TARGATT™ HEK293 Master Cell Line For Site-Specific Knock-in
TARGATT™ HEK293 Master Cell Line and Knockin Kit - A valuable research tool to generate stable knock-in cell lines and large, isogenic, mammalian cell libraries very efficiently!
The TARGATT™ Knockin Master Cell Line and Kit uses integrase-based integration of a transgene into a preselected intergenic and transcriptionally active genomic locus (hROSA26, hH11, hAAVS1 or other safe harbor loci) engineered with an integrase recognition “attP” docking site or “landing-pad"). Applied StemCell (ASC) provides landing-pad ready TARGATT™ Master Cell Lines and Kits.
The TARGATT™ HEK293 Master Cell Line and Knockin Kit includes a TARGATT™ cloning plasmid that contains an integrase-recognition “attB” sequence and can be used to generate the donor plasmid containing the gene of interest (transgene). When the donor plasmid is transfected into the master cell line along with the integrase expression plasmid (also provided in the kit), the integrase catalyzes the integration of the transgene at the attP-attB sites. This integration is unidirectional which results in a stable integrated knock-in cell line.
Of note, the landing pad in the TARGATT™ HEK293 master cell line is engineered into the well-defined, transcriptionally active, intergenic H11 locus (safe harbor locus/genomic hotspot). This locus enables high level expression of the integrated gene-of-interest without disruption of internal genes and gene silencing commonly seen with random integration.
Advantages of the TARGATT™ HEK293 Master Cell Line:
- High knock-in efficiency: with enrichment (up to 90%) or without (up to 40%)
- Site-specific integration into the H11 genomic hotspot well-defined safe harbor locus
- Single gene knockin: one variant - one locus - one cell line
- Unidirectional integration for stable knock-in cell lines
- Uniform, high level gene expression
- Fewer cell counts and library sizes needed to be required
- Permits non-viral high-throughput library screens
- Overcomes challenges such as random insertion, gene silencing, multiple copy gene integration, ablated gene expression.
The TARGATT™ HEK293 Master Cell Lines and Knockin Kit combines the scalability, affordability, and ease of use of bacterial/yeast systems and the advantages of using mammalian cells (closer to the human environment and post-translational modifications) for efficient and stable gene knockin into cell lines and for library generation. ASC can even engineer a landing pad into the cell line of your choice.
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Schematic Representation of the Transgene Integration in the TARGATT™ Master Cell Line
Figure 1. Schematic representation of TARGATT™ site-specific transgene integration mediated by integrase. The TARGATT™-HEK Master Cell Line was engineered with the attP landing pad at the hH11 safe harbor locus. The TARGATT™ plasmid containing the integrase recognition site, attB is used to clone the transgene. The integrase catalyzes an irreversible reaction between the attP site in the genome and attB site in the donor vector, resulting in integration of the gene of interest at the selected H11 locus. The cells containing the gene of interest can be enriched using the selection marker (gray box).
Confirmation of Site-Specific CMV-MCS Plasmid Integration
Figure 2. PCR gel electrophoresis to confirm the knockin of TARGATT™ 24 CMV-MCS-attB plasmid mediated by the TARGATT™ Integrase plasmid, after transfection into the TARGATT™ HEK293 Master Cell Line. Two sets of primers were used to confirm knockin: Upstream (512 bp) and Downstream primers (464 bp). The Human control primers (777 bp) was also used as a control to check the integrity of the cells and the genomic DNA (gDNA). Negative control (-) represents cells transfected with the TARGATT™ 24 CMV-MCS-attB plasmid and a mutant TARGATT™ integrase plasmid that is deficient for integration.
mCherry Expression After Transfection and Blasticidin Enrichment
Figure 3. The mCherry integration into the TARGATT™ HEK293 master cell line. Left: Integration mediated by the integrase 72 hours post-transfection. Cells were transfected with the mCherry positive control plasmid and either the provided TARGATT™ integrase plasmid (+Integrase) or a mutant TARGATT™ integrase plasmid deficient for integration (-Integrase). The mCherry plasmid has no promoter and requires the ubiquitous EF1 promoter in the landing pad after integration to express the reporter gene. The integration efficiency of mCherry knockin into landing pad was >40%, without selection. Right: Blasticidin enrichment of TARGATT™ HEK293 cells with a knocked-in mCherry-blasticidin plasmid. Cell pools (with 20x and 40x split ratio) were enriched in selection medium for 3 weeks (without cell sorting). The enrichment of mCherry was about 90% after blasticidin selection. Data represents the mean ± SE of two representative experiments done in triplicates.
