Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025044
Pigs, as crucial economic livestock species, possess remarkable reproductive traits and thus play a highly significant role in promoting the progress of the livestock industry. With the advent and application of CRISPR/Cas9 technology, researchers have explored genetic editing techniques to increase swine reproductive performance, flavour profiles, and nutritional attributes. Additionally, with respect to anatomy, physiology, immunology, and genomics as well as other traits, pigs exhibit remarkable similarities to humans. Genetically edited pigs play crucial roles in human disease models, xenotransplantation, breed improvement, vaccine development, and drug assessment. Common methods deployed in the preparation of genetically edited pigs include somatic cell nuclear transfer (SCNT), microinjection and sperm-mediated approaches. For example, Shen et al. [1] successfully generated P53-knockout Diannan miniature pigs using transcription activator-like effector nucleases combined with SCNT, offering a valuable resource for preclinical oncology research. In 2019, Chen et al. [2] employed microinjection to deliver Cas9 messenger ribonucleic acid (mRNA) and single guide ribonucleic acid (sgRNA) into the cytoplasm of fertilized eggs. These authors successfully obtained both the albinism phenotype and the combined phenotype of albinism and immunodeficiency in Tibetan miniature pigs. More recently, Tenihara et al. [3] introduced the CRISPR/Cas9 protein into fertilized porcine eggs via electroporation, enabling a simple, micromanipulation-free approach for generating gene-edited pigs. Among these methods, SCNT has gained extensive interest among researchers because of its reliability. An essential aspect of SCNT is the preparation of embryonic fibroblasts to serve as donor cells. Previously, the CRISPR/Cas9 plasmid editing system served as the predominant technique to generate genetically edited embryonic fibroblasts (Figure 1A) [4]. This approach, which is distinguished by its relative simplicity, high stability, and low cost, was formerly widely utilized in the production of gene-edited pigs. However, plasmid editing is associated with several notable limitations. First, it introduces resistance genes, posing risks of inaccurate gene editing, drug resistance and biosafety concerns. Second, during the CRISPR/Cas9 editing process, there is a possibility of ongoing editing due to deoxyribonucleic acid (DNA) integration. This continuous editing can increase the likelihood of off-target effects, random mutations, and interference with DNA repair mechanisms. Third, the acquisition of positive cell lines via the plasmid editing system typically demands an extended period of in vitro cultivation (lasting 3–4 weeks), which increases the risk of apoptosis and chromosomal aberrations. Consequently, plasmid-based transfection is now largely supplanted by ribonucleoprotein (RNP) systems for gene editing. RNP systems bypass plasmids, delivering the Cas9 protein and sgRNA directly into cells, reducing off-target effects and cytotoxicity [5]. In 2022, Xu et al. [6] developed the reporter RNA-enriched dual-sgRNA CRISPR/Cas9 ribonucleoprotein (RE-DSRNP) method, a transgene-free approach using CRISPR/Cas9 RNPs enriched with ATTO550-tracrRNA (IDT, Iowa, USA) as a fluorescent RNA probe (Figure 1B). This method reduced the time needed to generate donor cells from 3-4 weeks to one week, resulting in high-efficiency WIP1 gene knockouts and the production of pigs with male reproductive disorders. However, owing to genetic diversity, not all target genes achieve 95% editing efficiency, as demonstrated by the RE-DSRNP method, with some falling below 90%. For example, DOCK8, which belongs to the DOCK family, is an atypical guanine nucleotide exchange factor that plays a crucial role in immune responses. DOCK8 deficiency syndrome, a rare hereditary disorder, often leads to combined immunodeficiency and is characterized by elevated serum immunoglobulin E levels, increased eosinophil
Acta Biochimica et Biophysica Sinica•2024•DOI: 10.3724/abbs.2024121
