Effect Of Gene Knockout Research Articles

Effects of OsLPR2 Gene Knockout on Rice Growth, Development, and Salt Stress Tolerance

Effects of OsLPR2 Gene Knockout on Rice Growth, Development, and Salt Stress Tolerance

Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.

Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.

Comparison of the Effects of Inappropriate Meal Timing-Induced and Genetic Models of Circadian Clock Disruption on Uterine mRNA Expression Profiles

BackgroundAccumulating evidence reveals that inappropriate meal timing contributes to the development of lifestyle-related diseases. An underlying mechanism is thought to be the disruption of the intracellular circadian clock in various tissues based on observations in both systemic and tissue-specific clock gene-deficient mice. However, whether the effects of conditional clock gene knockout are comparable to those of inappropriate meal timing remains unclear. ObjectivesThis study aimed to compare the effects of a recently developed 28-h feeding cycle model with those of a core clock gene Bmal1 uterine conditional knockout (Bmal1 cKO) model on uterine mRNA expression profiles. MethodsThe models were generated by subjecting C57BL/6J mice to an 8-h/20-h feeding/fasting cycle for 2 wk and crossing Bmal1-floxed mice with PR-Cre mice. Microarray analyses were conducted using uterine samples obtained at the beginning of the dark and light periods. ResultsThe analyses identified 516 and 346, significantly 4-fold and 2-fold, up- or downregulated genes in the 28-h feeding cycle and Bmal1 cKO groups, respectively, compared with each control group. Among these genes, only 7 (1.4%) and 63 (18.2%) were significantly up- or downregulated in the other model. Moreover, most (n = 44, 62.9%) of these genes were oppositely regulated. These findings were confirmed by gene set enrichment analyses. ConclusionsThis study reveals that a 28-h feeding cycle and Bmal1 cKO differently affect gene expression profiles and highlights the need for considering this difference to assess the pathophysiology of diseases associated with inappropriate meal timing.

Transcriptome analysis of the effect of HERV-K env gene knockout in ovarian cancer cell lines.

Human endogenous retroviruses (HERVs) have been implicated in the pathogenesis of various diseases, particularly cancers. Previous investigations from our group demonstrated that targeted knockout (KO) of the HERV-K env gene led to a significant reduction in tumorigenic attributes, including proliferation, migration, and invasion of ovarian cancer cells. In this study, we aimed to elucidate the impact of HERV-K env KO on gene expression in ovarian cancer cell lines through comparative RNA sequencing (RNA-Seq) analysis with two distinct HERV-K env KO ovarian cancer cell lines, SKOV3 and OVCAR3. HERV-K env gene KO was achieved in SKOV3 and OVCAR3 ovarian cancer cell lines using the CRISPR-Cas9 system. Next-generation mRNA sequencing was employed to assess the gene expression profiles of both mock and HERV-K env KO ovarian cancer cells. Furthermore, comprehensive analyses involving gene ontology and pathway assessments were conducted. Transcriptome analysis revealed that 23 differentially expressed genes (DEGs) were upregulated and 17 DEGs were downregulated in SKOV3 cells. In OVCAR3 cells, 198 DEGs were upregulated, and 17 DEGs were downregulated. Notably, 53 DEGs exhibited statistically significant differences among the 1,612 DEGs identified. Our findings indicate that HERV-K env gene KO exerts a profound influence on gene expression patterns in OVCAR3 cells, while genetic alterations in expression were relatively modest in SKOV3 cells. Nevertheless, genes ND1, ND2, and CYTB displayed a common increase in expression, while ERRFI1 and NDRG1 exhibited a decrease in expression in both cell lines. Our study demonstrates that KO of the HERV-K env gene in ovarian cancer cell lines has a substantial impact on gene expression patterns and can be used to identify potential therapeutic targets for ovarian cancer and related diseases.

Century-long timelines of herbarium genomes predict plant stomatal response to climate change

Dissecting plant responses to the environment is key to understanding whether and how plants adapt to anthropogenic climate change. Stomata, plants’ pores for gas exchange, are expected to decrease in density following increased CO2 concentrations, a trend already observed in multiple plant species. However, it is unclear whether such responses are based on genetic changes and evolutionary adaptation. Here we make use of extensive knowledge of 43 genes in the stomatal development pathway and newly generated genome information of 191 Arabidopsis thaliana historical herbarium specimens collected over 193 years to directly link genetic variation with climate change. While we find that the essential transcription factors SPCH, MUTE and FAMA, central to stomatal development, are under strong evolutionary constraints, several regulators of stomatal development show signs of local adaptation in contemporary samples from different geographic regions. We then develop a functional score based on known effects of gene knock-out on stomatal development that recovers a classic pattern of stomatal density decrease over the past centuries, suggesting a genetic component contributing to this change. This approach combining historical genomics with functional experimental knowledge could allow further investigations of how different, even in historical samples unmeasurable, cellular plant phenotypes may have already responded to climate change through adaptive evolution.