Comparing TARGATT™ and Existing Gene Editing Technologies For Generation Stable Knock-in Cell Lines
Please Note: The TARGATT™ integrase-based knockin technology requires the master cell line to have a landing pad (docking site) engineered into the cell at the chosen locus to be able to knockin efficiently. We have master cell lines in HEK293, CHO, and hiPSC backgrounds. We can also custom engineer a landing pad and generate a Master Cell Line in the cell line of your choice. Please inquire for further details.
How is the TARGATT™ HEK293 Master Cell Line Different From Other Technologies For Purpose of Site-Specific Integration of Transgenes and Stable Cell Line Generation?
The TARGATT™ integrase system is designed based on serine integrases which catalyze irreversible site-specific transgene insertion via recombination between two distinct integrase-recognition/ attachment sites, attP and attB. The difference of the TARGATT™ HEK293 Master Cell Line is its high efficiency in gene insertion (up to 45% compared to 5% using other systems) and its specificity with very low off-target integration profiles.
Another advantage of the TARGATT™ HEK293 Master Cell Line is its fast Blasticidin enrichment and mCherry selection (2-3 days vs weeks) that allows you to purify the gene edited cells most effectively.
How do the TARGATT™ HEK293 Master Cell Lines compare to other HEK293 Master Cell Lines and Kits Using Similar Site-Specific Technologies?
- High basal integration efficiency (up to 40%) even without selection or enrichment. With selection, modified cells can be enriched up to or higher than 90%.
- Unidirectional recombination ensures stable knock-in cell lines
- Single-step transfection procedure – no need for re-targeting
- Stringent and low off-target profile for random integration
- Integration locus, H11 is a well defined, intergenic, and transcriptionally active safe-harbor locus
- The H11 locus expresses high level of protein, uniformly and consistently
- Enables the generation of isogenic cell lines efficiently
- With selection/enrichment, stable-line pools can be obtained within 2 weeks.
How does TARGATT™ technology Compare To Nuclease-Based (Cas9) and other systems such as Flp, Cre, etc.?
The TARGATT™ technology is based on serine integrases which catalyze efficient site-specific DNA cleavage, DNA strand exchange and ligation without help from outside proteins. This permits knockin efficiencies superior to those that are possible with nucleases like Cas9, which leave cleaved DNA to be dealt with by the host repair machinery.
Bi-directional recombinases such as Flp and Cre also permit avoidance of the cell DNA repair machinery but are highly inefficient for net-integration due to the extreme kinetic favorability of the excision reaction. I.e., while they can mediate the initial genomic-recombination event with the same efficiency, they then proceed to catalyze the excision reaction with high efficiency (as the substrates are now physically linked). Serine integrase attL and attR complexes do not synapse, so this subsequent excision is blocked, and thus a high net-integration efficiency can be achieved.
You can also review the table in the Application Notes section for more comparisons between the TARGATT™ and other commonly used technologies.
Potential Applications include but are not limited to:
Stem Cell Research
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Discovery, Selection & Screening.
TARGATT™ Master Cell Line
- Chi, X., Zheng, Q., Jiang, R., Chen-Tsai, R. Y., & Kong, L. J. (2019). A system for site-specific integration of transgenes in mammalian cells. PLOS ONE, 14(7), e0219842.
