Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of chicken Mda5 activity: regulation of IFN-β in the absence of RIG-I functionality.

doi: 10.4049/jimmunol.1003712

Figure Lengend Snippet: FIGURE 3. IFN treatment upregulates ChMda5 in chicken DF1 and HD11. Chicken HD11 macrophage-like cells (A) or DF1 chicken fibroblast cells (B) were treated with IFN-a or IFN-b for indicated times and then analyzed for ChMda5 expression by qRT-PCR. Experiments were per- formed in triplicate, and data are representative of two independent ex- periments. *p # 0.05 between IFN-treated or untreated control.

Article Snippet: The continuous chicken fibroblast cell line DF1 (CRL12203; American Type Culture Collection) was maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 1.5% sodium bicarbonate, penicillin (100 U/ml), and streptomycin (100 mg/ml).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of chicken Mda5 activity: regulation of IFN-β in the absence of RIG-I functionality.

doi: 10.4049/jimmunol.1003712

Figure Lengend Snippet: FIGURE 4. IFN-b is induced following pIC transfection in DF1 chicken fibroblasts. DF1 cells were transfected with the indicated doses of pIC for 2, 4, 8, or 24 h and then analyzed for IFN-b levels by qRT-PCR. Data represent fold increase relative to cells transfected without pIC. Experi- ments were performed in quadruplicate, and data are representative of two independent experiments. *p # 0.05 between treated and untreated control.

Article Snippet: The continuous chicken fibroblast cell line DF1 (CRL12203; American Type Culture Collection) was maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 1.5% sodium bicarbonate, penicillin (100 U/ml), and streptomycin (100 mg/ml).

Techniques: Transfection, Quantitative RT-PCR, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of chicken Mda5 activity: regulation of IFN-β in the absence of RIG-I functionality.

doi: 10.4049/jimmunol.1003712

Figure Lengend Snippet: FIGURE 5. Silencing of ChMda5 reduces pIC-induced IFN-b expres- sion in chicken DF1 cells. DF1 cells were transfected with 50 nM various siRNA targeted at ChMda5 or an irrelevant siRNA (siIrr) or transfected without siRNA (cells only) for 48 h. The cells were subsequently trans- fected with pIC (500 ng/ml) (black bars) or without pIC (open bars) for 4 h and then analyzed for ChMda5 and IFN-b expression. Data show the relative levels of ChMda5 expression (A) and IFN-b expression (B) in the ChMda5 knockdown cells relative to control cells (siIrr). Experiments were performed in quadruplicate, and data are representative of two in- dependent experiments. *p # 0.05 between siIrr and siMda5 conditions. siMda5.1, siMda5.2, and siMda5.3 are siRNA molecules directed at ChMda5 (see Materials and Methods); siMda5.1,2,3 combined siRNA targeted at ChMda5; siIrr, irrelevant siRNA.

Article Snippet: The continuous chicken fibroblast cell line DF1 (CRL12203; American Type Culture Collection) was maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 1.5% sodium bicarbonate, penicillin (100 U/ml), and streptomycin (100 mg/ml).

Techniques: Transfection, Expressing, Knockdown, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of chicken Mda5 activity: regulation of IFN-β in the absence of RIG-I functionality.

doi: 10.4049/jimmunol.1003712

Figure Lengend Snippet: FIGURE 7. Untreated or SAP-treated RNA differentially upregulates IFN-b expression in human and chicken cells. pIC or vRNAwere untreated or SAP-treated, then 250 ng/ml was transfected into HeLa (A) or DF1 (B) cells for 4 h. Data shown as the fold change of IFN-b (relative to untreated cells) as measured by qRT-PCR. Experiments were performed in duplicate, and data are representative of two independent experiments. *p # 0.05 between treatment and control, unpaired t test.