Gene regulatory network structure informs the distribution of perturbation effects.

Gene regulatory networks (GRNs) govern many core developmental and biological processes underlying human complex traits. Even with broad-scale efforts to characterize the effects of molecular perturbations and interpret gene coexpression, it remains challenging to infer the architecture of gene regulation in a precise and efficient manner. Key properties of GRNs, like hierarchical structure, modular organization, and sparsity, provide both challenges and opportunities for this objective. Here, we seek to better understand properties of GRNs using a new approach to simulate their structure and model their function. We produce realistic network structures with a novel generating algorithm based on insights from small-world network theory, and we model gene expression regulation using stochastic differential equations formulated to accommodate modeling molecular perturbations. With these tools, we systematically describe the effects of gene knockouts within and across GRNs, finding a subset of networks that recapitulate features of a recent genome-scale perturbation study. With deeper analysis of these exemplar networks, we consider future avenues to map the architecture of gene expression regulation using data from cells in perturbed and unperturbed states, finding that while perturbation data are critical to discover specific regulatory interactions, data from unperturbed cells may be sufficient to reveal regulatory programs.

The proliferation and differentiation of spermatogonial stem cells in the frist wave of spermatogenesis in rats with Trib3 gene knockout

This study explores the effects of Trib3 gene knockout on adult male rat spermatogenesis. Using CRISPR/Cas9, we knocked out the Trib3 gene in Wistar rats. Results indicate altered expression of PLZF, ID4, and c-KIT in knockout rats, suggesting impaired spermatogonial stem cell proliferation and differentiation. Histological analysis reveals reduced seminiferous tubule area and decreased spermatocyte numbers. Mating experiments demonstrate reduced offspring rates after the second self-mating in knockout rats. SYCP3, a meiosis marker, shows elevated expression in knockout rat testes at 14 days postpartum, suggesting an impact on reproductive processes. ELISA results indicate decreased testosterone, FSH, and FGF9 levels in knockout rat testicular tissues. In conclusion, Trib3 gene deletion may impede spermatogonial self-renewal and promote differentiation through the FSH-FGF9- c-KIT interaction and p38MAPK pathway, affecting reproductive capacity. These findings contribute to understanding the molecular mechanisms regulating spermatogenesis.

Effect of Spag11a gene knockout on the epididymis in mice: A histopathological and molecular analyses.

The sperm-associated antigen 11a (Spag11a) gene is exclusively expressed in the caput epididymis. Our previous studies demonstrated that small interfering RNA (siRNA)-mediatedablation of this gene resulted in increased proliferation of epididymal epithelial cells. Further, active immunization-mediated ablation of SPAG11A protein increased the susceptibility of male reproductive tract tissues to diethylnitrosamine (DEN)-induced tumorigenesis. In this study, we report that the caput epididymis of Spag11a knockout mice displayed hyperplasia and inflammation, while the caput epididymis of wild-type mice exhibited normal anatomical structure. Global transcriptome analyses in the caput epididymis of knockout mice indicated differential expression of genes involved in a variety of cellular processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested that the absence of Spag11a may activate microRNAs associated with cancer, chemical carcinogenesis-receptor activation, and chemical carcinogenesis-DNA adducts pathways; which may contribute to the promotion of tumorigenesis in the epididymis. The susceptibility of caput epididymis to chemically induced carcinogenesis in Spag11a knockout mice was analyzed. Histological analyses indicated that while the epididymis of wild-type mice did not show any signs of tumorigenesis, knockout mice displayed hyperplasia, anaplasia, dysplasia, neoplasia, and inflammation in the caput epididymis. Our results provide concrete evidence that deletion of Spag11a induces histopathological and molecular changes that contribute to tumorigenesis. It is possible that the expression of Spag11a gene could be one of the reasons for the rarity of epididymal cancers. The involvement of an epididymal gene in tumorigenesis is being demonstrated for the first time.

