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PubMed2025

Illuminating the mystery of thylacine extinction: a role for relaxed selection and gene loss.

Gene loss shapes lineage-specific traits but is often overlooked in species survival. In this study, we investigate the role of ancestral gene loss using the extinction icon-thylacine (Thylacinus cynocephalus). While studies of neutral genetic variation indicate a population decline before extinction, the impact of thylacine-specific ancestral gene losses remains unexplored. The availability of a chromosomal-level genome of the extinct thylacine offers a unique opportunity for such comparative studies. Here, we leverage palaeogenomic data to compare gene presence/absence patterns between the Tasmanian devil and thylacine. We discovered ancestral (between 13-1 Ma) loss of SAMD9L, HSD17B13, CUZD1 and VWA7 due to multiple gene-inactivating mutations, corroborated by short-read sequencing. The timing of gene loss mirrors the thylacine's shift towards hypercarnivory and increased body size. Notably, the loss of SAMD9 correlates with a carnivorous diet. Our genome-wide analysis reveals olfactory receptor loss and relaxed selection, aligning with reduced olfactory lobes in the thylacine, indicating olfaction is not its primary hunting sense. By integrating palaeogenomic data with comparative genomics, our study reveals ancestral gene losses and their impact on species survival and resilience to environmental changes. Our approach can be extended to other extinct and endangered species, helping to identify genetic factors for conservation efforts.

Animals↗
PubMedDate not supplied

[Construction and molecular cloning of hybrid plasmids containing specific fragments of Escherichia coli DNA].

In the paper a convenient procedure for the isolation of specific Eco RI-fragments of E. coli genome and their amplification on Km-resistance plasmid vector CKdelta11 is described. Plasmid CKdelta11 contains Col E1 replicon and has only one Eco RI site. The hybrid molecules were constructed in vitro using Eco RI-digestion followed by ligation. Then appropriated E. coli strain (polyauxotrophic strain E. coli K12 AB 2463) was transformed with ligated DNA mixture and hybrid plasmids, containing arg, leu, his and thr chromosomal markers were selected by molecular cloning and isolated from the obtained E. coli clones. The hybrid plasmids have two Eco RI sites and consist of one Eco RI-fragment of initial plasmid CKdelta11 and one Eco RI-fragment of El coli DNA. The method described allows to isolate and amplify on hybrid plasmids DNA fragments, containing any selectable genes or genes adjacent to the selectable ones.

Chemical Phenomena↗
PubMed1978

Irreversible gene repression model for control of development.

As the pluripotent cells of early embryos differentiate, each progressively loses the potency to develop into several phenotypes. Ultimately, each cell becomes irreversibly restricted to the expression of a single phenotype. Although in many instances details regarding those restriction events are well known, there is little information concerning the nature of the gene transcription changes involved. A model that accounts for the diminution of developmental potential as resulting from progressive, irreversible repression of previously active genes is presented. A scheme of progressive gene repression, rather than selective gene activation, is most consistent with observations from experimental embryology as well as from more recent biochemical experimentation.

Animals↗
PubMed2025

Multiomic Analyses Reveal the Molecular Mechanisms of Arid Adaptation in a Desert Rodent Species.

Organisms living in desert habitats face multiple simultaneous pressures, such as high temperatures and arid, and the population dynamics and community diversity of small rodents are strongly affected by climate extremes. However, the potential mechanisms by which desert rodents adapt to arid remain largely unexplored. Here, we assembled a 3.18 Gb genome, including 25,812 protein-encoding genes, for Orientallactaga sibirica, which is widely distributed across both arid and semihumid environments in Eurasia. Orientallactaga sibirica has longer ears and hind limbs to enhance heat dissipation, which may be related to the positively selected genes, such as Fgf10, Fgf11, Hoxc4, Hoxd1, and Bmp4. The renal transcriptome revealed increased fat and carbohydrate metabolism for metabolic water production in O. sibirica residing in arid habitats. Pathways such as material metabolism, oxidative stress response, osmoregulation, and water and salt reabsorption were enriched in candidate genes, such as Avp, Ang, and Ace, under positive selection in O. sibirica. Moreover, amino acid replacement was observed in the protein sequences of seven candidate genes, including Aldh7a1, Lnpep, Wnk4, C1qc, and Awat2, and these specific amino acid replacements of genes such as Umod and Scnn1a were related to unique osmoregulation, osmotic protection, and water retention compensation mechanisms. Water deprivation under laboratory conditions induced the upregulation of Umod and Aldh7a1 expression, further supporting the results observed in the wild population. These findings demonstrate that the positively selected genes related to limb development and specific amino acid replacements in the genes Umod and Scnn1a for unique osmoregulation in the renal vascular system may contribute to arid adaptation in the desert rodent species O. sibirica. This study provides novel insights into the adaptive evolution of desert small mammals and can serve as a reference for future research on renal damage-related diseases, such as human kidney stones and salt-sensitive hypertension.