Gene regulation refers to the precise regulation of gene expression in an organism, and transcription factors are proteins that bind to DNA and regulate gene expression by promoting or inhibiting the expressions of target genes. Since the late 1980s [1], scientists have studied special genes called Pax genes that control how genes function in organisms as they grow. There are nine Pax genes found in animals such as mice, zebrafish, and humans [2]. Based on the composition domain and homology of the sequence, the Pax family is divided into four subfamilies: Pax1/9, Pax2/5/8, Pax4/6, and Pax3/7 [3]. Pax7 plays a pivotal role in the implementation, protection, and repair of skeletal muscle. Pax7 helps to control the balance between self-renewal and differentiation of satellite cells, ensuring that they can proliferate when needed to generate new muscle cells and differentiate into mature muscle fibers when necessary for muscle development and repair. The expression of the Pax7 gene in nerve cells is critical for dorsal root and sensory ganglia development. The Pax7 gene serves as a primary controlling factor for skeletal muscle development while influencing different biological processes; however, its exact role in jawless vertebrates such as lamprey remains unclear, and extensive research is needed to elucidate the intricate underlying mechanisms involved. Given the unique status of lamprey as an ancient jawless fish, possessing an ancient lineage and distinctive biological features, it is rare to explore gene function across hundreds of millions of years of vertebrate evolution. The use of lamprey as a model system for gene function research represents an innovative approach in the fields of evolutionary and comparative genomics. In this study, we investigated the regulatory mechanism of Pax7 in lamprey via gene cloning, gene expression analysis, gene silencing and transcriptome data analysis. We also explored the interactions between genes with significant differences. Identification of Lr.Pax7 in lamprey tissues began with the retrieval of protein sequences that are similar to those of human Pax family members in sea lamprey (Petromyzon marinus) or zebrafish (Danio rerio) from the NCBI protein database (Supplementary Table S1) and the use of BLAST to identify corresponding homologs (Supplementary Table S2). Subsequently, we extracted the Pax sequences from our library. Lethenteron reissneri specimens were dissected to isolate various tissues. Primers targeting the pax domains were designed based on the Pax7 nucleotide sequence in the Lampreys cDNA library, and the aim was to verify the effectiveness of the Lampreys cDNA as a template for validation (Supplementary Table S3). Lr.Pax7 was successfully amplified via PCR in muscle tissue. Here, a variety of methods were used for bioinformatics analysis. The results showed that the amino acid sequence of Pax7 is highly similar among animals (Figure 1A), with a decreasing trend from higher to lower organisms, as revealed by sequence alignment. It can be observed from the evolutionary tree (Figure 1B) that Pax genes for each subfamily are present in ancestral chordate and that Pax genes are present in amphioxus. Petromyzon marinus, Lethenteron camtschaticum, and Lethenteron reissneri constitute a sister group and have become good models for the study of jawless vertebrates. Pax9, Pax2, Pax6, and Pax7 show high similarity to those of other higher vertebrates. Therefore, these genes were named Lr.Pax9, Lr.Pax2, Lr.Pax7, and Lr.Pax6. The results indicate that the Pax7 gene is significantly preserved across various species, from higher to lower. This suggests that the DNA sequence of the gene is remarkably similar among different species. Lr.Pax7 is positioned between vertebrates and invertebrates and is most closely related to P. marinus Pax7. This finding provides more insight into the original evolutionary position of the lamprey. Crystal structure prediction analysis revealed that Lr.Paxs and Hm.Paxs have highly homologous structures (Figure 1C). The Pax gene has a similar structure (Figure 1D), including a conserved DNA-binding structure called the pair-box domain. This structure contains approximately 128 amino acids and is responsible for binding with specific DNA sequences, regulating gene expression, and interacting with other proteins. To further investigate the evolutionary history of Pax7 in vertebrates, we compared the genetic environment of Pax7 with that of other vertebrates (Figure 1E). In addition, many Pax gene members also contain DNA-binding structures called homeodomains, which play important roles in development.
