Services

We produce batches of lentiviruses for research groups within LUMC or external preclinical research.

• Required: plasmid with gene of interest should be compatible with 3rd (or 2nd) generation lentivirus production system; please provide 15µg (or more if >10kb) plasmid DNA
• Transfection (3rd generation): co-transfection of your plasmid (see above) with 3 packaging plasmids pCMV-VSVG, pMDLG-RRE and pRSV-REV; transfection reagent: polyethylenimine (PEI)
• Producing cell line: 1x T175cm2 flask HEK 293T cells, cultured in DMEM with 8% FCS
• Harvest: 48h (15ml) and 72h (15ml); low protein binding 0.45µm filtered; pooled
• Aliquots: 3x 10ml (stored at -20°C or -70°C) and 1x 10µl which will be used for p24 ELISA
• p24 ELISA: 2 dilutions; 1:2000 and 1:8000 for 3rd generation
• Expected yield: titer range usually 200-500ng/ml for 3rd generation
• Quality control mycoplasma: 293T producer cell line mycoplasma screened
• Quality control titer: a p24 ELISA is always included for quality control of virus production
• Transduction guide (valid for most cell lines): to transduce cells with MOI 1 (1 virus/cell), you need 1ng virus (based on p24 ELISA) per 2500 cells  

We can concentrate lentivirus batches to achieve higher titers, purify the batch and/or remove serum.

• Required: to be concentrated virus batch can be produced by us (see section lentivirus production) or already produced by yourself.
• Ultracentrifuge: Beckman Coulter Optima XE-90, located at ML-II biosafety lab
• Rotor: Sw32Ti (standard; 30ml virus/tube) or Sw41Ti (volumes up to 10ml/tube)
• Gradient: 20% sucrose density gradient for purification (Sw32Ti: 4.5ml sucrose at bottom)
• Standard volume: 30ml virus (volume of 1 standard virus production)
• Scalability: when using up to 6 tubes per run, a higher concentrating factor of more than 100x can be reached
• Centrifugation: 2hr at 30000rpm
• Buffer: virus pellet dissolved in 600µl (or 1000µl if pooled from multiple tubes) serum-free buffer (50mM Tris-Cl; 130mM NaCl; 1mM EDTA; pH 7.8)
• Aliquots concentrated virus: 40/50µl and 10/15µl or other preferred volumes (stored at -70°C) and 1 aliquot of 10µl which will be used for p24 ELISA
• Quality control: a p24 ELISA is always included for quality control of concentrating virus
• Titration: p24 ELISA; 3 dilutions; dilution factor depending on expected titer  

We can determine titers of lentivirus batches by p24 ELISA. These batches can be produced by us (see sections lentivirus production and lentivirus concentrating) or you can ask us to measure the titer(s) of your own batch(es).

• Required: for samples of batches not produced by Viral Vector Facility: please provide at least 15µl
• p24 ELISA kit: ZeptoMetrix, Retrotek HIV type 1 p24 antigen ELISA 2.0
• Standard series: range of 7 concentrations and a blanc, all in duplo
• Platereader: Byonoy, Absorbance 96, wavelenght 450nm
• Quick (budget) titration: only 1 dilution of sample; usually 1:4000 (standard virus production); risc value is out of range standard series
• Titration standard virus production: 2 dilutions of sample; usually 1:2000 and 1:8000 (3rd generation)
• Titration concentrated virus: 3 dilutions of sample; usually 1:40000, 1:120000 and 1:360000; range depending on initial titer and concentrating factor
• Titration cultured transduced cells: undiluted sample in duplo; please provide 1ml; extended low range standard series; to test if supernatant of cultured transduced cells is free of virus (not detectable)  

We can produce batches of adenoviruses (wild type, 1st and 2nd generation replication deficient adenoviruses, or oncolytic adenoviruses), purify these batches, and determine titers by plaque assay.

• Producing cell lines: adenovirus vectors are generated and propagated on 911 and PER.C6 helper cell lines
• Standard scale of a large batch: 20x T175cm2 flasks
• Scalability: if desired, to reach higher titer and total yield, we can produce and pool multiple batches
• Harvest: virus is collected from cells around 42h (depending on virus) post-infection; additionally virus can be collected from medium my ammonium sulphate precipitation
• Purification: batches can be concentrated and purified by ultracentrifugation (CsCl gradients) followed by dialysis against 5% sucrose buffer
• Titration: infectious titers (pfu/ml) are determined by plaque assay on 911 cell line
• Quality control: optional, viral batches can be screened for presence of replication-competent adenoviruses (RCA) by PCR  

We can produce batches of reovirus (R124, JIN1, JIN3), purify these batches, and determine titers by plasque assay.

• Producing cell line: HER 911 cells
• Standard scale: 5x T75cm2 flasks
• Purification: batches can be purified by ultracentrifugation (CsCl gradient)
• Titration: infectious titers (pfu/ml) are determined by plaque assay on 911 cell line  

We obtained the human and mouse Mission shRNA libraries of Sigma-Aldrich (Merck). You can use these libraries to study gene knockdown. Clones from these libraries can be requested for research within LUMC, but may not be distributed to external groups. shRNA constructs from these libraries can be used to produce lentiviruses, by yourself or by us.

• Complete human and mouse TRC1 and TRC1.5 libraries are available and the main part of TRC2 (all validated shRNA constructs, and all constructs of target genes which are not already present in TRC1 or TRC1.5)
• 129,500 human shRNA clones (20,000 unique target genes)
• 118,000 mouse shRNA clones (21,200 unique target genes)
• Backbone: plasmids are based on pLKO.1-puro vector; TRC1 and TRC1.5 are identical; TRC2 has a minor modification (wPRE element included)
• Validated constructs: knockdown of at least 50% (high throughput qPCR); validation data can be found on the Sigma-Aldrich website
• To request constructs: please provide the accession/RefSeq number (NM_....../XM_......) of the target gene and/or specify the TRC numbers (TRCN0000......)
• Delivery: bacterial cultures grown overnight in 2ml LB medium with ampicillin  

We acquired the Human Sanger Arrayed Whole Genome Lentiviral CRISPR library from Sigma-Aldrich (Merck). You can use this library to study gene knock-out. Clones from this library can be requested for research within LUMC, but may not be distributed to external groups. gRNA constructs from this CRISPR library can be used to produce lentiviruses.

• 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)
• Backbone: gRNA sequences are cloned into pU6-gRNA-PGK-Puro-2A-BFP; these vectores aren’t all-in-one vectors; clones don’t contain a CAS9, so it needs to be administered separately
• CAS9: we suggest to use pLenti CAS9 Blast (Addgene #52962) to deliver CAS9
• To request constructs: please provide a list of genes (symbol and/or ENSG identification code)
• Delivery: bacterial cultures grown overnight in 2ml LB medium with ampicillin  

The department of Cel and Chemical Biology (CCB) obtained the human ORF library from Sigma-Aldrich (Merck). 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 research within LUMC.

• 16,000 human ORF clones; 1 clone per gene
• 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
• To request constructs: please provide gene symbol, GeneID and/or ENSG identification code
• Delivery: bacterial cultures grown in 2ml LB medium containing spectinomycin antibiotics