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The role of the CEBPB gene in porcine adipogenesis: a study using CRISPR/Cas9-edited mesenchymal stem cells
Abstract Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a powerful tool for gene editing and the regulation of gene expression. It enables the introduction of targeted mutations, thereby facilitating functional studies of specific genes in various cellular processes. In this study, we aimed to generate a deletion in the promoter region of the CEBPB gene, which encodes a transcription factor involved in adipogenesis, and to evaluate the impact of this modification on the adipogenic differentiation potential of porcine mesenchymal stem cells (MSCs). A 575-bp deletion was introduced in the target region, resulting in the generation of both homozygous and heterozygous mutant cells. Adipogenic differentiation was assessed by quantifying transcript levels of adipocyte marker genes ( GATA2 , CEBPA , PPARG , and FABP4 ) at days 0, 4, 6, 8, and 10 of the differentiation process. Disruption of CEBPB expression led to the downregulation of these adipogenic markers, indicating impaired adipocyte differentiation. Additionally, to assess the proliferative capacity of the edited cells, the expression levels of proliferation-associated genes ( CCND1 , MCM2 , and PCNA ) were measured. A reduction in their transcript levels was observed in both homozygous and heterozygous mutant cells. These findings indicate that both homozygous and heterozygous deletions in the CEBPB promoter completely block adipogenesis and alter MSC proliferation, highlighting the pivotal role of CEBPB not only in adipogenic differentiation but also in the regulation of cell proliferation in porcine mesenchymal stem cells. These results provide new insights into the molecular mechanisms underlying adipose tissue development and have implications for pig breeding strategies aimed at optimizing carcass composition, as well as for biomedical research focused on adipose tissue biology.
Droplet digital PCR quantification of selected microRNAs in raw mastitic cow’s milk from the west of Poland
Abstract Introduction MicroRNAs (miRNAs), a class of noncoding small RNAs, have been recognised as potential biomarkers of mammary gland conditions, including bovine mastitis diagnosis. The aim of this study was to quantify selected miRNAs in the milk of mastitic cows. Material and Methods Milk samples (n = 90) were collected from healthy and mastitic dairy cows originating from local dairy cattle farms located in the west of Poland. MicroRNAs of the miR-21a, miR-92a, miR-146a and miR-383 species were quantified using the highly sensitive droplet digital PCR method. Direct measurement of somatic cell count (SCC) was performed using a cell counter. Cows were divided into three groups: those with an SCC below 200,000/mL were designated Low (n = 25), those with an SCC between 200,000 and 999,999 were Medium (n = 34), and those with an SCC of 1,000,000 or higher were High (n = 31). Microbiological analyses were performed using standard culture testing. Results The level of miR-383 was very low and this miRNA was excluded from analysis. The miR-92a was used to normalise miR-21a and miR-146a expression levels. The obtained results of expression of miR-21a and miR-146a correlated with somatic cell number (R = 0.53 and 0.79, respectively). Conclusion These results show that ddPCR is a useful method for quantifying miRNAs in raw cow milk. It seems that miR-146a is a promising marker for bovine mastitis, although further studies are needed to select a panel of miRNAs that can be used in mastitis monitoring in Poland.
Droplet Digital PCR Quantification of Selected Intracellular and Extracellular microRNAs Reveals Changes in Their Expression Pattern during Porcine In Vitro Adipogenesis
Extracellular miRNAs have attracted considerable interest because of their role in intercellular communication, as well as because of their potential use as diagnostic and prognostic biomarkers for many diseases. It has been shown that miRNAs secreted by adipose tissue can contribute to the pathophysiology of obesity. Detailed knowledge of the expression of intracellular and extracellular microRNAs in adipocytes is thus urgently required. The system of in vitro differentiation of mesenchymal stem cells (MSCs) into adipocytes offers a good model for such an analysis. The aim of this study was to quantify eight intracellular and extracellular miRNAs (miR-21a, miR-26b, miR-30a, miR-92a, miR-146a, miR-148a, miR-199, and miR-383a) during porcine in vitro adipogenesis using droplet digital PCR (ddPCR), a highly sensitive method. It was found that only some miRNAs associated with the inflammatory process (miR-21a, miR-92a) were highly expressed in differentiated adipocytes and were also secreted by cells. All miRNAs associated with adipocyte differentiation were highly abundant in both the studied cells and in the cell culture medium. Those miRNAs showed a characteristic expression profile with upregulation during differentiation.