Chinese Journal of Tissue Engineering Research•2026•DOI: 10.12307/2026.21288
BACKGROUND: As crucial stabilizers of the knee joint, the medial collateral ligament and lateral collateral ligament play essential roles in restricting valgus and varus movements, respectively. However, the mechanical differences between the medial collateral ligament and lateral collateral ligament, the microstructure characteristics, and the effect of elastin degradation on their mechanical properties remain poorly understood. OBJECTIVE: To compare the mechanical differences between the medial collateral ligament and lateral collateral ligament, quantify the structural characteristics of the collagen fiber alignment, and investigate the effect of elastin degradation on the mechanical properties of both ligaments. METHODS: Left medial collateral ligaments and lateral collateral ligaments were harvested from adult pigs, frozen, and thawed. Quasi-static uniaxial tensile tests were performed to measure the mechanical properties of the medial collateral ligament and lateral collateral ligament, and the effects of repeated stretching on their mechanical properties were compared. Second harmonic generation imaging using a two-photon microscope was used to quantify the collagen fiber structure of the medial collateral ligament and lateral collateral ligament. After repeated stretching, the medial collateral ligament and lateral collateral ligament were incubated in elastase solution for 12 hours, followed by uniaxial tensile tests to determine the effect of elastin treatment on ligament mechanical properties. RESULTS AND CONCLUSION: (1) Quasi-static uniaxial tensile tests showed that the high-tension elastic modulus of the medial collateral ligament was higher than that of the lateral collateral ligament (P < 0.05), while there was no significant difference in the low-tension elastic modulus between the two groups (P > 0.05). Repeated stretching significantly reduced the low-tension elastic modulus of both the medial collateral ligament and lateral collateral ligament. (2) Elastase treatment significantly reduced the low-tension and high-tension elastic moduli of both the medial collateral ligament and lateral collateral ligament, and the decrease in the high-tension elastic modulus of the lateral collateral ligament was greater than that of the medial collateral ligament. After elastase treatment, both the low-tension and high-tension elastic moduli of the medial collateral ligament were higher than those of the lateral collateral ligament (P < 0.05). (3) Two-photon imaging showed that the collagen fibers of the medial collateral ligament maintained a crimped structure, and its fiber waviness was significantly higher than that of the lateral collateral ligament. (4) These results indicate that the medial collateral ligament has stronger elastic properties than the lateral collateral ligament, and elastase treatment has a greater effect on the mechanical properties of the lateral collateral ligament. These mechanical results may be related to the more crimped collagen fiber arrangement in the medial collateral ligament.
Chinese Journal of Tissue Engineering Research•2026•DOI: 10.12307/2026.21488
BACKGROUND: Mangiferin, a major bioactive compound derived from mango trees, is widely present in various traditional Chinese medicinal herbs and exhibits multiple biological functions including antibacterial, cholesterol-lowering, and anti-allergic effects. Existing studies have suggested that mangiferin may prevent and treat osteoarthritis pain. However, its specific mechanism of action remains unclear to date. OBJECTIVE: To systematically investigate the key targets and potential mechanisms of mangiferin in the treatment of osteoarthritis by integrating gene expression omnibus (GEO) microarray data analysis, network pharmacology, and molecular docking techniques, and to validate the findings in a rat model. METHODS: First, GEO microarray data were mined to identify potential therapeutic targets for osteoarthritis. Next, professional databases were integrated to predict the targets of mangiferin, and target information related to osteoarthritis was collected. A Venn diagram was generated using the Weishengxin platform, a protein-protein interaction network was constructed based on the STRING database, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed. Cytoscape 3.8.0 software was used to construct a drug-target-pathway-disease network, and molecular docking analysis and visualization were performed using the CBDOCK2 online docking platform. A rat model of osteoarthritis was established by anterior cruciate ligament transection of the left knee joint, and different concentrations of mangiferin were administered to observe and record the therapeutic effects. RESULTS AND CONCLUSION: A total of 144 potential targets of mangiferin were identified from multiple databases. Protein-protein interaction network analysis revealed important targets including interleukin-6, tumor necrosis factor, and nuclear factor kappa B1. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that 235 signaling pathways might be involved, including lipid and atherosclerosis-related pathways, advanced glycation end products-receptor, hypoxia-inducible factor 1, and estrogen, which are closely related to inflammation. In animal experiments, after 4 weeks of intervention with 40 μmol/L mangiferin, there was no significant difference in hindlimb weight-bearing compared with the sham-operated group. These findings suggest that mangiferin may exert therapeutic effects on osteoarthritis through a multi-target, multi-pathway mode of action, providing a new strategy and theoretical support for the treatment of osteoarthritis.
