Lentivirus Design & Production

Engineered Lentivirus vectors for gene expression and knockdown

Lentivirus vectors are the method of choice whenever a fast, efficient and cost-effective generation of stable cell models is required.

SIRION Biotech’s custom lentivirus service enables constitutive as well as inducible vectors for overexpression and gene knockdown. Our multicistronic vector design results in highly homogeneous, stable cell models avoiding time-consuming clonal selection posttransduction. With the all-in-one inducible vector system SIRION enables working with difficult, cytotoxic genes and shRNA strategies. We offer vectors in less than 4 weeks.

Custom Lentivirus at a glance:

Personal project consultancy with our specialists
Highly homogeneous stable cell pools through optimized vector design
Mono- and bicistronic vector design
Virion modification
Broad selection of promotor, selection marker and fluorescent tags
Reliable gene delivery into dividing and non-dividing cells
Fast, stable and strong expression
Inducible LV technology enabling characterization of toxic gene modulations
Potent transduction enhancers for hard-to-transduce primary cell types and increased tissue specificity.

Any project is thoroughly discussed with our customers to ensure that the resulting particles are feasibly designed and fit the final application.

Custom constitutive

To ensure the safety of Lentivirus vectors and prevent virus generation, 3rd-generation lentivectors exhibit the viral genome divided into separate plasmids. SIRION Biotech uses 3’SIN Technology (self-inactivative) of 3rd generation Lentivirus vector engineering. In 3’SIN Technology 400 bp deletion in the U3 region of the 3‘UTR inactivates promoter activity. During integration of the viral DNA into the host genome the 3‘SIN LTR is transferred to the 5‘end resulting in a provirus lacking U3 promoter activity at both ends. 3’SIN Technology enables controlled gene expression from internal promoter(s) which is a mandatory prerequisite for all inducible, cell type-specific or pathway reporter expression systems.

3’SIN Technology Lentivirus vectors:

No overlapping sequences between the co-transfected vectors to eliminate any homologous recombination
VSV-G protein pseudotyping for broad host tropism
Controlled gene expression from internal promoter(s)

In 3’SIN 3 packaging plasmids are used to express all protein components necessary for LV packaging and generation of infectious viral particles plus a plasmid carrying GOI. Lentivirus vectors are pseudotyped with VSV-G protein for mediation of fusion of the viral envelope with the host cell.

Constitutive Lentivirus

Depending on the project needs, it can be necessary to design a construct with strong constitutive gene overexpression or to induce expression at a particular time point. In this case it is important to design the construct which fits your needs the best. SIRION Biotech offers a broad portfolio of constitutive and inducible promoters for Lentivirus vectors.

Stable cell model generation to analyze long-term effects of gene overexpression and silencing in vitro
For dividing and non-dividing cells
Variety of strong and ubiquitous promoters (CMV, Ubi, EF1-α, etc.)
Custom promoters
IRES or 2A sequences for bicistronic vectors
Knockdown efficiency of at least 80% on mRNA level when combining with our RNAiONE service

Service Details

Cloning of customer cDNA/shRNA in SIRION’s lentiviral expression vector
Verification of cloning success by DNA-Sequencing
Lentivirus packaging in HEK293 cells
Purification, concentration and QC
Lentivirus titration by qRT-PCR

Scale	Infectious Units (IU)	Time
Standard	≥ 1 X10^7	4-6 weeks
Medium	≥ 1 X10^8	5-6 weeks
Large	≥ 5 X10^8	6-7 weeks
X-Large*	≥ 2.5 X10^9	7-8 weeks

*contact us directly for larger scales **the table represents timelines for constitutive Lentivirus generation, including cloning

Custom inducible

SIRION Biotech’s inducible expression platform is based on the latest 3G TET technology. This guarantees a high sensitivity without leakiness. SIRION offers inducible lentiviral ONE-vector and TWO-vector systems.

Fields of Application

Establish stable inducible cell models
Circumvent cell adaption to stable genetic modification
Enable characterization of toxic gene modulations

SIRION’s benefits

TET-inducible All-in-ONE vector backbone
TET-inducible TWO-vector technology for large constructs
Highly sensitive without leakiness
Knockdown efficiency of at least 80% on mRNA level when used in combination with RNAiONE
Custom lentivirus in less than 4 weeks

Service Details

Cloning of your cDNA/ shRNA into an inducible lentiviral expression vector
Verification of cloning success by DNA-Sequencing
Lentivirus production in Hek 293 cells
Purification, concentration and QC
Lentivirus titration by qRT-PCR and determination of the infectious titer [IU/ml]

Scale	Infectious Units (IU)	Time
Standard	≥ 1 X10^7	4-5 weeks
Medium	≥ 1 X10^8	5-6 weeks
Large	≥ 5 X10^8	TBD
X-Large	≥ 2.5 X10^9	TBD

*the table represents timelines for inducible Lentivirus generation, including cloning