Overview: Transient Mammalian Vectors

Different combinations of expression vector elements yield different protein expression levels. Vector behavior also depends on the expression cell line used. We have tested hundreds of combinations for their performance in HEK and CHO cells. You can select the best of these vectors from the interactive graph below. For other cell lines, we recommend a vector panel to determine the best performing vector for your system. We are always building and testing new vectors, and we’re happy to help you pick the vector that will work best for your application.

Transient Vector Performance Varies with Cell Type

DasherGFP Expression in Transiently Transfected HEK293 and CHO cells

DasherGFP expression in transiently transfected cells. HEK293 (adherent), Expi293® (suspension), CHO-K1 (adherent), Freestyle® CHO-S (suspension), and ExpiCHO® (suspension) cells were transfected at 2 x 10⁶ cells/ml using Lipofectamine 2000; transfections were carried out in triplicate and incubated for 72 hours post-transfection. Cells were lysed using M-PER and expression measured on a fluorimeter. Orange and grey data points are shown for comparison.

Available Vector Elements: Use the "Vector Selector" to find your choice

Overview - Stable Mammalian Vectors

The organization and orientation of functional elements in our stable expression vectors promote efficient stable cell-line generation and high protein expression levels. Vector elements include insulators, a variety of mammalian promoters (including EF1α, CMV, CAG, GAPDH and synthetic hybrid enhancer/promoter/intron combinations), post-transcriptional regulatory elements (HPRE, WPRE) and polyadenylation signals. Available markers include antibiotics neomycin/G418, puromycin, blasticidin or hygromycin, and auxotrophic markers glutamine synthetase or dihydrofolate reductase.

The stable vectors offered through our catalog are available as linearized DNA compatible with our Electra cloning system.

Expression of 2, 3 or 4 ORFs from a Single Vector

Two or more proteins can be expressed simultaneously from a single Leap-In transposon vector using two or more promoters. We have a set of compatible promoter elements that produce controlled protein expression ratios. These can be used to ensure optimal antibody heavy and light chain expression levels, or to express all the open reading frames for multi-chain bispecifics.

Available Vector Elements: Use the "Vector Selector" to find your choice

Overview - Lentiviral Vectors

ATUM’s 3rd generation lentiviral expression vectors (pD2100s) deliver efficient integration and high expression with EF1 or CMV promoters and post-transcriptional regulatory elements. Lentiviral expression vectors integrate a defined region of plasmid vector into a target cell line. They infect both dividing and non-dividing cells, producing homogenous levels of transgene expression across a population of cells. ATUM’s lentiviral vectors are available for gene synthesis orders, and as ready-to-use catalog vectors. Versions containing the gene for DasherGFP are also available as a positive control.

Lentiviral Vector Information

ATUM Lenti vectors are designed as 3rd generation vectors:

• No U3 sequence in the 5'UTR. In the 2100s we use a chimeric CMV enhancer / RSV LTR to drive expression in place of the U3 region.
• The U3 region is missing, which will make the virus self-inactivating. We use only the short 5'LTR at the 3' end.

References:

• Lentiviral Guide
• Miyoshi et. al., J. Virol, Oct 1998, p8150-5157

Lentiviral Packaging

ATUM does not supply packaging plasmids or cell lines, however ATUM Lenti vectors are compatible with both 2nd and 3rd generation lentiviral packaging systems. Minimally, plasmids encoding HIV-1 Gag, Pol, Rev and envelope (VSV-G is recommended) are necessary for co-transfection with Lenti vector for proper formation of infectious viral particles. HEK293 or a derivative (293T, etc.) cell line are recommended to allow for high efficiency simultaneous transfection of 3-4 plasmids.

See datasheet for an example of a protocol that has been effective for 2nd generation packaging systems.

Available Vector Elements: Use the "Vector Selector" to find your choice

Mammalian Vectors: Notes

Mammalian Vector Gene Synthesis Cost: Cloning of synthetic genes (encoding single open reading frames) into these vectors is available free of charge. Regular gene synthesis fees apply.

Positive control vectors carrying Dasher GFP are available from ATUM’s catalog at half price if ordered with gene synthesis orders.

Important Info: Mammalian Expression Vectors do NOT contain the multiple cloning sites.

Restriction Sites: The pJ and pD vectors do not contain restriction sites for excision of your gene. Any restriction site desired can be added to your synthetic gene insert, and must be included in your gene design if you wish to remove your gene with restriction enzymes.

Mammalian Expression Vectors are Research Use Only Products:

License for ATUM Research Use Only Vectors. Except as set forth and subject to the terms herein, if Customer purchases (1) any ATUM mammalian expression vector containing pD2100, pD2500, pH2500, pD2600, 3400, pD3500, 3600 or 3700 series vectors, a vector containing the FH8 solubility tag, or a transposon vector (the “ATUM Research Use Only Vectors”) (including Electra vectors, vector configurations for expression of multiple genes and other customized configurations of the ATUM Research Use Only Vectors, and Customer genes cloned into the ATUM Research Use Only Vectors) or (2) any ATUM Service that involves/utilizes ATUM Research Use Only Vectors, Customer is granted a limited, non-sublicensable, non-transferable, non-exclusive right to use the purchased amount of the ATUM Research Use Only Vectors to produce proteins for research and development purposes only. Customer shall not and shall not cause or permit any third party to (1) modify ATUM Research Use Only Vectors or any product containing ATUM Research Use Only Vectors in any way, including without limitation by replacing any protein-encoding sequence with any other protein-encoding sequence within the ATUM Research Use Only Vector and/or replacing the gene cloned into the ATUM Research Use Only Vectors; (2) directly or indirectly reverse-engineer the ATUM Research Use Only Vectors or any product containing the ATUM Research Use Only Vectors; (3) create any derivative(s) of the ATUM Research Use Only Vectors; (4) copy the ATUM Research Use Only Vectors (5) use the ATUM Research Use Only Vectors with any transposase unless Customer has obtained from ATUM a separate license for the use of Leap-In Transposase; or (6) use the ATUM Research Only Vectors for commercial purposes. Use of the ATUM Research Only Vectors for commercial purposes, including any use in manufacturing a product or service that is provided to a third party for consideration, requires a commercial license from ATUM.

pD600s. ATUM′s pD600 series of mammalian transient expression vectors are still available. These vectors use the strong CMV/IE enhancer/promoter and an SV40 replication origin to give high levels of protein expression in mammalian cells. Although most pD600s have mammalian selectable markers, we recommend the Bifunctional Leap-In® vectors for generating stable cell lines.

Zeocin Concentration: Reduced zeocin concentration is indicated for selection with pJexpress 602 and 612 vectors in mammalian cells. 10-30 µg/mL zeocin has been shown successful, with slow but consistent resistant outgrowth noted.

References

View Publications using ATUM Mammalian Expression Vectors

Search the ATUM Literature Database, containing over 2,660 scientific publications using ATUM technology for references relevant to your research.