Chinese Journal of Tissue Engineering Research•2026•DOI: 10.12307/2026.21624
BACKGROUND: Lower extremity deep vein thrombosis is a catastrophic complication after lumbar spine fusion, and previous studies have shown that some patients, such as those with prosthetic arthroplasty and abdominal surgeries, suffer from a combination of preoperative anemia, which predisposes them to postoperative deep vein thrombosis of the lower extremities. However, whether preoperative anemia increases the risk of lower extremity deep vein thrombosis after lumbar spine fusion is unclear. OBJECTIVE: To investigate the correlation between preoperative anemia and deep vein thrombosis of the lower extremities in patients after lumbar fusion. METHODS: The clinical data of 1 178 patients who underwent lumbar spinal fusion treatment admitted to Third Affiliated Hospital of Anhui Medical University from January 2020 to December 2023 were retrospectively analyzed. According to whether or not the patients developed lower extremity deep vein thrombosis after the operation, the patients were divided into the deep vein thrombosis group (ultrasound report suggestive of lower extremity deep vein thrombosis) and the non-deep vein thrombosis group (ultrasound report suggestive of no lower extremity deep vein thrombosis). The incidence of anemia was compared between the two groups, and risk factors for lower extremity deep vein thrombosis in lumbar fusion patients were determined by univariate analysis and multivariate logistic regression analysis. RESULTS AND CONCLUSION: Among 1 178 patients, there were 43 cases (3.7%) in the deep vein thrombosis group and 1 135 cases (96.4%) in the non-deep vein thrombosis group. The incidence of preoperative anemia in the deep vein thrombosis group was 32.6%, significantly higher than that in the non-deep vein thrombosis group (10.9%, P < 0.05). Univariate analysis showed significant differences between the two groups in preoperative hemoglobin (P < 0.001), preoperative red blood cell count (P=0.028), D-dimer positivity (P=0.029), hypertension (P=0.019), number of fused segments (P=0.023), anemia (P < 0.001), and blood transfusion (P=0.006) (P < 0.05). Multivariate analysis showed that preoperative anemia (OR=4.221, 95%CI: 1.198-14.802, P=0.025) and D-dimer positivity (OR=2.023, 95%CI: 1.065-3.844, P=0.031) were risk factors for lower extremity deep vein thrombosis after lumbar fusion. These findings suggest that the incidence of preoperative anemia is relatively high in patients undergoing elective lumbar fusion, and preoperative anemia is an independent risk factor for deep vein thrombosis after lumbar fusion. It is recommended that preoperative anemia should be actively managed and corrected before elective lumbar fusion to reduce the risk of lower extremity deep vein thrombosis.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025044
The generation of genetically edited pigs via somatic cell nuclear transfer (SCNT) has historically relied on plasmid-based CRISPR/Cas9 systems, which introduce resistance genes, risk off-target effects from prolonged editing, and require 3–4 weeks of in vitro selection. This study presents a transgene-free, rapid strategy using the IRE-DSRNP method to edit monoclonal porcine fetal fibroblasts. Three IgA-knockout cell lines were obtained with large deletions in the CH1-CH3 region: 1044 bp (heterozygous), 1043 bp (homozygous), and 1039 bp (homozygous). These cells were pooled and used as donor nuclei for SCNT. From 880 fresh oocytes, 660 mature oocytes were selected, 500 underwent enucleation and nuclear transfer, yielding 400 fused cells; 300 embryos were transplanted into a surrogate sow. Pregnancy was confirmed at 28 days, and after 143 days of gestation, six F0 piglets were born. Genotyping revealed two heterozygotes (4010#, 4015#) and four homozygous knockouts, with two piglets per genotype (1044, 1043, 1039 bp deletions). This approach eliminates plasmid integration, reduces off-target risks, and shortens the timeline for producing IgA-deficient Bama pig models, offering a robust platform for disease modeling and xenotransplantation research.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025094
Kainate receptors (KARs), members of the ionotropic glutamate receptor (iGluR) family, are established targets for neurological disorder therapeutics, yet their functional repertoire in jawless vertebrates remains undefined. This study identified four iGluR subunits in lamprey—Lr-GRIA2, Lr-GRIA4, Lr-GRIK1, and Lr-GRIN2B—with phylogenetic placement outside jawed vertebrate clades and conserved N-terminal ECDs. Functional interrogation of Lr-GRIK1 via siRNA silencing (5-, 2.3-, and 2.56-fold reduction at 24, 48, and 96 h post-transfection) followed by transcriptome sequencing revealed 9 genes with |log2 FC| > 1 and P < 0.05, enriched in signal transduction and immune pathways. Upregulated transcripts included MAP3K9, ANKRD1, HMOX2, ANKRD13B, NDKB, ADCY8, PKC, PCLO, NOG1L, and SDK2. Kidney tissue monitoring post-stimulation confirmed Lr-GRIK1 modulates immune-related molecule expression. These data establish a non-neurotransmission role for Lr-GRIK1 in lamprey immunity, demonstrating that KARs negatively regulate immune responses in this basal vertebrate lineage.