Transgenic Mouse Book Chapters
- Chen-Tsai, R. Y. (2020). Integrase-Mediated Targeted Transgenics Through Pronuclear Microinjection. In Transgenic Mouse (pp. 35-46). Humana, New York, NY.
- Chen-Tsai, R. Y. (2019). Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice. In Microinjection (pp. 71-86). Humana Press, New York, NY.
Description of the technology
- Zhu, F., Gamboa, M., Farruggio, A. P., Hippenmeyer, S., Tasic, B., Schüle, B., … Calos, M. P. (2014). DICE, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic Acids Research, 42(5), e34. http://doi.org/10.1093/nar/gkt1290.
- Tasic, B., Hippenmeyer, S., Wang, C., Gamboa, M., Zong, H., Chen-Tsai, Y., & Luo, L. (2011). Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proceedings of the National Academy of Sciences of the United States of America, 108(19), 7902–7907. http://doi.org/10.1073/pnas.1019507108.
Commentary, comparison with other transgenic methods
- Rossant, J., Nutter, L. M., & Gertsenstein, M. (2011). Engineering the embryo. Proceedings of the National Academy of Sciences, 108(19), 7659-7660.
Tet inducible mice generated by TARGATT™
- Fan, X., Petitt, M., Gamboa, M., Huang, M., Dhal, S., Druzin, M. L., … Nayak, N. R. (2012). Transient, Inducible, Placenta-Specific Gene Expression in Mice. Endocrinology, 153(11), 5637–5644. http://doi.org/10.1210/en.2012-1556.
Advantage of Hipp11 (H11) locus
- Hippenmeyer, S., Youn, Y. H., Moon, H. M., Miyamichi, K., Zong, H., Wynshaw-Boris, A., & Luo, L. (2010). Genetic Mosaic Dissection of Lis1 and Ndel1 in Neuronal Migration. Neuron, 68(4), 695–709. http://doi.org/10.1016/j.neuron.2010.09.027.
Applications for TARGATT™ technology
- Lindtner, S., Catta-Preta, R., Tian, H., Su-Feher, L., Price, J. D., Dickel, D. E., ... & Pennacchio, L. A. (2019). Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons. Cell reports, 28(8), 2048-2063.
- Wang, T. A., Teo, C. F., Åkerblom, M., Chen, C., Tynan-La Fontaine, M., Greiner, V. J., ... & Jan, L. Y. (2019). Thermoregulation via Temperature-Dependent PGD2 Production in Mouse Preoptic Area. Neuron, 103(2), 309-322.
- Clarke, B. A., Majumder, S., Zhu, H., Lee, Y. T., Kono, M., Li, C., ... & Byrnes, C. (2019). The Ormdl genes regulate the sphingolipid synthesis pathway to ensure proper myelination and neurologic function in mice. eLife, 8.
- Carlson, H. L., & Stadler, H. S. (2019). Development and functional characterization of a lncRNA‐HIT conditional loss of function allele. genesis, e23351.
- Chande, S., Ho, B., Fetene, J., & Bergwitz, C. (2019). Transgenic mouse model for conditional expression of influenza hemagglutinin-tagged human SLC20A1/PIT1. PloS one, 14(10), e0223052. doi:10.1371/journal.pone.0223052
- Hu, Q., Ye, Y., Chan, L. C., Li, Y., Liang, K., Lin, A., ... & Pan, Y. (2019). Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression. Nature immunology, 1.
- Matharu, N., Rattanasopha, S., Tamura, S., Maliskova, L., Wang, Y., Bernard, A., ... & Ahituv, N. (2018). CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science, eaau0629.
- Chen-Tsai, R. Y. (2019). Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice. In Microinjection (pp. 71-86). Humana Press, New York, NY
- Barrett, R. D., Laurent, S., Mallarino, R., Pfeifer, S. P., Xu, C. C., Foll, M., ... & Hoekstra, H. E. (2018). The fitness consequences of genetic variation in wild populations of mice. bioRxiv, 383240.
