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Flp in cho

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The Flp-In-CHO is a stable cell line expression system for recombinant protein production in Chinese Hamster Ovary (CHO) cells. The system allows for the integration of a gene of interest into a specific genomic locus, enabling consistent and high-level expression of the target protein.

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Lab products found in correlation

Flp in cho cells, by Thermo Fisher Scientific (1 mentions) Mycoalert, by Lonza (1 mentions) In fusion hd cloning kit, by Takara Bio (1 mentions) Pcdna3 clover, by Addgene (1 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions) Plus reagent, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Corning (1 mentions) Pen strep, by Corning (1 mentions) Ham s f12, by Corning (1 mentions)

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Flp-In CHO cells (36 citations) Flp-In-CHO cell line (10 citations) Flp-InTM-CHO cells (2 citations)

5 protocols using flp in cho

1

Isogenic cell line generation for GLP-1R

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Wildtype and mutant human GLP-1R were isogenically integrated into FlpIn-Chinese hamster ovary (FlpInCHO) cells (Invitrogen) and selection of receptor-expressing cells was achieved through treatment with 600 μg ml−1 hygromycin-B. Transfected and parental FlpInCHO cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and incubated in a humidified environment at 37 °C in 5% CO2. For all experiments cells passages 8–20 were used.
Wootten D., Reynolds C.A., Smith K.J., Mobarec J.C., Furness S.G., Miller L.J., Christopoulos A, & Sexton P.M. (2016). Key interactions by conserved polar amino acids located at the transmembrane helical boundaries in Class B GPCRs modulate activation, effector specificity and biased signalling in the glucagon-like peptide-1 receptor. Biochemical Pharmacology, 118, 68-87.
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2

Cell Culture Maintenance and Engineering

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BHK (ATCC), A431 (ATCC), MEF (as described in Meiring et al., 2019 (link)), and Flp-In-CHO (Invitrogen) cells were maintained in Dulbecco’s modified eagle medium (DMEM), supplemented with L-glutamine, and 10% fetal bovine serum at 37°C with 5% CO2. CHO cells stably expressing the human adenosine A1 G-protein-coupled receptor (A1AR) with C-terminally labelled APEX2 were generated as described previously (Baltos et al., 2016 (link)). Expression was maintained by addition of 500 μg/mL hygromycin-B to culture medium. All cells were subject to quarterly mycoplasma testing using the MycoAlert (Lonza) mycoplasma detection kit.
Rae J., Ferguson C., Ariotti N., Webb R.I., Cheng H.H., Mead J.L., Riches J.D., Hunter D.J., Martel N., Baltos J., Christopoulos A., Bryce N.S., Cagigas M.L., Fonseka S., Sayre M.E., Hardeman E.C., Gunning P.W., Gambin Y., Hall T.E, & Parton R.G. (2021). A robust method for particulate detection of a genetic tag for 3D electron microscopy. eLife, 10, e64630.
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3

Maintenance of Adherent Cell Lines

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BHK (ATCC), A431 (ATCC), MEF (as described in (Meiring et al., 2019) ) and Flp-In-CHO (Invitrogen) cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), supplemented with L-glutamine, and 10% fetal bovine serum (FBS) at 37 o C with 5% CO2. CHO cells stably expressing the human adenosine A1 G protein-coupled receptor (A1AR) with C-terminally labeled APEX2 were generated as described previously (Baltos et al., 2016) .
Expression was maintained by addition of 500 μg/mL hygromycin-B to culture medium.
Rae J., Ferguson C., Ariotti N., Webb R.I., Cheng H., Mead J.L., Riches J.D., Hunter D.J.B., Martel N., Baltos J., Christopoulos A., Bryce N.S., Cagigas M.L., Fonseka S., Hardeman E.C., Gunning P.W., Gambin Y., Hall T.E., & Parton R.G. (2020). APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy.
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4

Characterization of KCNC3 Mutants

Cited 2 times
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The human KCNC3 cDNA was provided by Dr. James L. Rae (Mayo Foundation). The p.Arg420His was generated as previously described [6 (link)]. The c.1746_54del (p.Pro583_Pro585del) mutant was generated in a pBKCMV vector with the In-Fusion HD Cloning kit (Clontech, Mountain View, USA). The KCNC3 cDNA and both mutants were subcloned into pcDNA3-Clover (Addgene no. 40259, provided by Michael Lin). Flp-In-CHO (Thermo Fisher Scientific, Waltham, USA) cells were cultured and transiently transfected with pBKCMV or Clover tagged KCNC3, KCNC3-c.1259G>A (p.Arg420His), and KCNC3-c.1746_54del (p.Pro583_Pro585del), as previously described [5 (link)]. Immunoblotting, immunofluorescence, and live cell imaging were performed as described previously [5 (link), 6 (link)].
Khare S., Galeano K., Zhang Y., Nick J.A., Nick H.S., Subramony S.H., Sampson J., Kaczmarek L.K, & Waters M.F. (2018). C-terminal proline deletions in KCNC3 cause delayed channel inactivation and an adult-onset progressive SCA13 with spasticity. Cerebellum (London, England), 17(5), 692-697.
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5

Transfection of Mammalian Cell Lines

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Flp-In-CHO (Chinese hamster ovary, Thermo Fisher Scientific) and human glioblastoma U87 cells (ATCC, HTB-14) were cultured in Ham’s F-12 or DMEM (Dulbecco’s modified Eagle’s medium) (Corning), respectively, with 10% fetal bovine serum (FBS), 25mM glucose, 4mM glutamine, and pen/strep (penicillin-streptomycin) at 37°C, 5% CO2. Transient transfections were carried out using Lipofectamine LTX with Plus reagent (Life Technologies). Total DNA was kept constant by adding control pcDNA3.1 plasmid.
Khare S., Nick J.A., Zhang Y., Galeano K., Butler B., Khoshbouei H., Rayaprolu S., Hathorn T., Ranum L.P., Smithson L., Golde T.E., Paucar M., Morse R., Raff M., Simon J., Nordenskjöld M., Wirdefeldt K., Rincon-Limas D.E., Lewis J., Kaczmarek L.K., Fernandez-Funez P., Nick H.S, & Waters M.F. (2017). A KCNC3 mutation causes a neurodevelopmental, non-progressive SCA13 subtype associated with dominant negative effects and aberrant EGFR trafficking. PLoS ONE, 12(5), e0173565.
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