Sirionbiotech Products & Services

RNAiONE™ algorithmic identification of shRNA sequences


RNAiONE™ algorithmic identification of shRNA sequences


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Products & Services | SIRION Biotech GmbH

Overview

SIRION Biotech offers RNAiONE™ as a guaranteed path to define the most effective small hairpin RNA (shRNA) sequences for target gene identification and validation which can be further translated into SIRION’s viral vector platform.

RNAiONE™ provides one solution for two challenges by identifying the most optimal shRNA sequence, and engineering a viral vector with the best performing validated shRNA. RNAiONE™ serves for effective gene knockdown with success rates above 90%.

Over the years SIRION Biotech has developed its base technology to the point where in 80% of recent projects it managed to identify targeted shRNA sequences with knockdown results of above 90%.

Identifying elusive shRNA sequences for effective gene knockdown

A short hairpin RNA (shRNA) is an artificial RNA molecule that is used to silence target gene expression via RNA interference (RNAi). shRNA has the following mechanism of action: shRNA binds to the complementary target mRNA in cell, inactivates or degrades the target mRNA leading to gene silencing. In gene therapy this can be relevant to certain types of cancer, where specific mRNAs were shown to be involved.

Expression of shRNA in cells is reached by delivery of shRNA to cells using viral vectors. Different viral vectors can be used to bring shRNA in cells including adeno-associated viruses (AAV), adenoviruses, and lentiviruses. AAV and adenoviruses do not integrate into genome, so gene expression remains relatively transient. Lentivirus provide a possibility to obtain stable gene expression due to viral genome integration allowing long-term knockdown of the target gene.

RNAiONE™ solution for uncovering best possible shRNA sequences

Designing and validation of the best performing shRNAs is essential for effective RNAi knockdown. The key challenge in designing of the effective shRNAs is to isolate the most optimal sequence from millions of possible combinations.

SIRION Biotech’s algorithm-based design of shRNA sequences allows successful identification of 10-15 most active shRNA sequences. The results are fully translatable to SIRION Biotech`s viral vector platforms and allow knockdown efficiency of more than 80 % on mRNA level.

RNAiONE™ can be applied whenever knockdowns are needed, including:

  • Gene loss of function studies
  • Gene expression silencing
  • Target identification & validation for early point drug discovery (e.g. in oncology)
  • Cell model generation

RNAiONE™ benefits: guaranteed results

The RNAiONE™ solution delivers a series of quantifiable benefits, including:

  • Knockdown guarantee of > 80% on mRNA level
  • Results are fully translatable into viral vector platforms
  • Full portfolio of RNAiONE compatible viral vector platforms (Lentivirus, Adenovirus, AAV)
  • RNAiONE validation of hard-to-knockdown targets with knock-down efficiency more than 90 %
  • Validated shRNA in less than three weeks
  • Documented project statistics
  • Customer Satisfaction Guarantee

Resources

Click on RNAiONE™ shRNA sequence algorithmic identification for other information.
Click on SIRION to contact the company directly.


Supplier Information
Supplier: SIRION Biotech GmbH
Address: Am Klopferspitz 19, 82152 Martinsried, Germany
Tel: +49 89 700 961 99 9
Fax: +49 89 700 961 99 8
Website: http://www.sirion-biotech.com/


Engineered lentiviral vectors for custom cell models


Engineered lentiviral vectors for custom cell models


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Products & Services | SIRION Biotech GmbH

For researchers looking to generate stable cell models in a fast, efficient and cost-effective way, lentiviral vectors are increasingly becoming the method of choice.

SIRION biotech offers a wide portfolio of customized lentiviral services:

  • lentiviral vectors for overexpression and gene knockdown ex vivo
  • constitutive and inducible promoters
  • multicistronic vector designs for highly homogeneous, stable cell models without time-consuming clonal selection post-transduction
  • SIRION’s All-in-ONE inducible vector system for easier handling with difficult cytotoxic genes and short hairpin (shRNA) strategies

The service allows vectors to be produced in less than 4 weeks.

Custom lentivirus features

The service offers cloning of customer cDNA/shRNA into lentiviral expression vectors with verification of cloning success by DNA-Sequencing. Lentivirus production is in HEK293 cells and includes purification, concentration and quality control (QC). Lentivirus titration is performed by qRT-PCR and determination of the infectious titer [IU/ml].

