following the gRNA target locus. This slightly limits possible target sites in each DNA.

CRISPR/Cas9 system produces double strand breaks (DSBs) in a gRNA-specific manner in DNA. DSBs can be repaired by either one of the endogenous DNA repair pathways: either by non-homologous end joining (NHEJ) or by homology directed repair (HDR) pathway. The two repair systems are error prone, and thus, NHEJ pathway results in small insertions and deletions (indel mutation), which can cause frameshifts or premature stop codons that knockout gene expression. For HDR pathway function, a homologous donor/repair template is required. CRISPR/Cas9-mediated DBSs greatly improve the chance of inserting transgenes or single nucleotide substitutions into the target DNA.

Besides gene expression/knockout studies, the CRISPR/Cas9 system has several other applications such as genome-scale functional screens, the creation of transgenic animals, somatic genetic modifications, transcriptional regulation, DNA labelling and cellular process tracking. Compared to other engineered endonuclease systems, such as ZFNs (Zinc-finger nucleases) and TALENs (transcription activator-like effector nucleases), the CRISPR/Cas9 system is highly specific, easier and cheaper to design and produce, efficient, well-suited for high-throughput platforms and useable in a variety of cell types and organism.

 

Human and mouse CRISPR/Cas9 gRNA LIBRARIES

Sigma-Aldrich's (Merck’s) arrayed human and mouse lentiviral CRISPR gRNA libraries have been developed in collaboration with the Welcome Trust Sanger Institute. Both human and mouse lentiviral CRISPR gRNA libraries contain two pre-cloned gRNA constructs per protein-encoding gene, a total of about 40,000 gRNA constructs per each library. The gRNAs have been designed to target the first half of the coding region of the target gene, avoiding the first 90 bases of the protein coding sequence. Additionally, the gRNAs target consensus genomic sequences, which should not contain SNPs (only human library).

The lentiviral vector does not express Cas9. This allows the researcher to choose the best Cas9 delivery and expression format (e.g. mRNA, DNA plasmid, protein or transgenic animal) depending on the experimental set up.

 

The formed FuGU Virus’ shRNA (TRC) libraries are maintained and overseen by Genome Biology Unit (GBU). For more information about the libraries please visit GBU web page or contact their customer support.

Genome-wide shRNA libraries

The RNAi Consortium (TRC) is a collaborative group of world-renowned academic and corporate life science research groups whose mission is to create comprehensive tools for functional genomics research and make them broadly available to scientists worldwide.

The TRC-1 human and mouse shRNA libraries were developed at the Broad Institute of MIT and Harvard and currently consist of ~159,000 pre-cloned shRNA constructs targeting ~16,000 annotated human genes (TRC-Hs 1.0) and ~15,950 annotated mouse genes (TRC-Mm 1.0) for RNA interference-mediated gene silencing.

TRC shRNA clones include hairpin sequences comprised of a 21 base stem and a loop consisting of six bases. Rules based design consisting of sequence, specificity, and position scoring were utilised to generate optimal shRNA sequences. The hairpin sequences were each cloned into the pLKO.1 vector and sequence verified. Typically, 3-5 shRNA constructs were created for each target gene to provide varying levels of knockdown and to target different regions of mRNA transcript. Two in five clones will typically provide at least 70 % knockdown of the gene target. Most target sets include an shRNA clone targeting the 3'UTR for use in phenotypic rescue studies using cDNA expression constructs.