fibroblast df1 cells  (ATCC)

Journal: Animal Bioscience

Article Title: Induction of germ cell-like cells from deleted in azoospermia-like enhanced green fluorescent protein gene knock-in chicken somatic cells via transgenic expression of pluripotency and germ cell-specific transcription factors

doi: 10.5713/ab.25.0233

Figure Lengend Snippet: Gene expression analysis in DF1 cells after transfer of each of the 10 selected transcription factor (TF) genes and generation of enhanced green fluorescent protein (eGFP) knock-in (KI) chicken DF1 cells into exon 1 of the DAZL gene. (A) Expression profiles of DDX4 and DAZL genes after transfection of each expression vector of the 10 selected transcription factor genes preferentially expressed in chicken germ cells. (B) Schematic diagram of the targeted site in the DAZL gene and KI vector with eGFP and puromycin-resistant gene conjugated by 2A sequences. Promoter-less of eGFP-2A-puro R KI vector was inserted into the start codon of the first exon in the chicken DAZL gene using a CRISPR/Cas9-mediated system. The expression of eGFP and puro R is controlled by the endogenous chicken DAZL promoter specifically in the germ cell lineage. The blue letters are the start codon of the DAZL gene. The red and bold letters are guide RNA (gRNA) and protospacer adjacent motif (PAM) sequences, respectively. (C) Morphology of eGFP-2A-puro R KI DF1 cells. Scale: 100 μm. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Chicken DF1 fibroblast cells (CRL-12203; ATCC) were cultured and regularly subcultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% foetal bovine serum (FBS; Hyclone) and a 1× antibiotic-antimycotic solution.

Techniques: Gene Expression, Knock-In, Expressing, Transfection, Plasmid Preparation, CRISPR

Journal: Animal Bioscience

Article Title: Induction of germ cell-like cells from deleted in azoospermia-like enhanced green fluorescent protein gene knock-in chicken somatic cells via transgenic expression of pluripotency and germ cell-specific transcription factors

doi: 10.5713/ab.25.0233

Figure Lengend Snippet: Induction of enhanced green fluorescent protein (eGFP)-expressing germ cells after transfection of 10 transcription factor (TFs) genes. (A) Experimental scheme for induction of germ cells from eGFP-2A-puro R knock-in (KI) DF1 cells through overexpression of 10 TFs. GFP expression was detected 7 days after transfection of the 10 TFs. (B) Flow cytometry analysis of eGFP expression 7 and 14 days after gene delivery of 10 TFs. (C) Immunostaining of the induced germ cell-like cells with germ cell-specific antibodies of SSEA-1 and chicken vasa. (D) Gene expression analysis of endogenous pluripotent and germ cell-specific genes during the induction period.

Article Snippet: Chicken DF1 fibroblast cells (CRL-12203; ATCC) were cultured and regularly subcultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% foetal bovine serum (FBS; Hyclone) and a 1× antibiotic-antimycotic solution.

Techniques: Expressing, Transfection, Knock-In, Over Expression, Flow Cytometry, Immunostaining, Gene Expression

Journal: Animal Bioscience

Article Title: Induction of germ cell-like cells from deleted in azoospermia-like enhanced green fluorescent protein gene knock-in chicken somatic cells via transgenic expression of pluripotency and germ cell-specific transcription factors

doi: 10.5713/ab.25.0233

Figure Lengend Snippet: Experimental scheme for induction of germ cells from enhanced green fluorescent protein (eGFP)-2A-puro R knock-in (KI) DF1 cells through overexpression of 10 transcription factors. GFP expression was detected 7 days after transfection of the 10 transcription factors. After 21 days of treatment, the induced germ cell-like cells were maintained with primordial germ cell (PGC) culture media.

Article Snippet: Chicken DF1 fibroblast cells (CRL-12203; ATCC) were cultured and regularly subcultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% foetal bovine serum (FBS; Hyclone) and a 1× antibiotic-antimycotic solution.