The SIRION custom lentivirus service is characterized by a number of distinctive features:

  • Personalized consultation on vector engineering. See contact form here
  • Optimized vector design for highly homogeneous stable cell pools
  • Reliable gene delivery into dividing and non-dividing cells
  • Fast, stable and strong expression
  • Inducible constructs enable characterization of toxic genes and shRNA
  • Scales from standard and X-large to XX-Large (on request)
  • Access in-house technology: virion modifications and potent transduction enhancers for hard-to-transduce cell types and increased tissue specificity

Custom constitutive lentivirus vectors

Custom constitutive Lentivirus vectors are useful in generating stable cell models to analyze long-term effects of gene overexpression and silencing in dividing and non-dividing cells.

SIRION’s Custom Constitutive Service offers:

  • Large promoter and marker gene selection
  • Variety of different mono- and bicistronic viral vector backbones
  • Combined together with SIRION’s RNAiONETM service, it guarantees flexibility in vector design with knockdown efficiency of at least 80% on mRNA level

Custom inducible lentivirus vectors

SIRION Biotech’s inducible expression platform is based on the latest 3G TET technology which allows to control gene expression reversibly by turning it on or off in the presence of doxycycline. The platform is beneficial for establishing stable inducible cell models, avoiding of silencing of promoter and enabling of toxic gene modulations.

SIRION’s Custom Inducible Service offers:

  • A variety of TET-based vector systems, including the SIRION All-in-One vector
  • High sensitivity without leakiness
  • Inducible knockdown efficiencies above 80% (on mRNA level) when used in combination with the RNAiONETM system

Lentivirus scale selection

SIRION provides custom constitutive as well as inducible lentiviral vectors in various scales.

Standard scales Titer in Viral Particles (IU) Timelines
Standard ≥ 1E7 IU 2-4 weeks
Medium ≥ 1E8 IU 3-5 weeks
Large ≥ 5E8 IU 6-7 weeks
X-Large ≥ 2.5E9 IU 7-8 weeks
XX-Large ≥ 5E9 IU On request

 

Resources

Click on SIRION custom-engineered Lentivirus vectors for other information.
Click on SIRION to contact the company directly.
Learn more about SIRION’s RNAiONETM system.


Supplier Information
Supplier: SIRION Biotech GmbH
Address: Am Klopferspitz 19, 82152 Martinsried, Germany
Tel: +49 89 700 961 99 9
Fax: +49 89 700 961 99 8
Website: http://www.sirion-biotech.com/


Customized adenoviral vectors (AVs)


Customized adenoviral vectors (AVs)


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Products & Services | SIRION Biotech GmbH

Overview

SIRION Biotech can supply a range of readily available adenoviral vectors (AVs) that can be customized to help overcome commonly encountered problems when using conventional lipid-based transfection methods on specialized and primary cell types.

Benefits

The SIRION AVs are customizable to specific application and target cell type and deliver distinct benefits:

Highly effective: Using the customized AVs can achieve close to 100% gene transfer into specialized cell types.
Easy to use: The fully customizable AV particles are also easy to handle. They are simply applied to the cells and washed after 12 to 24 hours.
Flexible and adaptable: SIRION offers full customization of adenovirus, lentivirus and AAV vectors to after full consultation with the client.

Strong gene expression

In a recent experiment, customized AV elicited particularly strong expression in in patient-derived retinal pigment epithelial (RPE) cells, which are a valuable target in ophthalmological research. RPE closely interact with photoreceptors to uphold visual function. Several forms of retinal degeneration can be traced to malfunction of this cell type.

RPE cells can be salvaged from tissue but genetic modification is difficult, with standard transfection methods reaching only some five per cent of all cells in any given population.

However, adenovirus vectors by SIRION Biotech outperformed standard transfection methods to elicit a robust expression in nearly all cells even six weeks after transduction , all while using the minimal dilution ratio of only one viral particle per cell.

Similar high performance is attainable with almost any adherent mammalian cell type and has been reported to extend to other species such as fish or birds.