- Ibrahim, L. A., Huang, J. J., Wang, S. Z., Kim, Y. J., Li, I., & Huizhong, W. (2018). Sparse Labeling and Neural Tracing in Brain Circuits by STARS Strategy: Revealing Morphological Development of Type II Spiral Ganglion Neurons. Cerebral Cortex, 1-14.
- Kumar, A., Dhar, S., Campanelli, G., Butt, N. A., Schallheim, J. M., Gomez, C. R., & Levenson, A. S. (2018). MTA 1 drives malignant progression and bone metastasis in prostate cancer. Molecular oncology.
- Jang, Y., Broun, A., Wang, C., Park, Y. K., Zhuang, L., Lee, J. E., ... & Ge, K. (2018). H3. 3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development. Nucleic acids research. https://doi.org/10.1093/nar/gky982
- Tang, Y., Kwon, H., Neel, B. A., Kasher-Meron, M., Pessin, J., Yamada, E., & Pessin, J. E. (2018). The fructose-2, 6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC. Journal of Biological Chemistry, jbc-RA118.
- Chen, M., Geoffroy, C. G., Meves, J. M., Narang, A., Li, Y., Nguyen, M. T., ... & Elzière, L. (2018). Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS. Cell Reports, 22(13), 3587-3597
- Kido, T., Sun, Z., & Lau, Y.-F. C. (2017). Aberrant activation of the human sex-determining gene in early embryonic development results in postnatal growth retardation and lethality in mice. Scientific Reports, 7, 4113. http://doi.org/10.1038/s41598-017-04117-6.
- Nouri, N., & Awatramani, R. (2017). A novel floor plate boundary defined by adjacent En1 and Dbx1 microdomains distinguishes midbrain dopamine and hypothalamic neurons. Development, 144(5), 916-927.
- Li, K., Wang, F., Cao, W. B., Lv, X. X., Hua, F., Cui, B., ... & Yu, J. M. (2017). TRIB3 Promotes APL Progression through Stabilization of the Oncoprotein PML-RARα and Inhibition of p53-Mediated Senescence. Cancer Cell, 31(5), 697-710.
- Jiang, T., Kindt, K., & Wu, D. K. (2017). Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells. eLife, 6, e23661.
- Booze, M. L., Hansen, J. M., & Vitiello, P. F. (2016). A Novel Mouse Model for the Identification of Thioredoxin-1 Protein Interactions. Free Radical Biology & Medicine, 99, 533–543. http://doi.org/10.1016/j.freeradbiomed.2016.09.013.
- Feng, D., Dai, S., Liu, F., Ohtake, Y., Zhou, Z., Wang, H., ... & Hayat, U. (2016). Cre-inducible human CD59 mediates rapid cell ablation after intermedilysin administration. The Journal of clinical investigation, 126(6), 2321-2333.
- Sun, N., Yun, J., Liu, J., Malide, D., Liu, C., Rovira, I. I., … Finkel, T. (2015). Measuring in vivo mitophagy. Molecular Cell, 60(4), 685–696. http://doi.org/10.1016/j.molcel.2015.10.009.
- Devine, W. P., Wythe, J. D., George, M., Koshiba-Takeuchi, K., & Bruneau, B. G. (2014). Early patterning and specification of cardiac progenitors in gastrulating mesoderm. eLife, 3, e03848. http://doi.org/10.7554/eLife.03848.
- Fogg, P. C. M., Colloms, S., Rosser, S., Stark, M., & Smith, M. C. M. (2014). New Applications for Phage Integrases. Journal of Molecular Biology, 426(15), 2703–2716. http://doi.org/10.1016/j.jmb.2014.05.014.
- Chen-Tsai, R. Y., Jiang, R., Zhuang, L., Wu, J., Li, L., & Wu, J. (2014). Genome editing and animal models. Chinese science bulletin, 59(1), 1-6.
- Park, K.-E., Park, C.-H., Powell, A., Martin, J., Donovan, D. M., & Telugu, B. P. (2016). Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins. International Journal of Molecular Sciences, 17(6), 810. http://doi.org/10.3390/ijms17060810.