Animals↗
PubMed2026

Research on identification of key genes and immune-metabolic mechanisms in atrial fibrillation through integrated multi-cohort transcriptomic analysis and machine learning.

This study aimed to integrate multiple datasets for the identification of atrial fibrillation (AF)-related differentially expressed genes (DEGs), analyze their underlying mechanisms through functional enrichment and machine learning, construct diagnostic models, and explore immune-metabolic interactions to provide novel biomarkers and theoretical foundations. Gene expression datasets were integrated and normalized, with batch effects removed using principal component analysis. Differential expression analysis, functional enrichment analysis (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways), and machine learning-based feature gene selection and model construction were performed. Shapley additive explanations analysis was utilized to interpret the constructed models, while gene set enrichment analysis, gene set variation analysis, and immune cell infiltration analysis were conducted to investigate the associations between feature genes and immune infiltration. After integrating and normalizing gene expression data and eliminating batch effects via principal component analysis, 6 DEGs were identified, including 4 upregulated and 2 down-regulated ones. Functional enrichment analysis showed these DEGs were significantly enriched in neuro-related biological processes and pathways, indicating their key roles in AF pathogenesis. Five key feature genes were selected using LASSO, random forest, and support vector machine-recursive feature elimination algorithms. They had significant expression differences between the AF and control groups (P&#x2005;<&#x2005;.001) and were located on distinct chromosomes. The constructed random forest and support vector machine models performed excellently (area under the curve&#x2005;&#x2265;&#x2005;0.85). Shapley additive explanations analysis revealed TNNI1 contributed most to model prediction, with its expression significantly positively correlated with immune cell infiltration. Gene set enrichment analysis and gene set variation analysis analyses further showed feature genes participated in AF pathogenesis by regulating immune modulation, metabolic pathways, and autophagy. Immune cell infiltration analysis found altered proportions of T-cell subsets and M0 macrophages in the AF group, along with complex links between feature gene expression and immune cell function. This study systematically elucidated the unique gene expression patterns and key regulatory pathways associated with AF, clarifying the crucial roles of feature genes in immune regulation, metabolic imbalance, and cellular dysfunction. These findings provide a theoretical basis and potential therapeutic targets for understanding AF pathogenesis and developing targeted treatment strategies.

Atrial Fibrillation↗
PubMed1979

Mechanisms of regulation of cell-mediated immunity. III. The characterization of azobenzenearsonate-specific suppressor T-cell-derived-suppressor factors.

Delayed type hypersensitivity to the hapten azobenzenearsonate (ABA) can be induced and suppressed by the administration of hapten-coupled syngeneic spleen cells by the appropriate route. Suppressor T cells stimulated by the intravenous administration of ABA-coupled spleen cells have been shown to produce a discrete subcellular factor(s) which is capable of suppressing delayed type hypersensitivity to azobenzenearsonate in the mouse. Such suppressor factors may be produced by the mechanical disruption of suppressor cells or by placing such suppressor cells in culture for 24 h. The suppressor factor(s) (SF) derived from ABA-specific suppressor cells exhibit biological specificity for the suppression of ABA delayed type hypersensitivity (DTH), but not trinitro-phenyl DTH, as well as the capacity to bind to ABA immunoadsorbents. Passage of suppressor factor(s) over reverse immunoadsorbents utilizing a rabbit anti-mouse F(ab')2 antiserum demonstrated that the antigen-specific T-cell derived SF does not bear conventional immunoglobulin markers. The suppressor factor(s) are not immunoglobulin molecules was further demonstrated by the inability of anti-ABA antibodies to suppress ABA DTH. Gel filtration of ABA suppressor factor(s) showed that the majority of the suppressive activity was present in a fraction with molecular weight ranging between 6.8 x 10(4) and 3.3 x 10(4) daltons. We also analyzed for the presence of determinants encoded by the H-2 major histocompatibility complex (MHC) and found that immunoadsorbents prepared utilizing antisera capable of interacting with gene products of the whole or selected gene regions of H-2 MHC, i.e., B10.D2 anti-B10.A and B10 anti-B10.A immunoadsorbents, retained the suppressive activity of ABA-SF. Elution of such columns with glycine HCl buffers (pH 2.8) permitted recovery of specific suppressive activity. Taken collectively such data supports the notion that suppressor T-cell-derived ABA suppressor factors have antigen-binding specificity as well as determinants controlled by the K end of the H-2 MHC. The distribution of strains capable of making SF has also been analyzed. The relationship of the antigen-binding specificity to VH gene products is discussed in this and the companion paper.