Highest Technological Standards

SIRION expertise in customizing AVs is derived from its position as a world leader in innovating virus vector technologies and providing custom services to academic and industrial partners as well as licensing unique virus technologies for pre-clinical and clinical development.

SIRION Biotech is the only company mastering all three major virus types used regularly in the life sciences; Adenovirus, Lentivirus and adeno-associated viruses (AAVs).

It can develop customized cell models and gene vectors in very short time frames, offering custom project management to fit different experimental setups.

Customized models can include control gene expression and knockdown, all-in-one lentivirus, AAV and adenovirus vectors, along with inducible tissue specific expression and transduction boosters.

Resources

Click on Customized adenoviral vectors (AVs) for other information.
Click on SIRION to contact the company directly.

E-cadherin staining (marked in red) shows AV vector treatment elicits GFP expression in almost every single cell of RPE cell-pool that is still evident six weeks after transduction

E-cadherin staining (marked in red) shows AV vector treatment elicits GFP expression in almost every single cell of RPE cell-pool that is still evident six weeks after transduction


Supplier Information
Supplier: SIRION Biotech GmbH
Address: Am Klopferspitz 19, 82152 Martinsried, Germany
Tel: +49 89 700 961 99 9
Fax: +49 89 700 961 99 8
Website: http://www.sirion-biotech.com/


Modifying iPS Cells With Lentivirus to build more robust disease models


Modifying iPS Cells With Lentivirus to build more robust disease models


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Products & Services | SIRION Biotech GmbH

Overview

SIRION Biotech can now use Lentivirus to modify induced Pluripotent Stem (iPS) cellsto enhance their usefulness in research. Modifying with gene markers can help researchers track each cell’s development and study their signaling properties. Inducing gene mutations in this way can help create new genetic models for different diseases.

Using viral vectors in research

Engineered iPS cells are essential biotech tools, used to develop new treatments for a long list of diseases and physiological conditions including diabetes, Parkinson’s and spinal cord injuries.

SIRION Biotech has worked with Dr. Alejandro Soto-Gutierrez, Assistant Professor of Pathology at the University of Pittsburgh and expert in working with different stem cell cultures, who uses viral vectors in his research to deliver more robust and consistent results.

Advanced lentiviral technologies have helped Dr.Soto-Gutierrez develop new strategies to grow and select iPS cells to understand hepatic tissue assembly and regeneration. His findings will be essential in the development of new therapies by modeling different liver pathologies, closer to the in-vivo situation.

Dr. Soto-Gutierrez has said that SIRION’s viral systems assist his research approach and his studies of human tissue biology.

Gene modification strategies

While iPS cells are seldom modified in the end-application, it can be tremendously helpful when researching their properties during therapy development. Gene markers help track the cells’ developmental status, localize them in in-vivo applications or study their genetic signaling properties. Inducing gene mutations leads to the creation of new disease models.

SIRION Biotech specializes in development of gene modification strategies for even the most difficult cell types. With a complete toolbox of high-end technologies, years of experience and more than 500 successful projects the laboratory caters to top academic and industrial scientists, such as Dr. Soto-Gutierrez, all around the world.

Resources

Click on Modifying iPS Cells With Lentivirus to build more robust disease models for other information.
Click on SIRION to contact the company directly.


Supplier Information
Supplier: SIRION Biotech GmbH
Address: Am Klopferspitz 19, 82152 Martinsried, Germany
Tel: +49 89 700 961 99 9
Fax: +49 89 700 961 99 8
Website: http://www.sirion-biotech.com/


ProVector™: Protecting production cells from problematic transgene expression


ProVector™: Protecting production cells from problematic transgene expression


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Products & Services | SIRION Biotech GmbH

Overview

SIRION Biotech has engineered ProVector™ as a new production cell to overcome problems caused by transgenic expression that can fatally compromise vector yield during virus production.

SIRION is using its new ProVector production technique to build Ad19 vaccination vectors whose properties can help elicit a strong and lasting immune response against target antigen.

Viral vector gene editing

Advanced viral vector technologies have become game changers for companies heavily invested in modern pharma R&DThese high-end gene editing tools form the bases for gene- and immunotherapy projects around the world and are predicted to play an crucial role in battles against a range of difficult to treat diseases, from HIV to Ebola.

