Services

The Viral Vector Facility produces batches of lentiviruses for research groups within the LUMC that don’t have the capacity to do so themselves.

• The Viral Vector Facility uses third-generation lentiviral vectors.
• The viruses are produced on HEK 293T cells by co-transfection of three packaging vectors (pCMV-VSVG, pMDLg-RRE and pRSV-REV) and a fourth plasmid containing your gene of interest.
• Virus containing medium is collected on both 48 h and 72 h post-transfection and filtrated by low-protein binding 0.45µm filters.
• If you need high titers, lentiviruses can be concentrated and purified by ultracentrifugation with 20% sucrose.
• Viral titers of batches are determined by p24 Elisa.

The Viral Vector Facility produces batches of adenoviruses for research groups within the LUMC that don’t have the capacity to do so themselves.

• Adenovirus vectors are generated and propagated on PER.C6 or 911 cells.
• Virus is collected from cells around 42 h post-infection. Additionally, virus can be collected from medium by ammonium sulphate precipitation.
• Adenoviruses are purified by CsCl gradients.
• Infectious titers (pfu/ml) are determined by plaque assay on 911 cells.
• Viral batches can be screened for presence of RCA (replication-competent adenoviruses) by PCR.

The Viral Vector Facility obtained the human and mouse Mission shRNA libraries of Sigma-Aldrich and The RNAi Consortium (TRC). You can use these libraries to study gene knockdown. Clones from these libraries can be requested for free for the purpose of research within the LUMC, but may not be distributed to external groups. shRNA constructs from these libraries can be used to produce lentiviruses.

The Viral Vector Facility acquired the Human Sanger Arrayed Whole Genome Lentiviral CRISPR library from Sigma-Aldrich. You can use this library to study gene knock-out. Clones from this library can be requested for free for research at LUMC. gRNA constructs from this CRISPR library can be used to produce lentiviruses.

• Acquired in 2016.
• 34,000 human gRNA clones (17,000 human target genes). 2 gRNA clones of each target gene.
• Bacterial glycerol stocks of all constructs are arrayed in 96-well plates (one clone per well).
• gRNA sequences are cloned into pU6-gRNA-PGK-Puro-2A-BFP. The vectors of the CRISPR library aren’t all-in-one vectors. The clones don’t contain a CAS9, so it needs to be administered separately. The vector pEF1a-CAS9-2A-Blasticidin can be used to deliver CAS9. An alternative method of CAS9 delivery is by an adenovirus containing CAS9.
• A non-target negative control and a human HPRT1 positive control are available. The vector backbone of both controls is pU6-gRNA-PGK-Puro-2A-BFP.
• The gRNA sequences are not shown on the website of Sigma-Aldrich. But the Viral Vector Facility has a database of this library.
• To request constructs from this CRISPR library, please provide a list of genes (symbol and/or ENSG identification code).
• CRISPR constructs are delivered as bacterial cultures (2 ml LB medium with ampicillin).
• This bacterial culture can be used to do a midi/maxi-prep and, if necessary, to generate lentiviruses.

The Viral Vector Facility obtained the human ORF (cDNA) library from Sigma-Aldrich. You can use constructs from this library for overexpression studies, to validate the results of gene knockdown (shRNA) or gene knock-out (CRISPR) experiments. Clones from this library can be requested for free for research at LUMC.

• 16,000 human ORF clones. 1 clone per gene.
• Acquired in 2017.
• Bacterial glycerol stocks of all constructs are arrayed in 96-well plates (one clone per well).
• Fully sequenced ORFs into entry vector pDONR223.
• The native stop codon is removed and replaced with a termination codon. There is no upstream promotor. For expression, it needs to be cloned into a Gateway-adapted expression vector.
• Clones are grown in 2ml LB medium containing spectinomycin antibiotics.

If you’re interested in one of the ORF clones, please contact Martijn Rabelink (LUMC, Department of Cell and Chemical Biology) for gene availability, sequence details, and clone picking.