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: Journal of Advanced Research

Article Title: The GOLM1-ACLY pathway regulates macrophage-secreted EFEMP1 via H3K27ac modifications to drive tumor progression

doi: 10.1016/j.jare.2025.07.003

Figure Lengend Snippet: GOLM1 expression is upregulated in macrophages and correlates with tumor progression (A) Representative H&E and immunofluorescence microscopy images of carcinoma and para -carcinoma tissue of lung cancer patients. Red, CD68; green, GOLM1; blue, DAPI. Upper row, scale bar = 500 μm; lower row, scale bar = 50 μm. (B) Analyzing the co-localization ratio of CD68 and GOLM1 using Pearson’s correlation coefficients via ImageJ with the JAcop plugin. 10 visual fields from 5 pairs of carcinoma and para -carcinoma tissues were examined. (C) Representative immunofluorescent staining of F4/80 and GOLM1 in liver sections from AAV- Golm1 or AAV-vector infected mice. Scale bar = 100 μm. (D) GOLM1 protein levels in BMDMs derived from AAV- Golm1 or AAV-vector infected mice. (E) Schematic diagram of orthotopic HCC tumor model established by injecting Hepa1-6 cells into the liver of AAV- Golm1 or AAV-vector infected mice. (F) Body weight growth curves of mice inoculated with tumor. n = 6 mice per group. (G) Photographs of liver organs with HCC nodules from AAV- Golm1 and AAV-vector mice at terminal growth size. Scale bar = 40 mm. (H) Analysis of the ratio of tumor area to tissue area. n = 6 mice/group. (I) Representative H&E and Ki67 staining images of liver tumor in AAV- Golm1 or AAV-vector infected mice. Red, Ki67; blue, DAPI. Scale bar = 60 μm. (J) Quantitative analysis of the Ki67 positive area. n = 5 mice/group. (K and L) Immunoblotting and statistical graph of the indicated protein levels in tumors of AAV- Golm1 mice and AAV-vector mice. n = 3 mice/group. The data are presented as the means ± SEMs. Statistical significance was determined by two-tailed Student's t test in (B, H, J, and L) and two-way ANOVA followed by Bonferroni's multiple comparisons test (F). ns, not statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human HCC cell lines Huh7 and HepG2; the human macrophage lines Thp1and HEK293T and the mouse liver cancer cell line Hepa1-6 were originally obtained from the ATCC.

Techniques: Expressing, Immunofluorescence, Microscopy, Staining, Plasmid Preparation, Infection, Derivative Assay, Western Blot, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: The GOLM1-ACLY pathway regulates macrophage-secreted EFEMP1 via H3K27ac modifications to drive tumor progression

doi: 10.1016/j.jare.2025.07.003

Figure Lengend Snippet: Macrophage-specific knockout of Golm1 impedes tumor proliferation (A) Schematic representation of orthotopic HCC tumor model established by injecting Hepa1-6 cells into the liver of Golm1 WT or Golm1 MKO mice. (B) Hepa1-6 tumors in Golm1 WT and Golm1 MKO mice at terminal growth size. Scale bar = 40 mm. (C) Analysis of the ratio of tumor area to tissue area. n = 6 mice/group. (D) H&E and Ki67 immunohistochemical staining of liver tumors in Golm1 WT and Golm1 MKO mice. Main image, scale bar = 200 μm; zoomed regions, scale bar = 40 μm. (E) Ki67 positive rate in tumors evaluated by immunohistochemistry analysis. n = 6 fields. (F-G) Representative bands (F) and quantitative analysis (G) of relative protein expression in tumors of Golm1 WT and Golm1 MKO mice. n = 3 mice/group. (H) GSEA enrichment analysis of cancer related pathways based on KEGG. n = 3 mice/group. (I) Schematic representation of the Thp1 nc -Huh7 or Thp1 −/− -Huh7 admixture tumor model on nude mice. (J) Tumor growth curves of subcutaneous models involving Huh7 cells cocultured with Thp1 nc or Thp1 −/− cells. n = 5 nude mice in each group. (K and L) Tumor images (K) and weight (L) of subcutaneous tumor in groups of nude mice. Scale bar = 20 mm. n = 5 nude mice in each group. (M and N) Representative bands (M) and quantitative analysis (N) of relative protein expression in tumors of Huh-7, Thp1 nc -Huh7 or Thp1 −/− -Huh7 nude mice. n = 3 mice/group. The data are presented as the means ± SEMs. Statistical significance was determined by two-tailed Student's t test (C, E, and G) and one-way ANOVA followed by Tukey's multiple comparisons test (J, L, and N). ns, not statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The human HCC cell lines Huh7 and HepG2; the human macrophage lines Thp1and HEK293T and the mouse liver cancer cell line Hepa1-6 were originally obtained from the ATCC.

Techniques: Knock-Out, Immunohistochemical staining, Staining, Immunohistochemistry, Expressing, Two Tailed Test