Objective To explore the polymorphism of tyrosinase (TYR) and melanocortin 1 receptor (MC1R) genes and their mRNA expression levels in relation to coat-color phenotypes in guinea pigs, providing genetic markers for locating dominant traits in guinea pigs. Methods A total of 57 self-bred ordinary-level guinea pigs were selected and divided into three groups based on coat color: white (n=22), variegated (n=22) and black (n=13). The guinea pigs were euthanized with an overdose of pentobarbital sodium via intraperitoneal injection. DNA was then extracted from the dorsal skin tissue. Polymorphism in the coding sequence (CDS) of the exons of the TYR and MC1R genes in each group was detected by cloning and sequencing. The mRNA expression of the two genes in skin tissues was detected by real-time fluorescent quantitative PCR to investigate the relationship between these genes and guinea pig coat color. Results A single nucleotide polymorphism (SNP) site was found in the CDS region of TYR exon Ⅰ, where the base A was replaced by G. All white guinea pigs had the G/G genotype for TYR, while no deep-colored (variegated and black) guinea pigs exhibited the G/G genotype for TYR. Most deep-colored guinea pigs had the A/A genotype, and a few had A/G genotype. The A/A genotype frequency in black guinea pigs was higher than in variegated guinea pigs. A 2 760 bp sequence deletion was identified in the exon of the MC1R gene, marked as the - gene, with non-deleted samples marked as N gene. Most white guinea pigs had the -/- genotype for MC1R, variegated guinea pigs mainly had the -/N genotype, and black guinea pigs mainly had the N/N genotype, with a few showing the -/N. The TYR gene expression level was higher in white guinea pigs, lower in variegated guinea pigs, and intermediate in black guinea pigs, but there was no significant difference among the three groups (P>0.05). The MC1R gene expression level in white guinea pigs was extremely low, while both variegated and black guinea pigs showed significantly higher levels than white guinea pigs (P<0.01). Black guinea pigs showed significantly higher levels than variegated guinea pigs (P<0.05). ConclusionThe TYR and MC1R genes synergistically regulate coat color of guinea pigs. The G-site mutation in the TYR gene may lead to albinism, and the change of N-site in the MC1R gene affects the depth of the coat color.

BACKGROUND:The traditional fixation method for femoral neck fractures is three hollow screws inverted triangle fixation,and the optimal fixation method for femoral neck fractures that have not achieved anatomical reduction is inconclusive. OBJECTIVE:To compare the biomechanical properties of cannulated screws internal fixation for sub-capitated femoral neck fracture with different reduction qualities based on finite element analysis. METHODS:The three-dimensional digital model was reconstructed using CT data of the proximal femur from a healthy male volunteer.The femur was modeled to sub-capitated femoral neck fractures.Fracture models were divided into anatomical reduction group,coxa vara group,and coxa valgus group.All fracture model groups were transferred using the standard group,screw depression group,and screw elevation group.A vertical downward stress of 1 400 N was applied to the femoral head at the top of the acetabulum.The displacement and stress distribution of the femur and internal fixator under different fixation methods were observed,and the maximum stress and displacement of the femur and fixator were compared. RESULTS AND CONCLUSION:(1)For anatomical reduction femoral neck fracture,the peak stress of fixation in the standard group,screw depression group and screw elevation group was 41.35,31.27 and 43.32 MPa,respectively.The maximum peak stress of the femur was found on the screw elevation group(28.58 MPa),and the standard group had the maximum peak displacement.(2)During hip varus,the stresses in the three subgroups were relatively dispersed and even.The peak stress of the femur in the standard group was the smallest,but the peak displacement was the largest.The stability of fixation might be poor.The peak displacement of the femur in the screw depression group was the smallest.(3)In the hip valgus,obvious screw stress concentration appeared in the screw depression group,and the peak displacement was the largest among the three subgroups,and an in-out-in phenomenon appeared.The peak stress of the screws in the screw elevation group was the largest among the three subgroups,but the peak displacement was the smallest.(4)It is concluded that for sub-capitated femoral neck fractures that are completely anatomically reduced,it is recommended to use standard inverted triangular nails for fixation.When the hip varus and hip valgus occur within the allowable range of the reduction standard,it is recommended to use the inverted triangle screw to fix it by rotating the corresponding angle in the same direction as the hip varus or valgus.

During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.
Humans ; Animals ; Gene Editing ; CRISPR-Cas Systems/genetics* ; HEK293 Cells ; Homologous Recombination ; DNA