Effects of MAL gene knockout on lung tissue morphology and on E-cad and α-SMA expression in asthma mouse models

Objectives To investigate the effects of myelin- and lymphocyte-associated protein (MAL) gene knockout on the morphological structure of lung tissue and the expression of E-cadherin (E-cad) and alpha-smooth muscle actin (α-SMA) in an asthmatic mouse model. Methods Twenty-four specific pathogen-free (SPF) C57BL/6J mice were divided into four groups: the wild-type normal (WT/SAL), wild-type asthmatic (WT/OVA), gene knockout normal (MAL-/-/SAL), and gene knockout asthmatic (MAL-/-/OVA) groups. The establishment of the asthma mouse models was confirmed by evaluating behavioral symptoms and histopathological H&E and Masson staining. Western blotting and RT–qPCR were used to measure E-cad and α-SMA expression levels in lung tissues. Results H&E staining of mouse lung tissues from WT/OVA, MAL-/-/SAL, and MAL-/-/OVA groups revealed a thickened bronchial wall, irregular lumen edge, locally fallen mucosal epithelium, and inflammatory cell infiltration compared with those of the WT/SAL group. In the WT and MAL-/- groups, the proportion of Masson-stained tissues in the OVA group was greater than that in the SAL group (p < 0.05). Compared with those in the WT/SAL group, the expression levels of α-SMA mRNA and protein were increased, while those of E-cad were decreased in the WT/OVA group (p < 0.01). Similarly, compared with those in the MAL-/-/SAL group, the expression levels of E-cad mRNA and protein were increased, while those of α-SMA were decreased in the MAL-/-/OVA group (p < 0.01). All these differences were statistically significant (p < 0.01). Conclusions The MAL gene contributes to EMT inhibition and the stability of the airway barrier under normal physiological conditions by regulating E-cad and α-SMA expression.

The effect of IGFBP3 gene knockout by the CRISPR/Cas9 system on the IGF-1 pathway in murine cells

BackgroundThe IGF-1 signaling pathway has been deeply involved in the aging mechanism. The insulin-like growth factor binding protein 3 (IGFBP-3) is a protein that binds to IGF-1 that regulates growth, survival, and aging. ObjectiveThe purpose of this study was to investigate the impact of the IGFBP3 gene knockout (KO) on the expressions of aging-related proteins and genes using the CRISPR/Cas9 system. MethodsThe IGFBP3 gene knockout (KO) was performed by the CRISPR/Cas9 system. Sanger DNA sequencing and Indel analyses were used to verify the induction of mutation. ResultsFirst, Sanger DNA sequencing was used to analyze the IGFBP3 gene knockout in murine cells (B16F1). The isolation of three colonies with the mutated DNA sequences in the IGFBP3 gene was validated. In addition, the expression levels of the IGFBP3 gene and protein in the edited B16F1 cells were lower than in those of normal B16F1 cells in western blot analysis as well as RT-PCR and qPCR. Moreover, IGFBP3 gene KO cells enhanced the level of SA-ß-gal staining and short telomere length compared to normal B16F1 cells. In particular, it was found that the expression levels of senescence-related proteins such as PI3K, AKT1, PDK1, and p53 were higher in IGFBP3 gene KO cells than in normal cells in both the absence and presence of IGF-1. ConclusionsTherefore, the above findings could provide a clue that IGFBP3 could play a key role in the aging mechanism.

Investigating the mechanisms underlying resistance to chemotherapy and to CRISPR-Cas9 in cancer cell lines

Cancer is one of the major causes of death worldwide and the development of multidrug resistance (MDR) in cancer cells is the principal cause of chemotherapy failure. To gain insights into the specific mechanisms of MDR in cancer cell lines, we developed a novel method for the combined analysis of recently published datasets on drug sensitivity and CRISPR loss-of-function screens for the same set of cancer cell lines. For our analysis, we first selected cell lines that consistently exhibit drug resistance across several classes of compounds. We then identified putative resistance genes for each class of compound and used inferred gene regulatory networks (GRNs) to study possible mechanisms underlying the development of MDR in the identified cancer cell lines. We show that the same method of analysis can also be used to identify cell lines that consistently exhibit resistance to the gene knockout effect of the CRISPR-Cas9 technique and to study the possible underlying mechanisms. In the GRN associated to the drug resistant cell lines, we identify genes previously associated with resistance (UHMK1, RALYL, MGST3, USP9X, and ESRG), genes for which an indirect association can be identified (SPINK13, LINC00664, MRPL38, and EMILIN3), and genes that are found to be overexpressed in non-resistant cancer cell lines (MRPL38, EMILIN3 and RALYL). In the GRNs associated to the CRISPR-Cas9 resistance mechanism, none of the identified genes has been previously reported in the admittedly sparse literature on the subject. However, some of these genes have a common role: APBB2, RUNX1T1, ZBTB7C, and ISX regulate transcription, while APBB2, BTG3, ZBTB7C, SZRD1 and LEF1 have a function in regulating proliferation, suggesting a role for these two pathways. While our results are specific for the lung cancer cell lines we selected for this work, our method of analysis can be applied to cell lines from other tissues and for which the required data is available.

