Fig. 5: Cytological function verification of LMBR1.
From: A dominant missense variant within LMBR1 related to equine polydactyly
a Construction and PCR validation of the LMBR1 site mutation cells. The gRNA is designed at the PAM sequence close to the target site and guided by Cas9 nucleic acid endonuclease for cleavage to break the DNA double strand. Then homologous recombination repair (HDR) is performed by exogenous oligo carrying the target site mutation as a template, and the target site mutation is recombined into the genomic target site to complete the LMBR1 gene site mutation vector construction. The control vector consisted of a segment of nonsense gRNA (Supplementary Fig. 3a, b). b Cells after 24 h transfection. The upper part is the control group, and the lower part is the mutant group. The dark field cell picture is in the middle and the bright field cell picture is on the right. c The mRNA expression of LMBR1 in the site mutant and control cells (n = 6). d Changes in apoptotic gene expression levels after LMBR1 site mutation (n = 6). e Cell scratch test; f CCK-8 cell proliferation assay (n = 4); g changes in expression levels of pathway key genes after LMBR1 site mutation. Error bars represent the standard deviation (SD) of the mean for each data point, and error bars denote the range of the data. *p-value < 0.05, **p-value < 0.01.