Animals↗
PubMed2026

Transcriptomic responses of Porphyrophora sophorae larvae during licorice root colonization reveal coordinated remodeling of translation, mitochondrial energy metabolism and defense-related genes.

BACKGROUND: Porphyrophora sophorae is a subterranean piercing-sucking scale insect that damages licorice (Glycyrrhiza uralensis) roots, but the molecular responses associated with larval root colonization remain insufficiently defined. METHODS: We compared non-parasitic larvae (NP) and root-colonizing larvae (RC) using six RNA-seq libraries, de novo transcriptome assembly, DESeq2-based differential expression analysis, GO/KEGG enrichment, annotation-based candidate gene screening, and RT-qPCR validation of selected genes. RESULTS: Sequencing yielded 260.91 million clean reads, and de novo assembly produced 60,794 non-redundant transcripts. DESeq2 identified 703 FDR-significant DEGs, including 49 upregulated and 654 downregulated genes in RC larvae. Upregulated genes were mainly associated with translation- and ribosome-related processes, whereas downregulated genes were enriched in mitochondrial, oxidation-reduction, energy metabolism, and oxidative phosphorylation-related functions. Annotation-based screening identified 75 FDR-significant candidate genes associated with chemosensation, defense-related responses, and energy metabolism, with mitochondrial energy metabolism-related genes forming the largest module. RT-qPCR validation based on the raw Ct data showed concordant expression directions for ten selected transcript targets. CONCLUSIONS: Root colonization in P. sophorae larvae was associated with coordinated transcriptional remodeling involving selective activation of translation-related processes, adjustment of mitochondrial energy metabolism, and changes in defense-related gene expression. These results provide candidate molecular targets for future functional studies of host contact, feeding establishment, and physiological adjustment in this subterranean scale insect.

Animals↗
PubMed1979

Hemoglobin synthesis in cultures of hepatic erythroid cells from the human fetus.

A recent theory of the control of human fetal hemoglobin synthesis, based on studies in cultured adult marrow, proposes that the phenotypic expression of fetal hemoglobin is largely dependent on the level of differentiation of the parental stem cells; that is, the earlier the progenitor, the greater the ability of its progeny to express fetal hemoglobin [Papayannopoulou, Th., Brice, M. & Stamatoyannopoulos, G. (1977) Proc. Natl. Acad. Sci. USA 74, 2923-2927]. To test this relationship with fetal tissue, we have studied hemoglobin synthesis in cultured human fetal liver, comparing gamma chain synthesis in the descendants of the early progenitors ("bursts") with that in the descendants of the later progenitors ("colonies"). Cells from the livers of midtrimester fetuses were cultured in methylcellulose with erythropoietin. The beta/(beta + gamma) globin synthetic ratio on days 5 to 7, when colonies predominated, was 0.09-0.11, a value characteristic of fetal reticulocytes, and on days 11 and 12, when bursts predominated, was 0.15-0.17. Thus, in fetal liver, the descendants of the earlier progenitor, the burst-forming unit, may be making more beta chains rather than more gamma chains, compared to descendants of the later progenitor, the colonyforming unit. Our data on fetal liver, taken together with the data on adult marrow by others, suggest that the erythroid colonies express the gene characteristic of the age of the organism to a greater degree than bursts, which express beta and gamma genes less specifically. Thus, the capacity for highly selective gene expression characteristic of differentiated cells appears to be less well developed in the burst-forming unit than in the colony-forming unit.

Cells, Cultured↗
PubMed2009

Similarities and differences between smoking-related gene expression in nasal and bronchial epithelium.

Previous studies have shown that physiological responses to cigarette smoke can be detected via bronchial airway epithelium gene expression profiling and that heterogeneity in this gene expression response to smoking is associated with lung cancer. In this study, we sought to determine the similarity of the effects of tobacco smoke throughout the respiratory tract by determining patterns of smoking-related gene expression in paired nasal and bronchial epithelial brushings collected from 14 healthy nonsmokers and 13 healthy current smokers. Using whole genome expression arrays, we identified 119 genes whose expression was affected by smoking similarly in both bronchial and nasal epithelium, including genes related to detoxification, oxidative stress, and wound healing. While the vast majority of smoking-related gene expression changes occur in both bronchial and nasal epithelium, we also identified 27 genes whose expression was affected by smoking more dramatically in bronchial epithelium than nasal epithelium. Both common and site-specific smoking-related gene expression profiles were validated using independent microarray datasets. Differential expression of select genes was also confirmed by RT-PCR. That smoking induces largely similar gene expression changes in both nasal and bronchial epithelium suggests that the consequences of cigarette smoke exposure can be measured in tissues throughout the respiratory tract. Our findings suggest that nasal epithelial gene expression may serve as a relatively noninvasive surrogate to measure physiological responses to cigarette smoke and/or other inhaled exposures in large-scale epidemiological studies.