Functional identification of two HMGB1 paralogues provides insights into autophagic machinery in teleost

High mobility group box 1 (HMGB1) is a multifunctional regulator that plays different roles in various physiological and pathological processes including cell development, autophagy, inflammation, tumor metastasis, and cell death based on its cellular localization. Unlike mammalian HMGB1, two HMGB1 paralogues (HMGB1a and HMGB1b) have been found in fathead minnow and other fish species and its function as an inflammatory cytokine has been well investigated. However, the role of fish HMGB1 in autophagy regulation has not been well clarified. In the present study, we generated HMGB1 paralogues single (HMGB1a−/− and HMGB1b−/−) and double knockout (DKO) epithelioma papulosum cyprini (EPC) cells from fathead minnow by CRISPR/Cas9 system, and the knockout efficiency of these genes was verified at both gene and protein levels. In this context, the effects of HMGB1 gene knockout on the protein expression of microtubule-associated protein 1 light chain 3 II (LC3-II), an autophagy marker, were determined, showing that single knockout of two HMGB1 paralogues significantly decreased the expression of LC3-II, and these inhibitory effects were further amplified in HMGB1 DKO cells under both basal and rapamycin treatment conditions, indicating the role of two HMGB1 paralogues in fish autophagy. In agreement with this notion, overexpression of HMGB1a or HMGB1b with Flag-tag markedly upregulated LC3-II protein expression. Interestingly, overexpressing two paralogues distributed in both cytoplasm and nucleus. Finally, the role of HMGB1-mediated autophagy was further explored, finding that HMGB1 could interact with Beclin1, a key initiation factor of autophagy. Taken together, these findings highlighted the role of HMGB1 paralogues as the autophagy regulator and increased our understanding of autophagic machinery in teleost.

Effect of RSN1 gene knockout on the adsorption of strontium ions by irradiated Saccharomyces cerevisiae

The irradiated Saccharomyces cerevisiae (Y-7) has good biosorption ability for strontium ions. To investigate the mechanism of strontium ion bioaccumulation in Y-7, we employed CRISPR/Cas9 gene editing technology to engineer Saccharomyces cerevisiae Y-7 and knock out the RSN1 gene, successfully constructing a RSN1 gene knockout strain (Y-7-rsn1Δ). When tested for strontium ion adsorption, the Y-7-rsn1Δ strain exhibited decreased capacity for adsorbing strontium ions and increased resistance to strontium ions. The results showed that RSN1 is involved in the transport of Sr2+, and observed significant decreases in intracellular Ca2+ of Y-7-rsn1Δ, indicating a strong correlation between bioaccumulation of Sr2+ and Ca2+. This demonstrated that the adsorption of strontium ions by Y-7 is regulated by the RSN1 gene. The knockout of the RSN1 gene resulted in the shift of the peak positions of carboxyl, amino, amide, hydroxyl, and phosphate groups on the cell surface.

Effect of Knocking Out HOXA5 Gene by CRISPR-Cas9-Mediated Gene Editing Technique on Proliferation of Acute Myeloid Leukemia Cells

To construct a acute myeloid leukemia (AML) cell line in which HOXA5 gene is stably knocked out by CRISPR-Cas9-mediated gene editing technique, so as to clarify the effect of HOXA5 gene knockout on the proliferation of AML cells, and preliminarily explore the role of HOXA5 gene in the pathogenesis of AML. The expression of HOXA5 in bone marrow mononuclear cells (BMMC) of non-tumor hematological patients and newly diagnosed AML patients was detected by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. The AML cell line KO-HOXA5-THP-1 was constructed in which HOXA5 gene was knocked out by CRISPR-Cas9-Mediated gene editing technique, and the knockout of HOXA5 gene was verified by qRT-PCR and Western blot, and the cell proliferation was detected by CCK-8 assay. Compared with non-tumor hematological patients, the levels of HOXA5 gene and protein in BMMC of newly diagnosed AML patients were significantly increased (P <0.05). The stable HOXA5 knockout cell line can be obtained by CRISPR-Cas9-Mediated gene editing technique, and the proliferation ability of THP-1 cells with HOXA5 gene knockout was significantly decreased (P <0.05). HOXA5 is highly expressed in AML cells, and knocking out HOXA5 can significantly affect the proliferation ability of AML cells, which provides a new potential therapeutic target for the precise treatment of AML.