Article Snippet: The continuous chicken fibroblast cell line DF1 (CRL12203; American Type Culture Collection) was maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 1.5% sodium bicarbonate, penicillin (100 U/ml), and streptomycin (100 mg/ml).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of chicken Mda5 activity: regulation of IFN-β in the absence of RIG-I functionality.

doi: 10.4049/jimmunol.1003712

Figure Lengend Snippet: FIGURE 6. IFN-b is upregulated to similar levels regardless of the length of pIC. A, Data show the fold increase in IFN-b levels in DF1 cells following transfection with 250 ng/ml unfractionated, long (6 kb), medium (3 kb), or small (1 kb) pIC molecules for 4 h. B, ChMda5-knockdown DF1 cells were transfected with long, medium, or small pIC molecules. Data show the IFN-b levels measured at 4 h after stimulation relative to the control knockdown (siIrr) cells transfected with long pIC. Experiments were performed in triplicate, and the data represent two independent experiments (A, *p # 0.05 between treatment and control, unpaired t test; B, *p # 0.05 between siIrr and siMda5.1 knockdown, unpaired t test).

Article Snippet: The continuous chicken fibroblast cell line DF1 (CRL12203; American Type Culture Collection) was maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 1.5% sodium bicarbonate, penicillin (100 U/ml), and streptomycin (100 mg/ml).

Techniques: Transfection, Knockdown, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Characterization of chicken Mda5 activity: regulation of IFN-β in the absence of RIG-I functionality.

doi: 10.4049/jimmunol.1003712

Figure Lengend Snippet: FIGURE 10. Knockdown of ChMda5 expression does not appear to impact on in vitro influenza virus proliferation. ChMda5-knockdown DF1 cells were infected with H3N2 influenza virus. HA titers were measured at 24, 48, and 96 h following infection. Data show HA titers following in- fection (A, moi 1.0; B, moi 0.1) at 48 and 96 h p.i. Experiments were performed in quadruplicate, and data represent three independent experi- ments.

Article Snippet: The continuous chicken fibroblast cell line DF1 (CRL12203; American Type Culture Collection) was maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 1.5% sodium bicarbonate, penicillin (100 U/ml), and streptomycin (100 mg/ml).

Techniques: Knockdown, Expressing, In Vitro, Virus, Infection

Journal: Poultry Science

Article Title: Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene

doi: 10.1016/j.psj.2026.106585

Figure Lengend Snippet: CRISPR/Cas9-mediated targeting of ACTB and GAPDH genes in chicken DF-1 cells. (A, F) Schematic diagrams of the ACTB (A) and GAPDH (F) gene structures, showing CRISPR/Cas9 targeting sites. (B–E) Validation of ACTB targeting vectors. (B, D) T7E1 assays and (C, E) Sanger sequencing of DF-1 cells transfected with CRISPR/Cas9 constructs targeting the 3′ region (B, C) or intron (D, E). (G–J) Validation of GAPDH targeting vectors. (G, I) T7E1 assays and (H, J) Sanger sequencing of DF-1 cells transfected with constructs targeting the 3′ region (G, H) or intron (I, J). gRNA sequences are shown in red or blue, PAM sequences in yellow. Deleted bases are indicated by strikethrough lines, substitutions by italics, and insertions by lowercase letters.

Article Snippet: Chicken DF-1 fibroblast cells (ATCC® CRL-12203, American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Cytiva, Marlborough, MA, USA) and 1 × antibiotic-antimycotic solution (Gibco).

