Purpose
The primary purpose of the Semmelweis University Cell Core Facility is to help all researchers at the university providing stable, cost-effective, and safe virus transduced cell lines. New, stable cell lines can facilitate research towards new directions and open new possibilities.
Background
Compared to transient expression, stable cell lines incorporate the target DNA sequence into the genom, allowing long-term, stable gene expression. These features make these cell lines reliable on long-term and make the experiments reproducible.
Aims
Our aim is to generate stabile cell lines with the help of lentivirus for labs who are not prepared for working with viruses or need our expertise in this field. We can generate cell lines to support gene overexpression (cDNA, CRISPR activation), knock-down (shRNA, CRISPR interference), or custom gene editing with the CRISPR/Cas9 system. Please contact us with any other type of genome editing plans, we can discuss the possibilities of custom-designed cell lines.
Workplan
Researchers deliver their plasmid of interest and the selected cell line to our lab. It is also possible to design the vector plasmid via consultation. The core will also help to select the right promoter, CRISPR technology and the most suitable vector design for in vivo or in vitro experiments. The Viral Vector Core Facility will start viral vector production which can take up 4 to 6 weeks (depending on cloning). With the RT-qPCR titrated lentivirus we perform an MOI (multiplicity of infection) test to determine the correct virus amount used for the specific cell type. With the determined MOI we transduce the cells and with FACS (Fluorescence-activated Cell Sorting) or antibiotic selection we can make a stable cell line. With single cell sorting, we can also prepare monoclonal cell lines. After a Mycoplasma test, we can hand over the stable, non-infectious cell lines for further validation or we can help with RT-qPCR expression verification.
Services
- Construct design and cloning strategie consultation
- Construct building / full cloning service
- Construct testing and validation e.g. restriction digestion, sequencing
- Maxiprep Service (or other DNA scale-up)
- Full service lentivirus production (Transfection of the vector plasmid into HEK 293T cells, collection and concentration of the supernatant by ultracentrifugation, titration with RT-qPCR).
- MOI test of the lentivirus on the given cell line
- Cell handling, Mycoplasma testing, expansion and transduction
- Antibiotic selection or FACS sorting of the cells
- Freeze Mycoplasma tested cells after one week expansion
Charges
The Cell Core Facility operates on a non-profit basis within the university. Our fees include the consumables and the actual salary costs.
Our services are available to all internal and external academic customers. Semmelweis University users have priority access. External and industrial customers please contact us for pricing.
First consultation and planning meeting is always free, further consultation and designs are charged 4000 Ft/hour.
One stable cell line production price is around 500.000 HUF
The final price of a cell line consists of the actual list of services:
- Full planning and construct design
- Molecular biology services:
MAXIPREP plasmid purification: 50.000 HUF
Construct building / full cloning service: 150 000 – 250 000 HUF (depending on cloning strategy)
- Lentivirus production: 150.000 HUF/batch
- FACS sorting: 20.000 HUF/h
- Cell line handling: 280.000 HUF (additional cell lines cost half price)
- RT-qPCR to validate gene expression changes: 75.000 HUF
All prices include taxes and university overheads.
To access our services, please contact us by email or phone.
Contact
Ágnes Varga, PhD
Phone: +36 1 459 1500 / 60369
Mobile: +36 30 016 4787
E-mail: varga.agnes1@semmelweis.hu
0.112 laboratory room
D part, ground floor
Basic Medical Science Center
Institute of Translational Medicine
Semmelweis University
1094 Budapest, Tűzoltó street. 37–47