Adult↗
PubMed2026

Genome-wide cis-expression Quantitative Trait Loci (eQTL) and transcriptomic signals reveal distinct molecular regulation across correlated feed efficiency traits.

INTRODUCTION: Feed efficiency (FE) is a complex trait which determines livestock production profitability, yet the molecular mechanisms behind it remain unclear. This study investigated the blood transcriptomic profile of lambs, alongside genotype data with the aim to uncover the genetic basis of FE traits such as absolute dry matter intake (DMIabsolute), DMI adjusted for body size (DMIadjusted), average daily live weight gain (ADG), and residual feed intake (RFI). MATERIALS AND METHODS: Bulk RNA-Seq and genotype data were analysed using three complementary approaches: differential gene expression (DGE) analysis, weighted gene co-expression network analysis (WGCNA), and cis-expression Quantitative Trait Loci (cis-eQTL) mapping. These methods were used independently to identify genes and regulatory networks associated with FE traits and to investigate evidence supporting multi-trait candidate gene selection. RESULTS: DGE analysis revealed 2, 24, 85 and 4 differentially expressed genes for DMIabsolute, DMIadjusted, ADG, and RFI (Padjusted < 0.05), functionally enriched in sensory perception, ATP-dependent chromatin remodeling, Notch signaling and immune response pathways. 9 gene modules significantly associated with the FE traits (P &#x2264; 0.05) with correlations ranging from r = -0.56 to 0.49, were identified using WGCNA. Single nucleotide polymorphism (SNP)-level cis-eQTL analysis identified 93 eSNPs associated with 74 genes (false discovery rate (FDR) < 0.05), while permutation-derived gene level analysis identified 280 eGenes (FDR < 0.2, empirical P < 0.03). Across the three analyses, applying thresholds of DGE (Padjusted < 0.05), WGCNA (correlation, P &#x2264; 0.05), and cis-eQTL gene-level significance (empirical P < 0.05), multiple overlapping genes were identified including DNMT3A, KANSL1, NCOR1 for DMIadjusted, ACOX2, FANCF, CIMIP2B, LOC101115106, ARMH2, LOC132657496 for ADG, and LOC114114576 for RFI representing regulators of variations in FE. DISCUSSION: The integration of DGE, WGCNA, and cis-eQTL analyses identified key genes and regulatory mechanisms associated with variation in FE traits. These results highlight that integrated multi-trait candidate gene identification approaches can reveal key genes that lower feed intake while maintaining animal growth, supporting breeding strategies aimed at improving efficiency and long-term economic sustainability in sheep.

average daily gain (ADG)↗
PubMed1978

In vitro immune response of spleen cells from mice genetically selected for high or low antibody production.

The aim of this study was the identification of the cell type in which genes selected for high or low response to SRBC express their functions. Spleen cells from high (H) and low (L) responder mice were immunized with SRBC in the Mishell and Dutton system. An antibody response of different magnitude was found in cultures of H and L spleen cells, the difference being at least as great as that observed in vivo. This finding under experimental conditions allowing the exclusion of any influence of the animal milieu during the immune response, suggest macrophages, B, and T lymphocytes as possible target cells of gene action. In vitro cell separation and recombination experiments in which spleen cells were immunized with SRBC, TNP-LPS, or TNP-HRBC indicate that the genetic differences between H and L responders brought about by selective breeding are expressed in lymphocytes to greater extent than in macrophages. The role of histoincompatibility in the recombination experiments in unlikely but cannot be excluded. Among lymphocytes, B cells but not helper T cells were found more responsive in cultures of spleen cells from H than from L mice.

Animals↗
PubMed2024

Repeated evolution on oceanic islands: comparative genomics reveals species-specific processes in birds.