Techniques: CRISPR, Biomarker Discovery, Sequencing, Transfection, Construct

Journal: Poultry Science

Article Title: Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene

doi: 10.1016/j.psj.2026.106585

Figure Lengend Snippet: Validation of Cas9-GFP knock-in at the ACTB and GAPDH loci in DF-1 Cells. (A) Schematic illustration of the 3′ region targeted and tagging CRISPR/Cas9 approaches. (B) Detection of GFP in ACTB and GAPDH targeted chicken DF-1 cells. Non-transfected wild-type (WT) DF-1 cells are shown as a control, appearing without fluorescence under standard and fluorescence microscopy. Cells successfully transfected with the knock-in vector constructs targeting ACTB and GAPDH genes exhibit green fluorescence, indicating expression of the reporter gene. Scale bar, 100 µm. (C) Knock-in-specific junction PCR of targeted sites. (D, F) Sequencing analysis of the 3′ region targeted knock-in in chicken DF-1 cells. The schematic illustrates the gene locus following CRISPR/Cas9-mediated insertion of a donor cassette at the 3′ region targeting site via non-homologous end joining (NHEJ). Sanger sequencing of the junction PCR products confirmed integration of the donor sequence in the adjacent genomic regions with indel mutations. (E, G) This schematic depicts the post-integration structure of each gene following CRISPR/Cas9-NHEJ-mediated targeted gene tagging. The donor plasmid was designed with GFP flanked by genomic homology arms corresponding to sequences adjacent to the targeted intron. Sanger sequencing of the junction PCR products confirmed integration of the donor sequence in the adjacent genomic regions with indel mutation.

Article Snippet: Chicken DF-1 fibroblast cells (ATCC® CRL-12203, American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Cytiva, Marlborough, MA, USA) and 1 × antibiotic-antimycotic solution (Gibco).

Techniques: Biomarker Discovery, Knock-In, CRISPR, Transfection, Control, Fluorescence, Microscopy, Plasmid Preparation, Construct, Expressing, Sequencing, Non-Homologous End Joining, Mutagenesis

Journal: Poultry Science

Article Title: Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene

doi: 10.1016/j.psj.2026.106585

Figure Lengend Snippet: Validation of Cas9 activity in ACTB and GAPDH knock-in (KI) chicken DF-1 cells. (A) Gene structure of the intergenic region between DMRT1 and DMRT3 is depicted, showing exons as boxes and introns as lines, with the gRNA target site indicated. (B) T7E1 assay for KI DF-1 cells ( ACTB 3′ KI, ACTB tagging, GAPDH 3′ KI, and GAPDH tagging) followed by transfection with gRNA expressing vector. (C) Sanger sequencing analysis of KI chicken DF-1 cells ( GAPDH 3′ KI, and GAPDH tagging) transfected with DMRT gRNA are shown. gRNA sequences are shown in red, PAM sequences in yellow. The strikethrough lines indicate regions where base pairs have been deleted.

Article Snippet: Chicken DF-1 fibroblast cells (ATCC® CRL-12203, American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Cytiva, Marlborough, MA, USA) and 1 × antibiotic-antimycotic solution (Gibco).

Techniques: Biomarker Discovery, Activity Assay, Knock-In, Transfection, Expressing, Plasmid Preparation, Sequencing

Journal: Poultry Science

Article Title: Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene

doi: 10.1016/j.psj.2026.106585

Figure Lengend Snippet: Generation and validation of single-cell clones with Cas9-GFP knock-in at the GAPDH locus in chicken DF-1 cells. (A) Bright-field (BF) and GFP fluorescence images obtained after subculture following single-cell seeding. Each panel represents a clonal population derived from a single genome-edited cell. A total of 16 single-cell-derived clones were identified from the 96-well plates, of which 12 maintained consistent growth after subculture. Clone numbers correspond to the original 16 identified clones, and images of the 12 viable clones are shown. Scale bar, 100 µm. (B) PCR analysis of 12 single-cell-derived clones following subculture. Intron-targeted knock-in alleles were confirmed by 5′ junction PCR using junction-specific primers. The presence of residual wild-type (WT) alleles in individual clones was assessed using WT allele–specific primers. GAPDH PCR served as a genomic DNA quality control. (C) Relative Cas9 copy number was estimated by quantitative PCR (qPCR) using genomic DNA from each clone, normalized to the endogenous GAPDH reference locus (two copies in diploid cells). Bars represent the mean ± SD of technical qPCR replicates ( n = 3).

Article Snippet: Chicken DF-1 fibroblast cells (ATCC® CRL-12203, American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Cytiva, Marlborough, MA, USA) and 1 × antibiotic-antimycotic solution (Gibco).

