Journal: Cellular & Molecular Biology Letters

Article Title: The multiple roles of Ggt1 -Cre in the generation of transgenic mice

doi: 10.1186/s11658-025-00844-1

Figure Lengend Snippet: Generation and validation of TEC-specific Fam134b knockout mice. ( A ) Gene-targeting strategy for generating Fam134b floxed mice. The CRISPR/Cas9 system was employed to insert loxP sites flanking exon 4 of the Fam134b gene in the same orientation, enabling conditional deletion of the targeted exon. ( B ) Breeding strategy for generating Fam134b cKO mice. Fam134b floxed mice were crossed with Ggt1 -Cre transgenic mice, which express Cre recombinase specifically in proximal TECs, to produce Fam134b f/f; Ggt1 -Cre offspring. ( C ) Genotyping results of transgenic mice. Agarose gel electrophoresis analysis of PCR products from genomic DNA. The red asterisk indicates the positive band confirming successful generation of Fam134b cKO mice. ( D ) Validation of FAM134B protein depletion in renal cortex. Western blot analysis demonstrated a significant reduction in FAM134B protein levels in renal cortical lysates from cKO mice compared with controls (Fam134b f/f, Con), confirming efficient and specific ablation of FAM134B in TECs

Article Snippet: Total protein extracts (20–40 μg) were resolved by SDS–PAGE under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBST) to minimize nonspecific binding of primary antibodies, including rabbit polyclonal anti-FAM134B (1:1000; Proteintech Group, China, cat. no. 21537–1-AP), rabbit monoclonal anti-Cre recombinase (1:1000; Cell Signaling Technology, USA, cat. no. 15036 T), and mouse monoclonal anti-GAPDH (1:5000; Absin, China, cat. no. abs830030), were incubated overnight at 4 °C, followed by three TBST washes.

Techniques: Biomarker Discovery, Knock-Out, CRISPR, Transgenic Assay, Agarose Gel Electrophoresis, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: The multiple roles of Ggt1 -Cre in the generation of transgenic mice

doi: 10.1186/s11658-025-00844-1

Figure Lengend Snippet: Genomic validation of exon 4 deletion in the Fam134b gene of systemic Fam134b knockout mice. ( A ) Sequencing chromatogram of PCR products from Fam134b −/− mouse tail genomic DNA. PCR amplification of genomic DNA extracted from Fam134b −/− mouse tails using the F1/R2 primer pair, generated a product with a single-peak sequencing chromatogram, indicating high specificity and the absence of nonspecific amplification artifacts. ( B ) Nucleotide sequence analysis of PCR products from Fam134b −/− mice. Sequencing of the amplified PCR product confirmed the deletion of exon 4 sequences between intron 3 and intron 4 of the Fam134b gene, with retention of a loxP site (the recognition sequence for Cre recombinase). ( C ) Graphic summary of sequence alignment. A schematic between the sequenced PCR product ( B ) and the wild-type Fam134b gene sequence demonstrated perfect matches exclusively at the 5′ end of intron 3 and the 3′ end of intron 4, with no alignment observed in the exon 4 region, confirming its deletion. ( D ) Detailed sequence alignment results. Further sequence alignment analysis verified the complete absence of exon 4 nucleotides between intron 3 and intron 4 in Fam134b −/− mice, providing definitive evidence of targeted genomic excision

Article Snippet: Total protein extracts (20–40 μg) were resolved by SDS–PAGE under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBST) to minimize nonspecific binding of primary antibodies, including rabbit polyclonal anti-FAM134B (1:1000; Proteintech Group, China, cat. no. 21537–1-AP), rabbit monoclonal anti-Cre recombinase (1:1000; Cell Signaling Technology, USA, cat. no. 15036 T), and mouse monoclonal anti-GAPDH (1:5000; Absin, China, cat. no. abs830030), were incubated overnight at 4 °C, followed by three TBST washes.

Techniques: Biomarker Discovery, Knock-Out, Sequencing, Amplification, Generated

Journal: Cellular & Molecular Biology Letters

Article Title: The multiple roles of Ggt1 -Cre in the generation of transgenic mice

doi: 10.1186/s11658-025-00844-1

Figure Lengend Snippet: Lineage tracing validates Ggt1 -Cre expression in mouse kidney and testis tissues. ( A ) Schematic representation of the B6/JGpt-H11em1Cin (CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato)/Gpt (B6-G/R) dual-fluorescent reporter system. A transgenic cassette containing ZsGreen, a transcriptional stop sequence (flanked by loxP sites), and tdTomato was inserted into the mouse H11 genomic locus. Under basal conditions, ZsGreen is ubiquitously expressed, producing green fluorescence. Upon Cre-recombinase-mediated loxP recombination, the ZsGreen sequence is excised, relieving transcriptional repression of tdTomato and enabling red fluorescence. ( B ) Breeding strategy for generating experimental cohorts. Heterozygous B6-G/R f/+ mice were inter-crossed to produce homozygous B6-G/R f/f offspring, the male offspring were subsequently mated with female Ggt1 -Cre mice to yield B6-G/R f/+ and B6-G/R f/+ ; Ggt1 -Cre progeny. ( C ) Genotypic validation of experimental cohorts. PCR-based genotyping confirmed the presence of loxP-flanked alleles and the Cre transgene in respective offspring. Representative gel images demonstrate successful identification of B6-G/R f/+ and B6-G/R f/+ ; Ggt1 -Cre genotypes. ( D ) Tissue-specific fluorescence expression patterns. Immunofluorescence analysis of kidney and testis tissues from 8-week-old B6-G/R f/+ and B6-G/R f/+ ; Ggt1 -Cre mice revealed tdTomato expression (red fluorescence) exclusively in Ggt1 -Cre-positive tissues, while ZsGreen (green fluorescence) persisted in Cre-negative controls. Scale bars: 50 μm. BM, basement membrane; SC, Sertoli cells; SS, secondary spermatocytes; SP, spermatozoa; PS, primary spermatocytes; SG, spermatogonia

Article Snippet: Total protein extracts (20–40 μg) were resolved by SDS–PAGE under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBST) to minimize nonspecific binding of primary antibodies, including rabbit polyclonal anti-FAM134B (1:1000; Proteintech Group, China, cat. no. 21537–1-AP), rabbit monoclonal anti-Cre recombinase (1:1000; Cell Signaling Technology, USA, cat. no. 15036 T), and mouse monoclonal anti-GAPDH (1:5000; Absin, China, cat. no. abs830030), were incubated overnight at 4 °C, followed by three TBST washes.

Techniques: Expressing, Transgenic Assay, Sequencing, Fluorescence, Biomarker Discovery, Immunofluorescence, Membrane