In modern vaccination studies, viral vectors are predominantly used with the intention of presenting key antigens to the immune system by reprogramming the patient’s own immune cells, so that the said antigens can express themselves directly.
While this technology has shown promising results in the past, several roadblocks can occur during development.

One such challenge is the unwanted expression of the antigen transgenes during production (“packaging”) of the virus particles. This can seriously compromise the vector yield and in worst cases can spell the end of otherwise well designed vector strategies, simply because the vector yields are too low for commercial production.

ProVector advantages

In collaboration with Prof. Peter Holst at the University of Copenhagen, SIRION Biotech has now successfully engineered a new production cell to address this obstacle.

Actively suppressing transgene expression protects the new ProVector™ cells from side effects during vector amplification. This increases virus yields by several factors, to levels that are comparable to standard, non-toxic gene vector productions. Transgene suppression is exclusively restricted to the ProVector cell line, leaving overall expression unaffected in post-transduction.

SIRION now plans to use the ProVector production technique to build Ad19 vaccination vectors in alliance with commercial partners. The Ad19 stereotype possesses promising properties that help elicit a strong and lasting immune-response against chosen antigens.

Resources

Click on ProVector™: Protecting production cells from problematic transgene expression for other information.
Click on SIRION to contact the company directly.

Transgene (GFP) suppression  during AV production in ProVector™ cells


Transgene (GFP) suppression during AV production in ProVector™ cells.
A) Normal Hek293 cells show strong GFP expression during AV production (left) while ProVector™ cells (right) suppress the transgene expression.
B) Once AV-CMV- GFP particles are produced and applied to a chosen experimental cell line (e.g. HT1080 cells) viral expression is no longer suppressed and becomes fully functional for standard virus vectors.


Supplier Information
Supplier: SIRION Biotech GmbH
Address: Am Klopferspitz 19, 82152 Martinsried, Germany
Tel: +49 89 700 961 99 9
Fax: +49 89 700 961 99 8
Website: http://www.sirion-biotech.com/


SIRION Biotech Adenovirus Services


SIRION Biotech Adenovirus Services


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Products & Services | SIRION Biotech GmbH

Overview

Recombinant adenoviruses have become the viral vectors of choice when transient and quantitative gene regulation is required. Adenoviruses from SIRION Biotech enable genetic engineering free of cell type limitations.

SIRION’s custom adenovirus construction service is based on a patented bacterial artificial chromosome (BAC) technology that enables construction in less than five weeks.

In addition, SIRION offers custom promoters and transduction enhancers for hard to transduce cell types as well as a range of off-the-shelf adenoviruses.

Background

Classical approaches to identifying and validating new targets in drug discovery use functional gene analysis using overexpression and knockdown studies. For many years, the field had to mainly rely on cancer cell lines that are easy to handle and manipulate.

More recently, the need for maximum authenticity has shifted the focus to primary cells and even patient derived cells. Now, however, classical transfection of those cells is also reaching its limits.

Adenoviruses provide an ideal new research channel for identifying target genes, for three main reasons.

  • Adenovirus transduction is transient due to episomal genome expression, thus circumventing the risk of site-specific mutagenesis
  • Adenoviruses are characterized by high cell type accessibility without species limitations
  • Adenoviruses exhibit excellent quantitative transduction efficiency and high titer productions

Product features

SIRION Biotech`s patented adenoviral vector platform allows for transient gene modification by offering a reliable system for highest gene overexpression or knockdown. It enables you to go from vector cloning to a functional tested and purified virus stocks in less than four weeks or ready-to-use frozen and functionally tested cell pools in less than six weeks.

A customized adenovirus vector generation service allows for virtually every desired expression construct. Beside the standard multicistronic expression systems, individual promoters or reporter genes can be chosen.

Adenovirus services

The standard SIRION Biotech adenovirus service comprises:

  • Cloning the cDNA of the Gene of Interest (GOI) up to 7.5 kb/ shRNA in an adenovirus shuttle plasmid
  • DNA-Sequencing verification of cloning success
  • Generation of recombinant adenovirus BACs
  • Production of low passage virus stocks and amplification in 293 cells
  • Purification and titration
  • Gene expression quantification on mRNA level by qRT-PCR

Optional services

In addition, customers can specify:

Transduction of customer cells and freezing 48h after transduction.
Gene expression quantification on protein level by Western Blot and quality control.
Shipment of ready-to-use viruses/ transduced cells along with detailed product documentation.