Techniques: Biomarker Discovery, Single Cell, Clone Assay, Knock-In, Fluorescence, Derivative Assay, Control, Real-time Polymerase Chain Reaction

Journal: Poultry Science

Article Title: Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene

doi: 10.1016/j.psj.2026.106585

Figure Lengend Snippet: Characterization of single-cell-derived Cas9-expressing DF-1 clones. (A) Flow cytometry analysis of GFP expression levels in GAPDH tagging clones. (B) Median fluorescence intensity (MFI) of GFP in each clone. Data represents n = 3 biological replicates; bars show mean ± SD. ⁎⁎⁎⁎ P < 0.0001. (C) Western blot analysis of Cas9 and GAPDH protein expression in each clone. α-tubulin was used as a loading control. (D–E) Functional validation of genome editing capability in single-cell-derived Cas9-expressing DF-1 clones. A guide RNA (gRNA) expression vector targeting an internal region between DMRT1 and DMRT3 was transfected into each clone. As a control, wild-type (WT) DF-1 cells were co-transfected with the same gRNA vector and a transient Cas9 expression plasmid. (D) Genome editing activity was assessed by T7 endonuclease I (T7E1) assay. (E) Sanger sequencing of the target site confirmed indel formation at the expected genomic locus. gRNA sequences are shown in red, PAM sequences in yellow. Deleted bases are indicated by strikethrough lines, substitutions by italics, and insertions by lowercase letters.

Article Snippet: Chicken DF-1 fibroblast cells (ATCC® CRL-12203, American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Cytiva, Marlborough, MA, USA) and 1 × antibiotic-antimycotic solution (Gibco).

Techniques: Single Cell, Derivative Assay, Expressing, Clone Assay, Flow Cytometry, Fluorescence, Western Blot, Control, Functional Assay, Biomarker Discovery, Plasmid Preparation, Transfection, Activity Assay, Sequencing

Journal:

Article Title: Endocytosis Is a Critical Step in Entry of Subgroup B Avian Leukosis Viruses

doi: 10.1128/JVI.76.24.12866-12876.2002

Figure Lengend Snippet: Entry assay of an HIV-1-based vector pseudotyped with the envelope protein of ALV-B. (A) Astrocytoma cells (U-251) and HEK 293 cells were stably transfected with TVBS3-expressing vector. Extracts from chicken embryonic fibroblasts (DF-1) and human cell lines transfected with or without TVBS3 were prepared, and 10 μg of protein was analyzed by Western blot analysis to detect TVBS3 expression. (B) HIV-1 vector (NLluc+env−) was pseudotyped with the envelope protein of ALV-B. Parental and HEK 293-TVBS3 cells were infected for 3 h with pseudotyped ALV-B viruses (10 ng of p24). Supernatant was removed and replaced with fresh medium, and luciferase activity was measured 48 h postinfection. (C) Parental and U-251-TVBS3 were infected with pseudotyped ALV-B (10 ng of p24) as described above for panel B. Experiments were performed in triplicates, and standard deviations are indicated for panels B and C.

Article Snippet: 293T cells and DF-1 chicken embryonic fibroblasts were obtained from the American Type Culture Collection.

Techniques: Plasmid Preparation, Stable Transfection, Transfection, Expressing, Western Blot, Infection, Luciferase, Activity Assay

Journal:

Article Title: Endocytosis Is a Critical Step in Entry of Subgroup B Avian Leukosis Viruses

doi: 10.1128/JVI.76.24.12866-12876.2002

Figure Lengend Snippet: Wild-type ALV-B is in clathrin-coated vesicles and endosome-like structures. Wild-type ALV-B viruses were prebound to DF-1 cells at 4°C for 1 h. Infection was initiated by shifting the temperature to 37°C. Thirty minutes after infection was initiated, samples were processed for ultrastructural analysis. Virus particles were observed (arrows) at a magnification of ×12,000 at the surface of DF-1 cells (A and B), in clathrin-coated vesicles (C and D), and in vesicles (E) while fusing to the vesicle membrane (F).

Article Snippet: 293T cells and DF-1 chicken embryonic fibroblasts were obtained from the American Type Culture Collection.

Techniques: Infection