A novel APP splice variant-dependent marker system to precisely demarcate maturity in SH-SY5Y cell-derived neurons.

SH-SY5Y, a neuroblastoma cell line, can be converted into mature neuronal phenotypes, characterized by the expression of mature neuronal and neurotransmitter markers. However, the mature phenotypes described across multiple studies appear inconsistent. As this cell line expresses common neuronal markers after a simple induction, there is a high chance of misinterpreting its maturity. Therefore, sole reliance on common neuronal markers is presumably inadequate. The Alzheimer's disease (AD) central gene, amyloid precursor protein (APP), has shown contrasting transcript variant dynamics in various cell types. We differentiated SH-SY5Y cells into mature neuron-like cells using a concise protocol and observed the upregulation of total APP throughout differentiation. However, APP transcript variant-1 was upregulated only during the early to middle stages of differentiation and declined in later stages. We identified the maturity state where this post-transcriptional shift occurs, terming it "true maturity." At this stage, we observed a predominant expression of mature neuronal and cholinergic markers, along with a distinct APP variant pattern. Our findings emphasize the necessity of using a differentiation state-sensitive marker system to precisely characterize SH-SY5Y differentiation. Moreover, this study offers an APP-guided, alternative neuronal marker system to enhance the accuracy of the conventional markers.

Prime editor-mediated correction of a pathogenic mutation in purebred dogs.

Canine hip dysplasia (HD) is a multifactorial disease caused by interactions between genetic and environmental factors. HD, which mainly occurs in medium- to large-sized dogs, is a disease that causes severe pain and requires surgical intervention. However, the procedure is not straight-forward, and the only way to ameliorate the situation is to exclude individual dogs with HD from breeding programs. Recently, prime editing (PE), a novel genome editing tool based on the CRISPR-Cas9 system, has been developed and validated in plants and mice. In this study, we successfully corrected a mutation related to HD in Labrador retriever dogs for the first time. We collected cells from a dog diagnosed with HD, corrected the mutation using PE, and generated mutation-corrected dogs by somatic cell nuclear transfer. The results indicate that PE technology can potentially be used as a platform to correct genetic defects in dogs.

Generation of genome-edited dogs by somatic cell nuclear transfer.

BACKGROUND: Canine cloning technology based on somatic cell nuclear transfer (SCNT) combined with genome-editing tools such as CRISPR-Cas9 can be used to correct pathogenic mutations in purebred dogs or to generate animal models of disease. RESULTS: We constructed a CRISPR-Cas9 vector targeting canine DJ-1. Genome-edited canine fibroblasts were established using vector transfection and antibiotic selection. We performed canine SCNT using genome-edited fibroblasts and successfully generated two genome-edited dogs. Both genome-edited dogs had insertion-deletion mutations at the target locus, and DJ-1 expression was either downregulated or completely repressed. CONCLUSION: SCNT successfully produced genome-edited dogs by using the CRISPR-Cas9 system for the first time.

Generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes.

Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT-(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues.

SMILE inhibits BMP-2-induced expression of osteocalcin by suppressing the activity of the RUNX2 transcription factor in MC3T3E1 cells.

Small heterodimer partner interacting leucine zipper protein (SMILE) is an orphan nuclear receptor and a member of the bZIP family of proteins. Several recent studies have suggested that SMILE is a novel co-repressor that is involved in nuclear receptor signaling; however, the role of SMILE in osteoblast differentiation has not yet been elucidated. This study demonstrates that SMILE inhibits osteoblast differentiation by regulating the activity of Runt-related transcription factor-2 (RUNX2). Tunicamycin, an inducer of endoplasmic reticulum stress, stimulated SMILE expression. Bone morphogenetic protein-2-induced expression of alkaline phosphatase and osteocalcin, both of which are osteogenic genes, was suppressed by SMILE. The molecular mechanism by which SMILE affects osteocalcin expression was also determined. An immunoprecipitation assay revealed a physical interaction between SMILE and RUNX2 that significantly impaired the RUNX2-dependent activation of the osteocalcin gene. A ChIP assay revealed that SMILE repressed the ability of RUNX2 to bind to the osteocalcin gene promoter. Taken together, these findings demonstrate that SMILE negatively regulates osteocalcin via a direct interaction with RUNX2.

ER stress-inducible ATF3 suppresses BMP2-induced ALP expression and activation in MC3T3-E1 cells.

Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts.