gfp cdna cassette  (Promega)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes

doi: 10.1016/j.omtm.2019.09.010

Figure Lengend Snippet: Dose-Dependent AAV5 - miHTT Transduction and miHTT Expression in HD71 Patient iPSC-Derived Neuronal Culture HD patient iPSC-derived neuronal culture was transduced with AAV5-miHTT at a MOI of 10 5 , 10 6 , or 10 7 . (A) Ten days post-transduction, DNA was isolated and AAV5 - miHTT vector DNA gc per μg gDNA was determined by SYBR Green qPCR. The dotted line reflects the lower limit of quantification for vector DNA. (B) Simultaneously, RNA was isolated and mature miHTT expression was determined relative to AAV5-GFP at a MOI of 10 7 , using U6 as a reference gene. The dotted line reflects the miHTT assay background.

Article Snippet: AAV5 vector encoding cDNA of the miHTT cassette and enhanced GFP was produced by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as described previously.

Techniques: Transduction, Expressing, Derivative Assay, Isolation, Plasmid Preparation, SYBR Green Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes

doi: 10.1016/j.omtm.2019.09.010

Figure Lengend Snippet: HTT mRNA and Mutant Htt Protein Lowering in HD71 Patient iPSC-Derived Neuronal Culture (A) HTT mRNA levels relative to the mean expression in control-treated cells (AAV5-GFP at a MOI of 10 7 ) were determined by gene-specific TaqMan qPCR. (B) Ultra-sensitive single molecule counting assay with 2B7 and MAB2166 antibodies to quantify human Htt protein (both wild-type and mutant), relative to AAV5-GFP at a MOI of 10 7 . Data were evaluated using a one-way ANOVA and corrected using a Bonferonni test. **p < 0.01, ***p < 0.001, ****p < 0.0001 versus controls.

Article Snippet: AAV5 vector encoding cDNA of the miHTT cassette and enhanced GFP was produced by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as described previously.

Techniques: Mutagenesis, Derivative Assay, Expressing, Single Molecule Counting

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes

doi: 10.1016/j.omtm.2019.09.010

Figure Lengend Snippet: qPCR Analysis of In Silico -Predicted AAV5-miHTT Off-Target Genes in HD71 and HD180 Patient iPSC-Derived Neuronal Cultures (A) BLAST top hits. (B) siSPOTR predicted off-target top list. HD71 and HD180 patient iPSC-derived neuronal cultures were transduced with AAV5-miHTT at a MOI of 10 7 (n = 3), AAV5-GFP at the same MOI (n = 3), or formulation buffer (n = 3). RNA was isolated and qPCR analysis was performed. Genes are normalized against the geometric mean of both housekeeping genes ( ACTB and HMBS ). Bars represent average ± SD of the percentage normalized expression with respect to formulation buffer-treated neuronal cultures. Data were evaluated using a paired Student’s t test versus formulation buffer control.

Article Snippet: AAV5 vector encoding cDNA of the miHTT cassette and enhanced GFP was produced by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as described previously.

Techniques: In Silico, Derivative Assay, Transduction, Isolation, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes

doi: 10.1016/j.omtm.2019.09.010

Figure Lengend Snippet: Confirmation of In Silico -Predicted AAV5-miHTT Target Genes from RNA-Seq Analysis HD71 iPSC-derived neuronal cultures were transduced with AAV5-miHTT at a MOI of 10 5 or 10 7 , formulation buffer as negative control, and AAV5-GFP at a MOI of 10 7 as viral transduction control (n = 4). RNA was isolated and unbiased RNA sequence analysis was performed. The analysis of the weighted proportions fold change and p value scores between the conditions were calculated using the Baggerley’s beta-binomial test. The housekeeping genes ACTB and HMBS are plotted in the graph to demonstrate equal RNA input between the treatment groups. Data were evaluated using a paired Student’s t test versus formulation buffer control.

Article Snippet: AAV5 vector encoding cDNA of the miHTT cassette and enhanced GFP was produced by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as described previously.

