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All Journal HAYATI Journal of Biosciences Jurnal Teknosains Indonesian Journal of Cancer Chemoprevention ANNALES BOGORIENSES JFIOnline Annales Bogorienses
Endah Puji Septisetyani, Endah Puji
Research Center for Biotechnology Indonesian Institute of Sciences Jl. Raya Bogor KM 46, Cibinong, Bogor, Indonesia 16911
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Civil Engineering, Building, Construction & Architecture Earth & Planetary Sciences Health Professions Immunology & microbiology Industrial & Manufacturing Engineering Mechanical Engineering Medicine & Pharmacology

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Recloning and Characterization of C2C12 Myoblast and Its Clonal Derivatives Prasetyaningrum, Pekik Wiji; Septisetyani, Endah Puji; Suyoko, Ahmad; Santoso, Adi
Indonesian Journal of Cancer Chemoprevention Vol 12, No 2 (2021)
Publisher : Indonesian Society for Cancer Chemoprevention

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14499/indonesianjcanchemoprev12iss2pp99-105

Abstract

The C2C12 myoblasts are adult murine muscle stem cells which isolated after injury to induce muscle regeneration. The cells are widely used in pharmaceutical and biological researches to represent skeletal muscle cells. In our laboratory, we utilize the cells for glucose uptake assay after insulin treatment and studying the muscle regeneration. In this study we conducted recloning of C2C12 cells by limiting dilution cloning (LDC) and investigated the biological properties incuding cell proliferation, adhesion and differentiation of the clonal cells in comparison to the parental cells. Cell proliferation rate had been determined by WST assay, cell adhesion had been observed after cell detachment by EDTA and cell differentiation into multinucleated myotube had been investigated after induction and incubation with horse serum. As results, two clonal derivatives of C2C12 myoblast cells had been retrieved by LDC and used for cell assays. Moreover, the results indicated that parental cells showed faster proliferation rate and better differentiation ability than that of clonal cells. In the contrary the parental cells exhibited weaker adhesion rate than clonal cells. To conclude, C2C12 parental cells are better for performing the glucose uptake or muscle regeneration assays since they showed better differentiation capability.Keywords: C2C12 cells, cells differentiation, myoblast, myotube, recloning.
Optimizaton of Cationic Lipid Mediated Transfection of pEGFP-c1 and pJ-EPO Plasmids in Chinese Hamster Ovary (CHO) Cells Attached Culture for Transient and Stable Recombinant Human Erythropoietin (rhEPO) Expression Septisetyani, Endah Puji; Kusumawati, Arizah; Santoso, Adi
Annales Bogorienses Vol. 19 No. 1 (2015): Annales Bogorienses
Publisher : BRIN

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Abstract

Cationic lipid is one of transfection agents which show high efficiency and low cytotoxicity. The transfection efficiencies can be varied depending upon the type or amount of cationic lipids, the cell line or DNA plasmid being used for transfection. The purpose of this study was to find optimal condition for transfection of CHO-K1 and CHO-S cells with pJ-EPO plasmid (containing human erythropoietin/ hEPO gene) compared with pEGFP-c1 plasmid (containing green fluorescence protein/gfp gene) by cationic lipid Lipofectamin 2000TM (lipofectamin) to generate stable transfectant expressing recombinant human erythropoietin (rhEPO). Optimization was carried out regarding the amount of lipofectamin, DNA concentration, and concentration of antibiotic Geneticin (G418) for selection of stable transfectants. By using standard amount of lipofectamin (10 μl/well) in 6-well plate, highest expression level of green fluorescent protein (GFP) was shown after transfection of CHO-K1 cells with 3 μg/well pEGFP-c1 while highest expression level of rhEPO was observed after transfection of CHO-K1 cells with 6, 8, or 10 μg/well pJ-EPO plasmid. The data also indicated that optimal transfection conditions of CHOK1 and CHO-S cells with pJ-EPO were shown with the use of 4 μg/well DNA in combination with 15 μl lipofectamin. Concentration of G418 used during cells selection also affected the expression where strongest rhEPO expression was shown at 750 ng/μl G418 concentration. Similar to GFP expression profile, rhEPO signal was detected very low during selection process sbased on Western blot data at day 9. Stronger rhEPO signal was observed after day 20 when the stable transfectants have been obtained.
Sequential Adaptation in Mammalian CHO-K1 Cells Producing Human Erythropoietin Wisnuwardani, Popi Hadi; Septisetyani, Endah Puji; Santoso, Adi
Annales Bogorienses Vol. 21 No. 1 (2017): Annales Bogorienses
Publisher : BRIN

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Abstract

The production of recombinant proteins for clinical applications using mammalian cell technology has become a prevalent system because of its capacity in assembling functional proteins. One of the main problems with CHO-K1 cells is that this cell has to grow in the presence of serum. However, the presence of serum will complicate the downstream step for protein production. Thus, protein produced in media without serum, theoretically, would be easier to purify. Technically, this type of cell can be produced by growing the CHO-K1 cells in serum-free media by using adaptation method in suspension condition. This research showed that through sequential adaptation using conditioned media, the CHO-K1 cell line that produces the human erythropoietin gene (hEPO) was able to grow in suspension culture using serum-free media. Based on Western blot analysis, it showed that the protein (hEPO) was able to be expressed in suspension culture with molecular mass of about 47 kDa.
Co-Authors Abdul Manaf Ali, Abdul Manaf Adi Santoso Alfi Yasmina Anam, M. Khairul Azzahra, Salsabila Edy Meiyanto Eti Yerizel Fauziah, Dita Harsan, Hayfa Salsabila Heldayani Heldayani, Heldayani Himawan, Alayna Lillahida Indri Kastian, Ria Fajarwati Komang Alit Paramitasari Kumara, Dennaya Kusumawati, Arizah Kusumawati, Arizah Lestari, Dinda Marzia Magdalena Tetelepta Mintarsih, Betty Mintarsih, Betty Mustofa Mustofa Muthi Ikawati Neng Herawati, Neng Novianti, Metta Popi Hadi Wisnuwardhani, Popi Hadi Prasetyaningrum, Pekik Wiji Prawesthi, Endang Rahmani, Mawardi Rahmani, Mawardi Ramadani, Ratna Dwi RATIH ASMANA NINGRUM Ratna Asmah Susidarti Sri Handayani Sukari, Mohd. Aspollah Sukari, Mohd. Aspollah Suyoko, Ahmad Wisnuwardani, Popi Hadi Yana Rubiyana, Yana