XX/XY Chimerism in Internal Genitalia of a Virilized Heifer
Five DSD heifers underwent genetic analysis in the present study. We cytogenetically analyzed in vitro cultured leukocytes and searched for SRY, AMELX/AMELY and ZFX/ZFY genes in leukocytes and hair follicles, finding that four of the studied heifers were freemartins (XX/XY leukocyte chimerism). The fifth case had an underdeveloped vulva localized ventrally and cranially to the mammary gland, a normal female sex chromosome complement (60,XX) in the leukocytes, and a lack of Y-chromosome-derived genes in the leukocytes and hair follicles. Postmortem anatomical examination of this heifer revealed the presence of normal ovaries with follicles, uterus, and oviducts, but molecular detection of the SRY, ZFX, ZFY,AMELX, and AMELY genes in these organs indicated the presence of a cell line carrying the Y chromosome. Further analysis of twelve microsatellite markers revealed the presence of additional variants at six loci in DNA samples derived from the reproductive organs; XX/XY chimerism was thus suspected in these samples. On the basis of the detection of AMELY (Y-linked) versus AMELX (X-linked) and SOX9 (autosomal) versus AMELY genes by droplet digital PCR (ddPCR), the Y/X and Y/autosome ratios were evaluated; they indicated the presence of XX and XY cell lines in the reproductive tissues. Our study showed that XX/XY chimerism can be present in the internal reproductive organs of the virilized heifers with a normal female set of sex chromosomes (60,XX) and a lack of Y-chromosome-derived genes in the leukocytes. The etiology of this phenomenon remains unknown.
Identyfikacja molekularnych zjawisk przyczynowych związanych z wczesnym rozwojem raka jelita grubego u genetycznie zmodyfikowanych świń w locus APC.
Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing
Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5′-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.
Genetic and epigenetic markers in the METTL21C gene associated with umbilical hernia in pigs
Abstract Background Hernias, particularly umbilical hernias (UH), are prevalent anatomical anomalies in swine, leading to significant welfare issues and economic losses. Besides environmental factors also genetic components contribute to the development of UHs, though the exact mechanisms remain unclear. This study employed a multiple approaches integrating RNA-seq, DNA methylation analysis, Sanger sequencing, droplet digital PCR and western blot analysis to investigate the genetic and epigenetic underpinnings of UH in pigs. Muscle tissue from affected and control pigs was examined to identify differentially expressed genes (DEGs) and associated pathways. Results We found 59 significant DEGs, including SIM1 , PITX1 , HOXA7 , METTL21C , PVALB , ALX1 , EYA2 , and TBX1 . Interestingly, RNA-seq analysis of METTL21C revealed its significant upregulation in UH-affected pigs. This was corroborated by epigenetic analysis, which identified hypomethylation at four CpG sites in the METTL21C within potential regulatory region, aligning with increased mRNA levels. Furthermore, Sanger sequencing uncovered an SNP (rs330073569) in the METTL21C regulatory region, which was significantly associated with UH condition. This SNP can potentially alter transcription factor binding leading to enhanced METTL21C transcription, and putatively contributing to the gene’s increased expression in UH pigs. Conclusions This study highlights potential genetic and epigenetic factors in UH etiology. The most significant result suggests that METTL21C plays an important role in the development of UH. This finding makes this gene a promising candidate for further research aimed at better characterizing umbilical hernia in pigs and at potentially eliminating undesirable variants from the gene pool.