Techniques: In Silico, RNA Sequencing Assay, Derivative Assay, Transduction, Negative Control, Isolation, Sequencing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes

doi: 10.1016/j.omtm.2019.09.010

Figure Lengend Snippet: qPCR Analysis of Possible Off-Target Genes in Two Different HD Patient iPSC-Derived Neuronal Cultures (A) HD71. (B) HD180. HD patient iPSC-derived neuronal cultures were transduced with AAV5-miHTT at a MOI of 10 5 or 10 7 , formulation buffer as negative control, and AAV5-GFP at a MOI of 10 7 as viral transduction control (n = 3). RNA was isolated and qPCR analysis was performed. Genes are normalized against the geometric mean of both housekeeping genes ( ACTB and HMBS ). Bars represent average ± SD of the percentage normalized expression with respect to formulation buffer-treated neuronal cultures. Data were evaluated using a paired Student’s t test versus formulation buffer control.

Article Snippet: AAV5 vector encoding cDNA of the miHTT cassette and enhanced GFP was produced by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as described previously.

Techniques: Derivative Assay, Transduction, Negative Control, Isolation, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes

doi: 10.1016/j.omtm.2019.09.010

Figure Lengend Snippet: qPCR Analysis of In Silico -Predicted Off-Target Genes Analyzed in iPSC-Derived Astrocytes iPSC-derived astrocytes were transduced with AAV5-miHTT at a MOI of 10 7 (n = 3), AAV5-GFP at a MOI of 10 7 (n = 3), and formulation buffer (n = 3). RNA was isolated and qPCR analysis was performed. Data were evaluated using a paired Student’s t test versus formulation buffer control. HKG, housekeeping genes.

Article Snippet: AAV5 vector encoding cDNA of the miHTT cassette and enhanced GFP was produced by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as described previously.

Techniques: In Silico, Derivative Assay, Transduction, Isolation

Journal: Molecular Therapy

Article Title: AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington’s Disease Minipig Model

doi: 10.1016/j.ymthe.2018.06.021

Figure Lengend Snippet: TgHD Minipig AAV5-miHTT Efficacy Study (A) Experimental outline and performed bioanalytical procedures per treatment group. Brain slices of group 1 were used for immunohistochemical analysis. (B) From groups 2–4, corticostriatal and corticothalamic slices were obtained and tissue punches were taken from putamen, caudate nucleus, thalamus, and cortex. One animal died 2 days post-surgery because of complications related to the surgical procedure; thus, only two animals could be subsequently analyzed of the 1 × 10 13 gc AAV5-miHTT group. (C) From left (L) and right (R) hemisphere punches DNA and total RNA were isolated for vector DNA, mature miHTT expression, and HTT mRNA quantification. From right hemisphere punches, lysate was used for mutant huntingtin protein quantification.

Article Snippet: AAV5 vector encoding cDNA of the enhanced GFP transgene and miHTT cassette was packaged into AAV5 by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as previously described., The complete transcription unit was flanked by two non-coding AAV-derived inverted terminal repeats, and expression was driven by a combination of the cytomegalovirus early enhancer element and chicken β-actin promoter.

Techniques: Immunohistochemical staining, Isolation, Plasmid Preparation, Expressing, Mutagenesis

Journal: Molecular Therapy

Article Title: AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington’s Disease Minipig Model

doi: 10.1016/j.ymthe.2018.06.021

Figure Lengend Snippet: AAV5-miHTT Distribution and miHTT Expression 3 Months after Putaminal and Thalamic Injection in tgHD Minipig Brain: Putamen, Caudate Nucleus, Thalamus, and Cortex One punch was taken per hemisphere per brain structure. Punches from left and right are displayed separately. (A) Vector DNA copies (gc/μg DNA) in the tgHD minipig brain. (B) Mature miHTT expression was determined by custom TaqMan RT-qPCR; values are represented as molecules per cell. (C) Correlation analysis graph plotting vector DNA and miHTT expression from matching brain region. Data from dissected regions from AAV5-miHTT-treated animals were evaluated with non-linear regression log-log line with ordinary fit and Pearson correlation. Formulation buffer samples were excluded from the correlation and fit. Dotted lines represent lower limit of quantification.

Article Snippet: AAV5 vector encoding cDNA of the enhanced GFP transgene and miHTT cassette was packaged into AAV5 by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as previously described., The complete transcription unit was flanked by two non-coding AAV-derived inverted terminal repeats, and expression was driven by a combination of the cytomegalovirus early enhancer element and chicken β-actin promoter.