Techniques: Knock-In, Over Expression, Expressing, Transfection

Journal: Functional & Integrative Genomics

Article Title: Systematic evaluation of CrRNA design parameters for optimized Cas13d-mediated RNA targeting in chicken cells

doi: 10.1007/s10142-025-01776-x

Figure Lengend Snippet: RfxCas13d-mediated knockdown of DsRed in chicken fibroblast DF1 cells. a Schematic of RfxCas13d-mediated targeted recognition and degradation of DsRed mRNA ( b ) Representative fluorescence microscopy images of RfxCas13d targeting of DsRed using five different crRNA in stable RfxCas13d-expressing DF1 cell line. c DsRed fluorescence knockdown determined by flow cytometry. Data points in the graph are averages of the normalised mean fluorescence intensity from technical triplicates. Error bars show SD

Article Snippet: The chicken fibroblast DF1 cells (American Type Culture Collection number: CRL–12203) were maintained in Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FCS), 2mM L-glutamine, 10mM Hepes, 1.5% (w/v) sodium bicarbonate, 100 U/mL penicillin, and 100 μg/mL of streptomycin.

Techniques: Knockdown, Fluorescence, Microscopy, Expressing, Flow Cytometry

Journal: Functional & Integrative Genomics

Article Title: Systematic evaluation of CrRNA design parameters for optimized Cas13d-mediated RNA targeting in chicken cells

doi: 10.1007/s10142-025-01776-x

Figure Lengend Snippet: Comparison of on-target and collateral effects of RfxCas13d and HfCas13d in chicken cells. a DF1-RfxCas13d and DF1-HfCas13d were each co-transfected with vectors encoding DsRed and five different crRNA (targeting DsRed mRNA) or NT crRNA (negative control). DsRed fluorescence knockdown by different crRNAs was determined by flow cytometry. b DF1-RfxCas13d and DF1-HfCas13d were each co-transfected with vectors encoding GFP and individual crRNA (targeting GFP mRNA) or NT crRNAs (negative). The percentage GFP fluorescence knockdown by different crRNAs was determined by flow cytometry. c Assessment of collateral activity of RfxCas13d and HfCas13d in chicken cells. The corresponding cell lines were transfected with crRNA targeting DsRed, along with expression vectors for DsRed (on-target) and GFP (serves as collateral reporter). Representative microscopy images of both DsRed and GFP fluorescence degradation in DF1-RfxCas13d cells (top panel) compared with DF1-HfCas13d cells (bottom panel). d The percentage DsRed (on-target activity) and GFP (collateral activity) fluorescence knockdown measured by flow cytometry. Values shown as the mean ± Sd. Data points in the graph are averages of the normalised mean fluorescence intensity from technical triplicates

Article Snippet: The chicken fibroblast DF1 cells (American Type Culture Collection number: CRL–12203) were maintained in Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FCS), 2mM L-glutamine, 10mM Hepes, 1.5% (w/v) sodium bicarbonate, 100 U/mL penicillin, and 100 μg/mL of streptomycin.

Techniques: Comparison, Transfection, Negative Control, Fluorescence, Knockdown, Flow Cytometry, Activity Assay, Expressing, Microscopy

Journal: Functional & Integrative Genomics

Article Title: Systematic evaluation of CrRNA design parameters for optimized Cas13d-mediated RNA targeting in chicken cells

doi: 10.1007/s10142-025-01776-x

Figure Lengend Snippet: Transcriptome-wide collateral activity of RfxCas13d and HfCas13d in chicken cell lines. a Venn diagram of overlapping differentially expressed genes (DEGs) upregulated or downregulated. b Volcano plot of the RNA-seq analysis comparing RfxCas13d (left) or HFCas13d (right) DF1 transgenic cells transfected with DsRed and GFP, and either DsRed-crRNA or NT-crRNA. Lines denote a > 1.25-fold change (FC). c Gene ontology (GO) and biological process (BP) term analysis for the DEGs in (b)

Article Snippet: The chicken fibroblast DF1 cells (American Type Culture Collection number: CRL–12203) were maintained in Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FCS), 2mM L-glutamine, 10mM Hepes, 1.5% (w/v) sodium bicarbonate, 100 U/mL penicillin, and 100 μg/mL of streptomycin.

Techniques: Activity Assay, RNA Sequencing, Transgenic Assay, Transfection