Uncovering the essential roles of glutamate carboxypeptidase 2 orthologs in Caenorhabditis elegans.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans to genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. The present study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to physiology of this protease group and in extension to human GCP2 involvement in pathophysiological processes.

Stress granule-related genes during embryogenesis of an invertebrate chordate.

Controlling global protein synthesis through the assembly of stress granules represents a strategy adopted by eukaryotic cells to face various stress conditions. TIA 1-related nucleolysin (TIAR), tristetraprolin (TTP), and Ras-GTPase-activating protein SH3-domain-binding protein (G3BP) are key components of stress granules, allowing the regulation of mRNA stability, and thus controlling not only stress responses but also cell proliferation and differentiation. In this study, we aimed at investigating the roles of tiar, ttp, and g3bp during embryogenesis of the solitary ascidian Ciona robusta under both physiological and stress conditions. We carried out CRISPR/Cas9 to evaluate the effects of gene knockout on normal embryonic development, and gene reporter assay to study the time and tissue specificity of gene transcription, together with whole-mount in situ hybridization and quantitative real time PCR. To induce acute stress conditions, we used iron and cadmium as "essential" and "non-essential" metals, respectively. Our results highlight, for the first time, the importance of tiar, ttp, and g3bp in controlling the development of mesendodermal tissue derivatives during embryogenesis of an invertebrate chordate.

Effect of LSDV008 gene knockout on the cultural properties of recombinant lumpy skin disease virus

Lumpy skin disеаsе virus (LSDV) is а poxvirus thаt cаusеs а sеvеrе systеmic disеаsе in cаttlеаnd is rаpidly еxpаnding its gеogrаphic boundаriеs. Lumрy dеrmаtitis (LD) is chаrаctеrizеd by fеvеr, nоdulеs оn thе skin, mucоus mеmbrаnеs, аnd intеrnаl оrgаns. Thе disеаsе cаn cаusееmаciаtiоn with swоllеn lymрh nоdеs аnd sоmеtimеs dеаth. In rеcеnt yеаrs, thе disеаsе hаs bеcоmееndеmic in vаriоus раrts оf Аsiа, cаusing significаnt еcоnоmic dаmаgе tо livеstоck рrоductiоn. Sincе thеrеаrе nо sреcific trеаtmеnts fоr LSD, vаccinаtiоn is thе mоst еffеctivе wаy tо cоntrоl аnd еrаdicаtе thе disеаsе. Thе mоst cоmрlеtе immunity cаn bеоbtаinеd by vаccinаtiоn with livеаttеnuаtеd vаccinеs. Аttеnuаtiоn thrоugh lоng рassagеs is assоciatеd with randоm mutatiоns in thе gеnоmе, and thе mеchаnism оf аttеnuаtiоn rеmаins unclеаr. Tаrgеtеd rеmоvаl оf virulеncе gеnеs in thе virаl gеnоmе by gеnеtic еnginееring is thе mоst рrоmising dirеctiоn in thе crеаtiоn оf аttеnuаtеd роxvirusеs. Thе LSDV008 gеnееncоdеs арrоtеin similаr tо thе γ-intеrfеrоn rеcерtоr аnd is ароtеntiаl virulеncе gеnе fоr thеLSD virus. In thеsе studiеs, wе studiеd thееffеct оf dеlеtiоn оf thе LSDV008 gеnеоn thе culturаl рrореrtiеs оf thе rеcоmbinаnt Аtyrаu-B virus. Thераrеntаl LSD virus Dеrmаtitis nоdulаrеs/2016/Аtyrаu/KZ (Аtyrаu-KZ) wаs usеd аs а cоntrоl. Аs а rеsult оf thе studiеs, it wаs fоund thаt thе gеnе knоckоut did nоt аffеct thе rерlicаtiоn аctivity оf thе rеcоmbinаnt Аtyrаu-B virus in vitrо. Thе rеcоmbinаnt virus аccumulаtеd in cеll culturеs in thе sаmе titеrs аs thераrеnt virus. Thе mоst sеnsitivе cеll systеms fоr thе rерrоductiоn оf Аtyrаu-KZ аnd Аtyrаu-B LSD virusеs аrе lаmb tеsticlе (LT), bull kidnеy (MDBK) аnd sаigа kidnеy (SK) cеll culturеs, which cаn bе usеd tооbtаin virаl mаss in furthеr sciеntific rеsеаrch.