- Guenther, C. A., Tasic, B., Luo, L., Bedell, M. A., & Kingsley, D. M. (2014). A molecular basis for classic blond hair color in Europeans. Nature Genetics, 46(7), 748–752. http://doi.org/10.1038/ng.2991.
- Villamizar, C. A. (2014). Characterization of the vascular pathology in the acta2 r258c mouse model and cerebrovascular characterization of the acta2 null mouse. UT GSBS Dissertations and These (Open Access). Paper 508 (2014)
Does TARGATT™ 24 CMV-MCS cloning plasmid (AST-3064) express mCherry after recombination?
Yes, the TARGATT™ 24 CMV-MCS cloning plasmid (AST-3064) contains mCherry and will express mCherry after recombination.
Do you share the sequence of the AST-3064 cloning plasmid used in the AST-1305 kit?
We can share the redacted plasmid map and sequence for promoter, MCS, the 3‘UTR, and the antibiotic selection marker of the AST-3064 cloning plasmid with a signed Material Transfer Agreement (MTA). We will not be able to provide the full length sequences of the plasmid for proprietary reasons.
What is the antibiotic resistance marker to amplify the AST-3065 mCherry positive control and AST-3201 integrase Plasmid?
We cannot disclose the growth and purification protocols of the AST-3065 and AST-3201 plasmids for proprietary reasons. The amount of the plasmids provided in the kit is sufficient for 9 transfections (3 triplicate transfections in a 24-well plate, according to the provided protocol). Additional plasmids (AST-3065 and AST-3201; $550/15 µg each plasmid) are available for those who have previously purchased the AST-1305 TARGATT™ HEK293 Master Cell Line and Knockin Kit.
Can I use DH5-alpha competent cells for transforming the donor plasmid instead of NEB® 10-beta cells?
The E. coli DH5-alpha cells are only good for small plasmids. We recommend they should be avoided for most mammalian vectors. The NEB® 10-beta cells have a higher efficiency for transforming large plasmids. If you are trying other competent cells, the efficiency and results may not be the same as what we see with the 10-beta cells.
Can the AST-3064 TARGATT™ cloning plasmid be used for generating mammalian cell libraries with the TARGATT™ HEK293 Master Cell Line?
No. The TARGATT™ 24 (CMV-MCS-attB) plasmid is meant only for single transgene integration to generate stable knockin cell lines. It cannot be used for mammalian library construction as it would lead to expression of many variants from non-integrated plasmids for an extended and undetermined period of time and would require diluting out the plasmids.
For library construction, we have two TARGATT™ library cloning plasmids (with mCherry for FACS enrichment/ blasticidin resistance for drug enrichment) that can be used with our TARGATT™ HEK293 Master Cell Line to efficiently build mammalian cell libraries. Please inquire for details.
How can I design primers to confirm insertion of the gene of interest for the TARGATT™ HEK293 Master Cell Line?
The datasheet (product manual) provides sequence for universal primer sets (upstream primers, downstream primers) to confirm site-specific insertion of the gene of interest (GOI) in the appendix section. The datasheet also includes sequence for a human control primer which we recommend you use to check the integrity of the cells and genomic DNA (gDNA).
Do you have TARGATT™ Master Cell Lines in other cell line backgrounds in addition to the TARGATT™ HEK293 Master Cell Line?
In addition to the TARGATT™ HEK293 Master Cell Line and Knockin Kit, Applied StemCell also provides TARGATT™ master cell lines in the Chinese Hamster Ovary (CHO) cell and induced pluripotent stem cell (iPSC) backgrounds. The different cell line backgrounds enable the use of TARGATT™ site-specific integration technology to efficiently generate stable knockin cell lines for a multitude of purposes such as protein screening, bioproduction, cellular differentiation and directed-differentiation of cell lineages, immuno-compatible cell line generation, and more.
We also offer custom TARGATT™ Master Cell Line Generation Service where we can engineer the TARGATT™ landing pad into any cell background and locus of your choice and design the corresponding plasmids.
