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 functional genomics. Viruses are already 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

FuGU 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 Functional Genomics Unit by co-transfecting a highly transfectable cell line with a transfer vector, which carries the shRNA, cDNA, gRNA etc. of interest, together with packaging constructs. The two packaging constructs produce the proteins important for viral capsid structure, assembly and function as well as the proteins that determines the viral tropism (see below). The virus particles will be assembled in the cells and 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. The target cells need to be completely virus-free before taking out from the virus lab and this can be verified with the RCV test also offered as service by BVC facility of Helsinki Virus Core.

 

Principles of recombinant lentivirus production

Biomedicum Functional Genomics Unit uses a standard three-plasmid lentiviral system to produce high-titer VSV-G pseudotyped lentiviral particles. The recombinant lentivirusparticles are produced by transient transfection of the gene/shRNA transfer vector along with two packaging plasmids to an optimized derivative of 293T cells. At the time of transfection, the confluency of the cells should be approximately 50-60%. The 293T and derivative cells are highly transfectable and can be efficiently transfected by many standard methods, including liposomes and Calcium Phosphate. FuGU recommends e.g. Lipofectamine (Invitrogen) or the cationic polymer jetPEI™ (polyplus) transfection reagent. After transfection, the cells are incubated for 4 h at 37°C, after which new medium is added.

After 72 h incubation, the virus-containing supernatant is collected and filtered through 0,45 um PES filter. Virus supernatant is then aliquoted in cryovials and stored in -80°C, or concentrated by ultracentrifugation.

Titer of the lentiviral supernatant is measured by ELISA p24 test, which is carried out for both concentrated and unconcentrated samples. With every virus batch produced, FuGU libraries uses internal controls to ensure the high quality of the process.

Lentiviral cell culture medium can be concentrated by ultracentrifugation to increase viral particle titer (approx. 100-fold). Crude lentiviral media preparations often include cell debris and proteins that are toxic to target cells and can cause immunogenic reactions in vivo. Sucrose centrifugation separates viruses in sucrose density gradient due to differences in particle densities makes viruses consistently non-toxic for use in animal experiments.

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 in order to produce recombinant retroviruses.

Retroviral particles are cannot be concentrated because of unstability 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 and 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. FuGU libraries pseudotypes recombinant lentiviruses with 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, FuGU libraries 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 (for full list of host cells, contact Biomedicum FuGU Libraries).

 

Biosafety

The safety practises and procedures followed in our core units:

Recombinant virus particles are BSL2 material, and need to be maintained at BSL2 premises. Infected cells can be transferred into normal laboratory conditions only after the replication incompetence of the virus products in the target cells has been tested.

BSL2 safety practices should be followed when preparing and handling lentiviral particles. Personal protective clothing should be worn at all times. Use plastic pipettes in place of glass pipettes or needles. Liquid waste should be decontaminated with at least 10% bleach. Laboratory materials that come in contact with viral particles should be treated as biohazardous waste and autoclaved. Please follow all safety guidelines from your institution for work in a BSL2 facility.

If you have any questions about what safety practice to follow, please contact your institution’s safety office.

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 his/her own virus vectors for lentiviral produciton or ask for a full service from FuGU libraries.

Use of Lentiviral Products

The recombinant viral particles are biohazardous material and must be handled appropriately at BSL2 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 (BSL1). Alternatively, cell lysates or fixed cells can be brought to BSL1 laboratories without RCV testing.

RCV test as a service is performed from unfiltered cell culture media by the in house BVC facility.

The following transduction protocol is suitable for FuGU-produced mini-, midiscale lentiviral as well as ecotropic and amphotropic retroviral particles. We generally use two methods for transduction (=infection):

  • classic transduction
  • centrifugation-based spin transduction.

Classic transduction procedure is gentler to the cells than spin transduction. However, spin transduction maximizes the infection efficiency by low-speed centrifugation. The following 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

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 optimized Cas9 endonuclease and a single guide RNA (gRNA, fusion of bacterial crRNA and tracrRNA). The gRNAs Watson-Crick 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 a given 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 labeling and the follow-up of cellular processes. 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 arrayed human and mouse lentiviral CRISPR gRNA libraries have been developed in collaboration with the Wellcome Trust Sanger Institute. Both human and mouse lentiviral CRISPR gRNA libraries contain 2 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.

Validation
In-house validation of about 100 customer-selected gRNA constructs in human library were sequenced with correct clone coming up in 99% of the cases.

 

What kind of selection do the gRNA constructs have?

gRNA construct are in LV04 vector that includes pyromycin selection cassette and a blue fluorescent protein (BFP) tag for positive-cell visualization. 

Ampicillin selection is used for the growth and maintenance of the constructs in bacterial cells in LB medium.

Where can I find the full gRNA vector map?

LV04 full vector map and sequence can be found here.

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, designed and developed by the RNAi Consortium at the Broad Institute of MIT and Harvard, include hairpin sequences comprised of a 21 base stem and a 6 base loop. Rules based design consisting of sequence, specificity, and position scoring were utilized 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.

shRNA constructs can provide varying levels of knockdown in different systems and cell lines, and therefore FuGU strongly recommends validating the knockdown achieved in your system.

While the TRC shRNA clones were designed to minimise any recombination events, all genome-scale libraries of this nature have inherent errors due to spontaneous mutations occurring during plasmid propagation and purification. For this reason, FuGU offers sequencing of all clones ordered at an affordable price. Please contact us for more information.

What kind of selection do the shRNA constructs have?

The constructs are in pLKO.1 vector with puromycin selection.
Carbenicillin selection is used for the growth and maintenance of these low copy number constructs in bacterial cells in Terrific-Broth (TB) medium.

We recommend to use LB + Amp for DNA maxipreparations to avoid overgrowth.

Where can I find the pLKO.1 construct map?

Full TRC library pLKO.1 construct map and sequence is available here.