Journal: bioRxiv
Article Title: Dyrk1a gene dosage controls bipolar cell development and retinal connectivity
doi: 10.64898/2026.03.15.710015
Figure Lengend Snippet: (A) Schematic diagram illustrating the genomic organisation of the Dyrk1a tm1Jdc/J allele ( ENSMUSG00000022897 , Chr16:94570010 – 94695517 bp). The gene comprises 12 coding exons (black boxes) and gives rise to six mRNA isoforms. The longest transcripts ( ENSMUST00000023614 , ENSMUST00000119878, ENSMUST00000122284) encode a protein of approximately 90 kDa. LoxP sites (yellow triangles) flank exons 5 and 6 and facilitate Cre -mediated recombination of the kinase domain in the translated protein. Oligonucleotides used to assess the extent of allele recombination were designed to amplify across reference exon 4 (R4F and R4R, black arrows) and target exon 6 (T6F and T6R, red arrows). (B – E) Representative lineage tracing analysis of Dyrk1a +/f :ROSA26R R/R (B), Lhx2-Cre:ROSA26R R/R (C), Lhx2-Cre:Dyrk1a +/f :ROSA26R R/R (D) and Lhx2-Cre:Dyrk1a f/f :ROSA26R R/R (E) at P0. Robust β-galactosidase activity is detected throughout the retina of all animals that carry the Lhx2-Cre transgene (C – E) confirming Cre -mediated recombination of the ROSA26R R/R reporter allele. (F) Quantitative analysis of Dyrk1a tm1Jdc/J allele recombination efficiency in genomic DNA harvested from the retina of Lhx2-Cre ( n = 4), Lhx2-Cre:Dyrk1a +/f ( n = 4) and Lhx2-Cre:Dyrk1a f/f ( n = 4) mice at P0. A significant genotype-dependent loss of the Dyrk1a LoxP region was observed in heterozygous and homozygous animals, respectively. Quantification is based on the level of product generated by the LoxP target primer pair normalised against the level of product amplified by the reference primer pair for each biological replicate. (G) Representative immunoblots showing the main 90 kDa Dyrk1a band and the 19/17 kDa cleaved Caspase3 bands with β-actin as a normalisation control in soluble protein extracts prepared from the retina and pigment epithelium of Lhx2-Cre , Lhx2-Cre:Dyrk1a +/f and Lhx2-Cre:Dyrk1a f/f mice at P0. (H) Quantitative analysis of Dyrk1a levels in soluble protein extracts prepared from the retina and pigment epithelium of Lhx2-Cre ( n = 12), Lhx2-Cre:Dyrk1a +/f ( n = 12) and Lhx2-Cre:Dyrk1a f/f ( n = 12) mice at P0. Dyrk1a levels were significantly reduced in a genotype-dependent manner in heterozygous and homozygous animals. Quantification is based on the 90 kDa Dyrk1a protein band normalised to β-actin within the same biological replicate. (I) Quantitative analysis of cleaved Caspase3 levels in soluble protein extracts prepared from the retina and pigment epithelium of Lhx2-Cre ( n = 12), Lhx2-Cre:Dyrk1a +/f ( n = 12) and Lhx2-Cre:Dyrk1a f/f ( n = 12) mice at P0. A genotype-dependent increase in cleaved Caspase3 levels was observed in heterozygous mice with significance reached in homozygous animals. Quantification is based on the 19/17 kDa cleaved Caspase3 protein band normalised to β-actin in the same biological replicate. All data represents the mean ± SEM values. Statistical differences were calculated using one-way ANOVA followed by a Tukey’s multiple comparison test. p-values are denoted as follows: ***p ≤ 0.001 and ****p ≤ 0.0001. Scale bar: (B – E) 50 μm. Abbreviations: bp, base pair; Casp3, cleaved Caspase3; Ch16, chromosome 16; GCL, ganglion cell layer; INBL, inner neuroblastic layer; ONBL, outer neuroblastic layer; R, reference primer pair; RPE, retinal pigment epithelium; T, target primer pair.
Article Snippet: The following primary antibodies and dilutions were used: β-actin (1:20000, Sigma Aldrich, #A3854); cleaved Caspase3 (Asp175) (1:1000, Cell Signalling, #9661S); Dyrk1a (1:1000, Abnova, #H00001859).
Techniques: Activity Assay, Generated, Amplification, Western Blot, Control, Comparison
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