Additional services

SIRION Biotech also offers off-the-shelf premade and validated adenoviruses, an adenovirus transduction enhancer and products summarized under the AdenoONE™ brand.

All included, SIRION Biotech offers the most comprehensive portfolio for adenovirus applications.

Adenovirus Applications

SIRION Biotech adenovirus products have been successfully used in two main applications:

Transient Gene Overexpression
Transient expression of the GOI in U87MG cells transduced with an Ad-CMV-TargetX vector, typically using Western Blot to show gene expression three to five days after transduction, relative to cells transduced with an empty control vector (Ad-ctrl).

This application of adenoviruses delivers the following benefits:

  • Quantitative gene delivery and uniform expression in most cell types
  • Highest possible gene expression levels
  • Gene expression adjustable over a wide range by virus amount (copy number)
  • Cells tested to be virus-free allowing handling in S1 laboratories
  • From cDNA sequence to functional gene expression cells in under six weeks
  • Functionality tested frozen cells ready to use

Transient Gene Silencing
This uses transient knockdown of CDK5 in HUVEC cells transduced with the Ad-U6-shRNA vector. Again, Western Blot or qRT-PCR is used to show CDK5 expression four to eight days after transduction relative to NT-shRNA expressing control cells.

Use of adenoviruses in this application delivers benefits that include:

  • Quantitative shRNA delivery to and uniform expression in most cell types
  • Highest possible target gene knockdown
  • Degree of Knockdown adjustable over a wide range by virus amount
  • Guaranteed knockdown of at least 80% on mRNA when combined with RNAiONETM
  • Cells tested to be virus-free allowing handling in S1 laboratories
  • Delivery of functionality tested knockdown cells in less than six weeks
  • Functionality tested frozen cells ready to use

In Vivo Applications
Adenoviruses can be used in vivo in cases that include:

  • Stereotactic injection to specific brain regions
  • Intravenous liver injections
  • Ex-vivo transduction and re-implantation in regenerative medicine

Hard to transduce cell types
Challenging cell types such as NIH-3T3 can be transduced with Ad-GFP either with or without the SIRION Biotech transduction enhancer AdenoBOOST. This can enhance adenovirus transduction up to 50-fold by bridging adenoviruses with the cell surface.

Resources

Click on SIRION Biotech Adenovirus Services for other information.
Click on SIRION to contact the company directly.


Supplier Information
Supplier: SIRION Biotech GmbH
Address: Am Klopferspitz 19, 82152 Martinsried, Germany
Tel: +49 89 700 961 99 9
Fax: +49 89 700 961 99 8
Website: http://www.sirion-biotech.com/


Webinar by Dr Carl J Christel for all your Virus Vector needs


Webinar by Dr Carl J Christel for all your Virus Vector needs


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Products & Services | SIRION Biotech GmbH

Sirion Biotech’s Manger, Dr Carl J Christel to give free webinar to share his knowledge on the next generation of genetic cell modification.

He will be talking on the three main topics of;

  • Custom lentivirus for full transgene expression control in transduced cell pools
  • Specialized adenovirus services to efficiently transduce close to any cell
  • AAV as superior tools for in-vivo gene manipulations

Dr Christel has said “With our experience working with Virus vectors, we consider ourselves to be experts within this field; the sophisticated viral vectors we supply are able to be used in gene function analysis – target research – gene therapy and novel vaccines. With more than 400 successful projects and more than 150 repeat customers, we now want to share the information we have found on these projects with the industry.”

Resources

Click on Webinar by Dr Carl J Christel for all your Virus Vector needs for other information.
Click on SIRION Biotech Webinar Registration USA to register.
Click on SIRION Biotech Webinar Registration Europe to register.
Click on SIRION to contact the company directly.


Supplier Information
Supplier: SIRION Biotech GmbH
Address: Am Klopferspitz 19, 82152 Martinsried, Germany
Tel: +49 89 700 961 99 9
Fax: +49 89 700 961 99 8
Website: http://www.sirion-biotech.com/