Can I use other TARGATT™ Plasmids such as AST-3050 or AST-3051 with the TARGATT™ HEK293 Master Cell Line?
No. the plasmids AST-3050 and AST-3051 cannot be used with the TARGATT™ HEK293 Master Cell Line. If you are interested in a custom plasmid other than the ones provided in the kit, please inquire.
Can I use Hygromycin or G418 in my cell culture system?
No, you cannot use hygromycin or G418 in your cell culture system.
We also would like to use hygromycin to select for TARGATT cells inserted with a hygromycin resistance gene. On the website, it says we cannot use hygromycin on TARGATT HEK. Why is that?
Hygromycin and G418 were used to construct the line. All unrelated drugs should be usable. E.g. puromycin, zeocin, etc.
Will the integration of the plasmid backbone into the TARGATT™ HEK293 Master Cell Line affect expression of the gene of interest (transgene)?
No. The integration of the plasmid backbone should not affect the expression of your gene of interest in the TARGATT™ HEK293 Master Cell Line (internal test with hundreds of transgenes). The TARGATT™ 24 Cloning Plasmid has a proprietary design and the backbone has been engineered with regulatory elements to ensure site-specific integration into a transcriptionally active safe harbor locus and reduce random integration and gene silencing.
Can you generate a TARGATT™ Master Cell Line with the cell line I am interested?
Yes, we can generate a TARGATT™ Master Cell Line in your cell line of interest.
Can you generate a TARGATT™ Master Cell Line with docking site at a different locus?
Yes, we can generate a TARGATT™ Master Cell Line with a docking site at your locus of choice. We have currently master cell lines in HEK293, CHO, and hiPSC backgrounds, with docking site in the proprietary H11 and ASC2 safe harbor loci.
What is the size of the transgene that can be integrated into the TARGATT™ HEK293 Master Cell Line with the specified efficiency?
We have successfully integrated plasmids up to 8 kb with more than 40% efficiency without enrichment/ selection with the current system (considerably higher than similarly available technologies). With selection/ enrichment, the size of the integrated plasmid should not be affected by efficiency. There is strong literature evidence that supports high efficiency and stringent integration of large transgenes (up to 200 kb) using serine integrases in a variety of cell lines, yeast, drosophila, and animal models.
Below are a few references:
- Tasic B, Hippenmeyer S, Wang C, Gamboa M, Zong H, Chen-Tsai Y, Luo L. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7902-7. doi: 10.1073/pnas.1019507108. Epub 2011 Apr 4. PubMed PMID: 21464299
- Bischof, J., Maeda, R. K., Hediger, M., Karch, F., & Basler, K. (2007). An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proceedings of the National Academy of Sciences of the United States of America, 104(9), 3312–3317. doi:10.1073/pnas.0611511104
- Venken, K. J., He, Y., Hoskins, R. A., & Bellen, H. J. (2006). P [acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science, 314(5806), 1747-1751.
- Duportet, X., Wroblewska, L., Guye, P., Li, Y., Eyquem, J., Rieders, J., ... & Weiss, R. (2014). A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic acids research, 42(21), 13440-13451.
AST-1305: I ordered your kit AST-1305. In 5.4 of the manual, after 72 hours transfection, is it OK to plant the 20 fold diluted cells in blasticidin selection medium directly without growth medium first then medium replacement after 24 hours.
Yes, diluting in 10 μg/mL blas medium does work.
AST-1305: Does the landing pad have both attP and EF1 promoter? Or only attP? If EF1 a promoter is present, how big is the promoter?
The landing pad has both attP and the EF1-alpha promoter. The size of the promoter is 1.34 kb.
AST-1305: Does the landing pad only integrate into one of the 2 chromosomes? How did you verify it is a single copy instead of 2 copies?
The landing pad was only placed on one chromosome. We verified this by co-transfecting TARGATT integrase with an equal mix of GFP and mCherry split-cassette (no promoter) donor plasmids. No cells were obtained that express both proteins above background levels. I.e., if there were two or more copies of the landing-pad, then cells that express both should have been observed, but they were not.
