df 1 cells  (Procell Inc)

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: Genes

Article Title: lncRNA-803 Suppresses Apoptosis in DF-1 Cells via the miR-6555-3p/MDM4/p53 Axis

doi: 10.3390/genes17040440

Figure Lengend Snippet: The effect of lncRNA-803 on the apoptosis pathway of DF-1 cells. ( A ) DF-1 cells were infected with lentivirus to overexpress or knockdown lncRNA-803. ( B ) Statistical analysis of the infection efficiency of lentiviruses engineered to overexpress or knockdown lncRNA-803. ( C ) Relative expression levels of lncRNA-803 in DF-1 cells. ( D ) Effect of lncRNA-803 on the middle and late apoptosis rates of DF-1 cells. ( E ) Effect of lncRNA-803 on the late apoptosis rate of DF-1 cells. ( F ) Effect of lncRNA-803 on the mRNA expression levels of genes involved in the apoptosis pathway in DF-1 cells. ( G ) Effect of lncRNA-803 on the expression of apoptosis pathway-related genes in DF-1 cells. * represents a significant difference compared with lncRNA-803 overexpression control group. ** represents an extremely significant difference compared with lncRNA-803 overexpression control group. # represents a significant difference compared with lncRNA-803 knockdown control group. ## represents an extremely significant difference compared with lncRNA-803 knockdown control group. Ns means the difference is not significant. ( n = 3).

Article Snippet: HEK-293T cells and DF-1 cells were purchased from Procell Company (Wuhan, China).

Techniques: Infection, Knockdown, Expressing, Over Expression, Control

Journal: Genes

Article Title: lncRNA-803 Suppresses Apoptosis in DF-1 Cells via the miR-6555-3p/MDM4/p53 Axis

doi: 10.3390/genes17040440

Figure Lengend Snippet: Identification of miRNAs related to the p53 pathway that interact with lncRNA-803. ( A ) Effect of lncRNA-803 on the mRNA level of genes of tomor protein p53 (p53) pathway in DF-1 cells. ( B ) Effect of lncRNA-803 on the protein level of genes of p53 pathway in DF-1 cells. ( C ) Effect of lncRNA-803 on miRNAs expression in DF-1 cells. * represents significant difference compared with lncRNA-803 overexpression control group. ** represents an extremely significant difference compared with lncRNA-803 overexpression control group. # represents significant difference compared with lncRNA-803 knockdown control group. ## represents an extremely significant difference compared with lncRNA-803 knockdown control group. ns represents no significant difference. ( D ) DF-1 cells co-transfected with miR-6555-3p mimics and dual luciferase reporter vector. ( E ) Effect of pmiR-GLO transfection on firefly luciferase and renilla luciferase activities in DF-1 cells. ( F ) The effect of co-transfection with dual luciferase reporter vector and miR-6555-3p mimics on relative luciferase activity in DF-1 cells. ( G ) DF-1 cells co-transfected with miR-6555-3p mimics and dual luciferase reporter vector. * represents a significant difference. ** represents an extremely significant difference. ( n = 3).

Article Snippet: HEK-293T cells and DF-1 cells were purchased from Procell Company (Wuhan, China).

Techniques: Expressing, Over Expression, Control, Knockdown, Transfection, Luciferase, Plasmid Preparation, Cotransfection, Activity Assay

Journal: Genes

Article Title: lncRNA-803 Suppresses Apoptosis in DF-1 Cells via the miR-6555-3p/MDM4/p53 Axis

doi: 10.3390/genes17040440

Figure Lengend Snippet: The effect of miR-6555-3p on the apoptosis pathway of DF-1 cells. ( A ) DF-1 cells transfected with miR-6555-3p mimic or inhibitor. ( B ) Statistical results on the transfection efficiency of miR-6555-3p mimic or inhibitor. ( C ) The relative expression of miR-6555-3p in DF-1 cells with miR-6555-3p overexpression or knockdown. ( D ) Effect of miR-6555-3p on middle and late apoptosis rate of DF-1 cells. ( E ) Effect of miR-6555-3p on late apoptosis rate of DF-1 cells. ( F ) Effect of miR-6555-3p on the mRNA level of genes of apoptosis pathway in DF-1 cells. ( G ) Effect of miR-6555-3p on the protein levels of genes of apoptosis pathway in DF-1 cells. * represents significant difference compared with miR-6555-3p overexpression control group. ** represents an extremely significant difference compared with miR-6555-3p overexpression control group. # represents significant difference compared with miR-6555-3p knockdown control group. ## represents an extremely significant difference compared with miR-6555-3p knockdown control group. ( n = 3).

Article Snippet: HEK-293T cells and DF-1 cells were purchased from Procell Company (Wuhan, China).

Techniques: Transfection, Expressing, Over Expression, Knockdown, Control

Journal: Genes

Article Title: lncRNA-803 Suppresses Apoptosis in DF-1 Cells via the miR-6555-3p/MDM4/p53 Axis

doi: 10.3390/genes17040440

Figure Lengend Snippet: Identification of murine double minute 4 ( MDM4 ) related to the p53 pathway that interact with miR-6555-3p. ( A ) Effect of miR-6555-3p on the mRNA level of genes of p53 pathway in DF-1 cells. ( B ) Effect of miR-6555-3p on the protein level of genes of p53 pathway in DF-1 cells. * represents significant difference compared with lncRNA-803 overexpression control group. ** represents an extremely significant difference compared with lncRNA-803 overexpression control group. # represents significant difference compared with lncRNA-803 knockdown control group. ## represents an extremely significant difference compared with lncRNA-803 knockdown control group. ns represents no significant difference. ( C ) DF-1 cells co-transfected with miR-6555-3p and dual luciferase reporter vector. * represents a significant difference. ** represents an extremely significant difference. ( n = 3).

Article Snippet: HEK-293T cells and DF-1 cells were purchased from Procell Company (Wuhan, China).

Techniques: Over Expression, Control, Knockdown, Transfection, Luciferase, Plasmid Preparation