EDITING CDKN1A AND RASD1 IN A CELL CULTURE SYSTEM

Abstract As an individual ages, there is a higher incidence of cancer and infertility. Testicular germ cell cancer rates have been rising year after year. According to the American Cancer Society 1 of every 250 males will develop testicular cancer during their lifetime. In this research project, we aim to develop a molecular toolbox to analyze gene function in male germinal cells using a CRISPR/Cas9 cell culture system. This biotechnology allows for the targeted alteration and cutting of specific DNA strands. To achieve this, we will utilize cancer cell lines (SUSA and NT2/D1 testicular cancer cell lines) and knock out two genes of interest: cyclin-dependent kinase inhibitor 1 (CDKN1A) and ras-related dexamethasone-induced 1 (RASD1). Our objective is to investigate the effects of gene knockout on cell proliferation. For this purpose, we designed primers and single-guide RNAs using bioinformatic databases: Benchling and Chop Chop. The tools we used were employed to amplify human genomic DNA and assess the effectiveness of the guide RNAs through in vitro cutting of our genes of interest, followed by gel electrophoresis observations. Based on the results obtained from these experiments, our next step is to electroporate the guide RNA and Cas9 complex to alter gene function in our cell culture system. Through a better understanding of gene function in testicular cancer cell lines, we hope to inform future therapeutic targets for both testicular cancer and age-related infertility.

Effects of secretin gene knockout on the diversity, composition, and function of gut microbiota in adult male mice.

The gut microbiota plays a vital role in maintaining gastrointestinal homeostasis, however, whether it is influenced by gut hormones remains unknown. Secretin is a well-known gastrointestinal hormone produced by enteroendocrine S cells. This study utilized 16S rRNA amplicon sequencing to characterize the effect of SCT deficiency on the gut microbiota. Our results show that systemic SCT knockout alters the composition and abundance of the mouse gut microbiota but does not affect fecal short-chain fatty acids and lipids concentrations. At the genus level, the abundance of Turicibacter, Bacteroides, Ruminococcu, Romboutsia, Asaccharobacter, and Parasutterella increased in SCT-/- mice, whereas the abundance of Akkermansia and Escherichia decreased. Functional prediction results showed that lack of SCT reduced the abundance of carbohydrate metabolism-related pathways but increased the abundance of linoleic acid metabolism and branched-chain amino acid degradation. Overall, systemic SCT knockout had only minor effects on gut microbiota composition and function in adult male mice fed a standard chow diet.

Silk Fibroin-Modified Liposome/Gene Editing System Knocks out the PLK1 Gene to Suppress the Growth of Lung Cancer Cells.

Polo-like protein kinase 1 (PLK1) plays a key role in lung cancer cell mitosis. The knockout of PLK1 gene by the CRISPR-Cas9 system can effectively inhibit the proliferation of tumor cells, but there is no suitable vector for in vivo delivery. In this study, CRISPR-Cas9 gene knockout plasmids encoding sgRNA, Cas9 and green fluorescent protein were constructed. Then, the plasmids were packaged with liposome (Lip) and cholesterol-modified Antheraea pernyi silk fibroin (CASF) to obtain the CASF/Lip/pDNA ternary complex. The CASF/Lip/pDNA complex was transfected into lung cancer cells A549 to investigate the transfection efficiency, the PLK1 gene knockout effect and the inhibitory effect on lung cancer cells. The results showed that the transfection efficiency of the CASF/Lip/pDNA complex was significantly higher than that of the Lip/pDNA binary complex, and the expression of PLK1 in cells transfected with CASF/Lip/pDNA complexes was significantly lower than that in cells transfected with Lip/pDNA complexes. The CASF/Lip/pDNA complex significantly increased the apoptosis rate and decreased the proliferation activity of lung cancer cells compared with Lip/pDNA complexes. The cytotoxicity of the complexes was evaluated by coculture with the human bronchial epithelial cells BEAS2B. The results showed that CASF/Lip/pDNA complexes exhibited lower cytotoxicity than Lip/pDNA complexes. The fibroin-modified liposome/PLK1 gene knockout system not only effectively inhibited the growth of lung cancer cells but also showed no obvious toxicity to normal cells, showing potential for clinical application in lung cancer therapy.