Elevated serum concentration of anti‐Mullerian hormone and its association with SNP variants in the AMH gene in a tortoiseshell tomcat with a disorder of sex development (38,XX; SRY-negative)
AbstractTesticular disorders of sex development (DSD) in cats with XX sex chromosomes and the absence of the SRY gene are rare congenital abnormalities. A Maine Coon tomcat with a normal penis, gonads in the scrotum, low serum testosterone concentration, and an elevated level of anti‐Müllerian hormone (AMH) was subjected to genetic analyses due to an unusual tortoiseshell coat color for males. Primary studies revealed the presence of XX sex chromosomes, the lack of SRY and the presence of two copies of the candidate SOX9. The DSD tomcat and its parents were analyzed using whole genome sequencing. Candidate SNPs in AMH, ORC1, DOCK8, PRKAR1A, and TMEM186 genes, as well as a known intronic 5‐kb deletion in X‐linked ARHGAP36 gene, which is responsible for orange coat, were identified. Potentially pathogenic homozygous genotypes were observed in all candidate genes; however, only in AMH and ORC1 were these genotypes rare in a control cohort. Further studies were focused on two SNPs located in the 5′‐and 3′‐untranslated regions (UTRs) of AMH. It has been experimentally demonstrated that only a short AMH transcript is present in feline testes. In silico analysis revealed that the SNP located in the 3′UTR of AMH occurs within a sequence that partially matches the canonical binding site for human miR‐5571‐5p. This microRNA is expressed in mammalian testes, which we confirmed in feline testicular tissue. We concluded that SNP in the 3′UTR of AMH is associated with elevated expression of the encoded hormone; however, it is not the cause of the testicular DSD phenotype in the studied Maine Coon tomcat.
Cytogenetic and molecular insight into the genetic background of disorders of sex development in seventeen cats
AbstractThe genetic background of feline disorders of sex development (DSDs) is poorly understood. We performed comprehensive cytogenetic, molecular, and histological studies of 17 cats with abnormal external genitalia, unusual behavior, or tricolor coats (atypical in males). The DSD phenotype of three cats was associated with sex chromosome abnormalities: X/Y translocation (38,XXSRY+), 37,X/38,XY mosaicism, and XX/XY leukocyte chimerism. The remaining 14 affected cats were classified as XY DSD (SRY-positive). In this group and 38 normal males, we analyzed a priori selected candidate genes (SRY, TAC3, CYP11B1 and LHCGR). Only a previously reported nonpathogenic variant was found in SRY. Moreover, SRY gene copy number was determined, and three variants were observed: 6, 5 (modal), and 4 copies in a single DSD case. The known variants in TAC3 and CYP11B1, responsible for testicular hypoplasia, persistent primary dentition or congenital adrenal hyperplasia, were not found in the study group. Nine novel polymorphisms were identified in the LHCGR gene, one of which, a potentially regulatory indel variant in 5′UTR, was significantly associated (p = 0.0467) with XY DSD. Our report confirmed that abnormalities of sex chromosomes are important causes of feline DSDs. We also showed that the indel variant of LHCGR can be considered a promising marker associated with XY DSD phenotype.
Transcript Patterns of Bovine CYP21A2 and Its Pseudogene in Adrenal and Ovarian Tissues
Background: The cytochrome P450 family 21 subfamily A member 2 gene (CYP21A2) encodes 21-hydroxylase, a key enzyme in adrenal steroid biosynthesis. Despite its physiological importance, the diversity of CYP21A2 transcript variants and their tissue-specific expression in domestic animals, including cattle, remains largely unexplored. This study aimed to characterize CYP21A2 transcription in adrenal glands and ovaries and assess the potential transcriptional activity of its pseudogene, CYP21A1P. Methods: CYP21A2 transcription was investigated in adrenal and ovarian tissues of 12 healthy cows using semi-quantitative PCR and Sanger sequencing. Real-time PCR was performed to confirm expression levels. Melting curve analysis and electrophoresis were used to validate distinct amplicons corresponding to different transcript variants. Extended amplicons were sequenced to identify transcripts corresponding to reference sequences and potential pseudogene products. Results: A single transcript variant (NM_001013596.1) was consistently detected in adrenal glands, whereas ovaries expressed two variants: NM_001013596.1 and XM_024983378.2. Semi-quantitative analysis showed significantly higher CYP21A2 expression in adrenal glands compared to ovaries (p < 0.01). In ovarian samples, the NM_001013596.1 variant was more abundant than the XM_024983378.2 (p < 0.01). Sanger sequencing revealed two products matching CYP21A2 reference transcripts and an additional, longer product containing sequence motifs specific to the pseudogene CYP21A1P, indicating its transcriptional activity. Conclusions: These results provide the first evidence of tissue-specific expression and differential abundance of CYP21A2 transcript variants in cattle and suggest the transcription of the CYP21A1P pseudogene. The findings reveal the complexity of CYP21A2 expression in steroidogenic tissues and suggest potential regulatory roles for transcript and pseudogene variants in bovine physiology.