Techniques: Expressing, Injection, Plasmid Preparation, Quantitative RT-PCR

Journal: Molecular Therapy

Article Title: AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington’s Disease Minipig Model

doi: 10.1016/j.ymthe.2018.06.021

Figure Lengend Snippet: AAV5-miHTT Treatment Demonstrates a Dose-Dependent Human Mutant Huntingtin Target Engagement in tgHD Minipig Brains (A) Human HTT-specific RT-qPCR of mutant HTT mRNA expression relative to an average of formulation buffer-treated animals in the tgHD minipig brain: putamen, caudate nucleus, thalamus, and cortex. Primer-probe against GAPDH used as reference gene. (B) SMC quantification of soluble human mutant huntingtin protein levels in brain punches from the right hemisphere. (C) Pearson correlation of mutant HTT mRNA and mutant huntingtin protein levels showing a positive correlation between mutant HTT mRNA with mutant huntingtin protein levels in punches taken from the right hemisphere. Formulation buffer-injected punches were not taken along for correlation. (D) Human mutant HTT mRNA expression in the left hemisphere injected with AAV5-miHTT in the striatum. Values relative to an average of same brain punch from control tgHD animals (n = 3). (E) SMC quantification of soluble human mutant huntingtin protein levels in brain punches from the left hemisphere injected with AAV5-miHTT in the striatum. (F) Pearson correlation of mutant HTT mRNA and mutant huntingtin protein levels showing a positive correlation between mutant HTT mRNA with mutant huntingtin protein levels in punches injected with 3 × 10 13 gc/brain of AAV5-miHTT in the striatum. Data were evaluated using two-way ANOVA with Tukey’s multiple comparison test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: AAV5 vector encoding cDNA of the enhanced GFP transgene and miHTT cassette was packaged into AAV5 by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as previously described., The complete transcription unit was flanked by two non-coding AAV-derived inverted terminal repeats, and expression was driven by a combination of the cytomegalovirus early enhancer element and chicken β-actin promoter.

Techniques: Mutagenesis, Quantitative RT-PCR, Expressing, Injection

Journal: Molecular Therapy

Article Title: AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington’s Disease Minipig Model

doi: 10.1016/j.ymthe.2018.06.021

Figure Lengend Snippet: Single Molecule Counting Quantification of Soluble Human Mutant Huntingtin in CSF of tgHD Minipigs before and after AAV5-miHTT Treatment (A) Correlation plot of age versus human mutant huntingtin levels in measurements at −14, 7, and 0 days pre-treatment. (B) Longitudinal mutant huntingtin levels of individual CSF samples. Data were evaluated using two-way ANOVA with repeated measurements: *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: AAV5 vector encoding cDNA of the enhanced GFP transgene and miHTT cassette was packaged into AAV5 by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as previously described., The complete transcription unit was flanked by two non-coding AAV-derived inverted terminal repeats, and expression was driven by a combination of the cytomegalovirus early enhancer element and chicken β-actin promoter.

Techniques: Single Molecule Counting, Mutagenesis

Journal: Molecular Therapy

Article Title: AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington’s Disease Minipig Model

doi: 10.1016/j.ymthe.2018.06.021

Figure Lengend Snippet: Cytokine Multiplexing Luminex in CSF tgHD Animals before and after AAV5-GFP, Formulation Buffer, and AAV5-miHTT Treatment (A) IFN-α. (B) IL-10. (C) IL-8. Data were evaluated using two-way ANOVA with repeated measurements (*p < 0.05; **p < 0.01; ***p < 0.001) to day 0.

Article Snippet: AAV5 vector encoding cDNA of the enhanced GFP transgene and miHTT cassette was packaged into AAV5 by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as previously described., The complete transcription unit was flanked by two non-coding AAV-derived inverted terminal repeats, and expression was driven by a combination of the cytomegalovirus early enhancer element and chicken β-actin promoter.

Techniques: Multiplexing, Luminex

Journal: Molecular Therapy

Article Title: AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington’s Disease Minipig Model

doi: 10.1016/j.ymthe.2018.06.021

Figure Lengend Snippet: Histological Examination of tgHD Minipig Cortico-Striatal Brain Slices after Injections of AAV5-GFP, Formulation Buffer, and AAV5-miHTT (A) Microglial marker IBA1 immunohistochemical staining in representative sections of all groups. (B) Striatal medium-sized spiny neuronal marker DARPP-32 immunohistochemical staining in representative sections of all groups. Magnification factor between ×1 and ×1.25. *Biopsies taken for biomolecular analyses. Scale bars, 2 mm. (C) Quantification of total IBA-1 relative to whole section area. (D) Quantification of local IBA-1 optical density, relative to the activated area. (E) Quantification of DARPP-32 expression relative to manual selected striatal area. Data were evaluated using a one-way ANOVA with Bonferroni’s multiple comparison test: **p < 0.01; ***p < 0.001.

Article Snippet: AAV5 vector encoding cDNA of the enhanced GFP transgene and miHTT cassette was packaged into AAV5 by a baculovirus-based AAV production system (uniQure, Amsterdam, the Netherlands) as previously described., The complete transcription unit was flanked by two non-coding AAV-derived inverted terminal repeats, and expression was driven by a combination of the cytomegalovirus early enhancer element and chicken β-actin promoter.

Techniques: Marker, Immunohistochemical staining, Staining, Expressing

Journal: American Journal of Translational Research

Article Title: Cathepsin D protects renal tubular cells from damage induced by high glucose independent of its enzymatic activity

doi:

Figure Lengend Snippet: Expression of CatD in renal tissues. Renal tissue sections from 105 diabetes mellitus (DM) patients and five non-DM patients were immunohistochemically stained with anti-cathepsin D antibody. Representative images for DM and non-DM patients are provided.

Article Snippet: Transduction of CatD into HK2 cells using lentiviral vectors Lentiviral plasmids containing CatD cDNA and a GFP expression cassette were produced by GeneChem Corporation (Shanghai, China).

Techniques: Expressing, Staining

Journal: American Journal of Translational Research

Article Title: Cathepsin D protects renal tubular cells from damage induced by high glucose independent of its enzymatic activity

doi:

Figure Lengend Snippet: No effects of CatD upregulation on HK2 proliferation under culture with normal glucose. (A) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h, then cultured for additional 48 h. Transfection efficiency was analyzed by measurement of GFP+ positive cells under a fluorescence microscopy, and representative images are provided. (B) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h, western blot was performed using the indicated antibodies. (C) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Viable cells were counted using trypan blue exclusion experiment daily. (D) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Cell proliferation was analyzed using RTCA in a real-time pattern. (E) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. DNA synthesis was analyzed using the EdU incorporation assay, and representative images are provided. (F) Total and EdU-positive cells in (E) were counted, and the rate of EdU incorporation was calculated. (G) HK2 cells were infected with lentiviral vectors containing empty or CatD construct, and cell cycle progression was analyzed using flowcytometry. N.S., not significant.

Article Snippet: Transduction of CatD into HK2 cells using lentiviral vectors Lentiviral plasmids containing CatD cDNA and a GFP expression cassette were produced by GeneChem Corporation (Shanghai, China).

Techniques: Infection, Construct, Cell Culture, Transfection, Fluorescence, Microscopy, Western Blot, DNA Synthesis

Journal: American Journal of Translational Research

Article Title: Cathepsin D protects renal tubular cells from damage induced by high glucose independent of its enzymatic activity

doi:

Figure Lengend Snippet: Suppression of high-glucose-mediated damage by CatD upregulation in HK2 cells. A: HK2 cells transduced with empty or CatD-containing lentivirus were cultured with 25 mM glucose, and cell proliferation was analyzed using RTCA in a real-time pattern. B: HK2 cells transduced with empty or CatD-containing lentivirus were cultured with 5 mM (NG) or 25 mM (HG) glucose, and DNA synthesis was analyzed using EdU incorporation experiments. C: HK2 cells transduced with empty or CatD-containing lentivirus were treated with various concentrations of AGEs or a comparable amount of BSA for 24 h, and cell viability was measured using MTT assay. D: HK2 cells transduced with Empty or CatD-containing lentivirus were treated with various concentrations of AGEs or a comparable amount of BSA for 24 h, and apoptotic cells were measured using flow cytometry. *, P<0.05.

Article Snippet: Transduction of CatD into HK2 cells using lentiviral vectors Lentiviral plasmids containing CatD cDNA and a GFP expression cassette were produced by GeneChem Corporation (Shanghai, China).