AST-1305: How is the plasmid for library cloning different from AST-3064? Does it still have mCherry and Blasticidin resistant gene? Is the GOI still under CMV promoter? Also, does it have an ampicillin marker?
1) We have two library plasmids, one co-expresses mCherry with the GOI variants, the other co-expresses the blasticidin-resistance protein (we can clone a custom plasmid if these are not desirable markers). The major difference is that the library plasmids do not have a promoter, so a single GOI variant per cell is expressed after site-specific integration (via the EF1a promoter in the landing-pad). 2) If AST-3064 is used, then non-integrated GOI variants (100’s to 1,000’s per cell) will continue to be present until enough cell division has occurred for their plasmids, mRNA and protein to be diluted out. We don’t know how to provide guidance on when this timepoint is reached (it depends on the GOI variants, transfection efficiency, etc.), so the library plasmids are strongly recommended for variant screening. 3) The bacterial marker is kanamycin. We provide this and other needed information (e.g. restriction cloning sites) in the datasheet.
AST-1305: Do you provide a marker to detect transfection efficiency?
No. Please reference the product datasheet for information on the separate GFP plasmid that can be used but is not provided in the kit (See "Material Required but not Provided"). You can also use any CAG or CMV-driven GFP plasmid with a size <7kb.
AST-1305: Can this kit be used to build libraries?
No, but it can be used to express a single antibody.
AST-1305: When are mCherry and blas expressed?
mCherry and blas are only expressed in cells where integration has occurred. They are not part of the same transcript, so there would be no physical connection between the markers and the GOI (e.g., antibody chain).
AST-1305: Could you please share the protocols for cloning of monocistronic (single gene) and bicistronic (IgG) constructs?
Please reference the AST-1305 datasheet for the detailed protocol (Transfection procedure included).
Are the TARGATT™ HEK293 cells adherent?
Our TARGATT™ HEK293 cells are adherent so you will need to adapt them to suspension. However, we also have TARGATT™ CHO cells, which are suspension cells.
Do you have data that shows the inserted copy number is actually a single copy?
Supporting data can be found on the product datasheet. For more details, please contact us.
Could you let me know the ratio of the single copy number/ total inserted copy number?
TARGATT™ allows for single-copy insertion.
Which HEK293 cell line is the original Cell Line for this kit (there are some kind of type (e.g. HEK293T, HEK293S)?
Our TARGATT™ HEK293 line was derived from plain HEK293.
If you are looking for HEK293T with our TARGATT™ landing pad, contact us.
Could let me know the efficiency of insertion with genes that have 20 kb and 10 kb lengths respectively?
Larger plasmid molecules have a bigger mass than smaller ones, and the mass that can be transfected is limited, so fewer molecules of large plasmids will be delivered into cells. This results in a lower integration efficiency for large plasmids. We have not measured the efficiencies of plasmids over ~8 kb, but for an idea of how quickly efficiency decreases with size, you can consult the integration efficiency figure in our datasheet (which shows how the integration efficiency of an ~8 kb plasmid compares to one's of smaller sizes).
After the transgene integration, the attR and attL sequence are left completely or sometimes have indels?
The attR and attL sites are left completely. I.e. the TARGATT™ enzyme being used forms a tetramer complex that completes the full recombination reaction (four-strand cleavage, 180-degree rotation, ligation) without any help from the host cell's machinery.
Can you confirm whether attP and attB sites are always divided at the same sequence part?
Yes, the attP and attB sites that we use are always cleaved and ligated at the exact same positions.
If I would like to use a different promotoer, what should I do?
The CMV can be removed by digesting with AscI and an enzyme in the multiple-cloning site (MCS) -- e.g. AscI + BsiWI, AscI + HindIII, etc. If you need additional help figuring out how to clone your promoter of interest, please send us the promoter's sequence. We can reply with advice about how to clone it or propose some other strategy.