Techniques: Transduction, Cell Culture, DNA Synthesis, MTT Assay, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Cathepsin D protects renal tubular cells from damage induced by high glucose independent of its enzymatic activity

doi:

Figure Lengend Snippet: Protection of HK2 cells by CatD, independent of its catalytic activity. (A, B) CatD-transduced HK2 cells were cultured with 5 mM (A), or 25 mM (B) glucose in the absence or presence of pepstatin A, and cell proliferation was analyzed using RTCA in a real-time manner. (C) CatD-transduced HK2 cells were cultured with 5 mM (NG), or 25 mM (HG) glucose in the absence or presence of pepstatin A, and DNA synthesis was measured using EdU incorporation experiments. (D) HK2 cells were treated with AGEs in the absence or presence of pepstatin A, and cell viability was measured using the MTT assay. (E) HK2 cells were infected with empty lentivirus vector, lentivirus containing wild-type CatD (WT-CatD), or lentivirus containing an inactive mutant of CatD (D295N-CatD), and transduction efficiency was observed using fluorescence microscopy. (F) HK2 cells were infected with empty lentivirus vector, lentivirus containing wild-type CatD (WT-CatD), or lentivirus containing an inactive mutant of CatD (D295N-CatD), and CatD expression was analyzed using western blot analysis. (G) HK2 cells were infected with empty lentivirus vector, lentivirus containing wild-type CatD (WT-CatD), or lentivirus containing an inactive mutant of CatD (D295N-CatD) for 72 h, and treated with AGEs for another 24 h, following which cell viability was measured using the MTT assay. *, P<0.05; N.S., not significant.

Article Snippet: Transduction of CatD into HK2 cells using lentiviral vectors Lentiviral plasmids containing CatD cDNA and a GFP expression cassette were produced by GeneChem Corporation (Shanghai, China).

Techniques: Activity Assay, Cell Culture, DNA Synthesis, MTT Assay, Infection, Plasmid Preparation, Mutagenesis, Transduction, Fluorescence, Microscopy, Expressing, Western Blot

Journal: American Journal of Translational Research

Article Title: Cathepsin D protects renal tubular cells from damage induced by high glucose independent of its enzymatic activity

doi:

Figure Lengend Snippet: Prevention of AGE-mediated LMP and MMP loss by CatD independent of its catalytic activity. A: Empty, WT-CatD, or D295N-CatD vector-transduced HK2 cells were treated with BSA or AGEs for 24 h, LMP was measured by AO staining, and representative images are presented. B: Empty, WT-CatD, or D295N-CatD vector-transduced HK2 cells were treated with BSA or AGEs for 24 h, MMP was measured by TMRE staining, and representative images are presented.

Article Snippet: Transduction of CatD into HK2 cells using lentiviral vectors Lentiviral plasmids containing CatD cDNA and a GFP expression cassette were produced by GeneChem Corporation (Shanghai, China).

Techniques: Activity Assay, Plasmid Preparation, Staining

Journal: PLoS ONE

Article Title: Differences in Transcription Patterns between Induced Pluripotent Stem Cells Produced from the Same Germ Layer Are Erased upon Differentiation

doi: 10.1371/journal.pone.0053033

Figure Lengend Snippet: A , Immunostaining of established miPS cell clones for pluripotency markers. AP, alkaline phosphatase. Red staining from PE-conjugated human monoclonal antibodies, green from GFP-expressing BJ1 feeder, and blue for DAPI. Scale bars = 100 µm. B , DNA methylation status of CpG dinucleotides in the OCT3/4 and NANOG gene promoter regions in parental (P) and miPS cells. C , Expression levels of pluripotency marker and reprogramming genes in parental (P) cells, iPS cells and iPS-derived MSC of fibroblast (f) and myoblast (m) lineages. Data were extracted from the 44K+ Agilent transcriptome array and scaled. Y axis, expression levels normalized against β-actin gene expression. The data are representative of 3–4 independent experiments. Expression levels of pluripotency marker and reprogramming genes in parental cells (lanes 1–5), iPS cells (lanes 6–11) and iPS-derived MSC (lanes 12–17). Data were extracted from the 44K+ Agilent transcriptome array and scaled. Y axis, expression levels normalized against β-actin gene expression. The experiments were carried out in duplicate.

Article Snippet: iPS cell lines from primary human fibroblasts were previously obtained and characterized at the Institute for Stem cell Therapy and Exploration of Monogenic Diseases, (I-Stem, Evry France) . iPS cell lines from primary human myoblasts were generated in this work in collaboration with I-Stem (Evry France) by infection with retroviral cassettes harbouring the cDNA encoding OCT4 , SOX2 , c-MYC and KLF4 , and GFP under transcriptional control of its promoters (Addgene,Cambridge, MA) (Addgene plasmids 17220, 17225, 17226, 17227).

Techniques: Immunostaining, Clone Assay, Staining, Bioprocessing, Expressing, DNA Methylation Assay, Marker, Derivative Assay, Gene Expression