Understanding the interplay between genetic drift, natural selection, gene flow, and demographic history in driving phenotypic and genomic differentiation of insular populations can help us gain insight into the speciation process. Comparing patterns across different insular taxa subjected to similar selective pressures upon colonizing oceanic islands provides the opportunity to study repeated evolution and identify shared patterns in their genomic landscapes of differentiation. We selected four species of passerine birds (Common Chaffinch Fringilla coelebs/canariensis, Red-billed Chough Pyrrhocorax pyrrhocorax,&#xa0;House Finch&#xa0; Haemorhous mexicanus and Dark-eyed/island Junco Junco hyemalis/insularis) that have both mainland and insular populations. Changes in body size between island and mainland populations were consistent with the island rule. For each species, we sequenced whole genomes from mainland and insular individuals to infer their demographic history, characterize their genomic differentiation, and identify the factors shaping them. We estimated the relative (Fst) and absolute (dxy) differentiation, nucleotide diversity (&#x3c0;), Tajima's D, gene density and recombination rate. We also searched for selective sweeps and chromosomal inversions along the genome. All species shared a marked reduction in effective population size (Ne) upon island colonization. We found diverse patterns of differentiated genomic regions relative to the genome average in all four species, suggesting the role of selection in island-mainland differentiation, yet the lack of congruence in the location of these regions indicates that each species evolved differently in insular environments. Our results suggest that the genomic mechanisms involved in the divergence upon island colonization-such as chromosomal inversions, and historical factors like recurrent selection-differ in each species, despite the highly conserved structure of avian genomes and the similar selective factors involved. These differences are likely influenced by factors such as genetic drift, the polygenic nature of fitness traits and the action of case-specific selective pressures.

Animals↗
PubMed2026

Comparative genomics and full-length transcriptome profiling of wing morphs in Tetrix grossus (Orthoptera: Tetrigidae).

Wing polymorphism represents a paradigmatic dispersal-reproduction trade-off, yet its molecular basis remains uncharacterised in the phylogenetically distant pygmy grasshoppers (Tetrigidae). Here we integrate comparative genomics across ten orthopteran species with full-length transcriptomics of long-winged (FL) and short-winged (FS) Tetrix grossus. OrthoFinder recovered 118 orthogroups specific to T. grossus. Against a backdrop of pronounced gene-family contraction (36 expansions versus 222 contractions; net -186, mirrored at the ancestral Tetrix node, +37/-140), we identified an ancestral, Tetrix-specific expansion of hormone-regulation (12 genes; fold enrichment 7.93) and lipid/carbohydrate-metabolic families organised into syntenic clusters, alongside 513 positively selected genes enriched for integrin-mediated cell adhesion (6 genes), a process relevant to epithelial and appendage morphogenesis. Full-length transcriptomics of one long-winged (FL) and one short-winged (FS) adult female detected 7530 (FL) and 7515 (FS) expressed genes, with 794 FL- and 776 FS-restricted transcriptome-derived SNP-associated genes. The FL morph was enriched for an EGFR/Ras-Rho developmental-patterning axis and neuromuscular flight genes, whereas the FS morph was enriched for insulin/peptide-hormone response and growth-regulatory loci. Overall, we present genomic resources and testable hypotheses concerning the evolution and regulation of wing morphs in Tetrigidae rather than a validated genetic architecture of wing-morph determination.

Animals↗
PubMed2026

Multi-omics identification and functional validation of signal regulatory protein gamma as a prognostic biomarker and immune regulator in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) comprises biologically diverse tumors, and durable responses to immune-checkpoint blockade are achieved by only a subset of patients. There remains a need for markers that connect clinical outcome with malignant-cell phenotypes and tissue-level immune organization. METHODS: We integrated The Cancer Genome Atlas HNSCC cohort (TCGA-HNSC), five Gene Expression Omnibus (GEO) validation cohorts, single-cell RNA sequencing, Visium spatial transcriptomics, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq)-informed protein-potential inference, pharmacogenomic screening, genetic-risk analysis and experimental validation. A reconstructed 296-pipeline survival modelling framework was used to prioritize prognostic hub genes across validation-cohort-specific analyses. RESULTS: SIRPG was repeatedly ranked among the top ten selected genes in all five validation cohorts. At single-cell resolution, SIRPG-high tumor cells showed stronger malignant-cell features, immune-inhibitory and metabolic programs, Scissor-positive risk association, CLCA2/P53-related perturbation signals and inferred SIRPG-CD47/signal regulatory protein (SIRP) communication. Spatial analyses placed this axis within an immune-checkpoint-coupled niche, supported by Maxspin/multiview intercellular spatial modelling (MISTy) spatial coupling, communication analysis by optimal transport (COMMOT)-inferred CD47-SIRPG communication and scProTrans-inferred CD47/SIRPG protein-potential overlap. Functionally, SIRPG knockdown reduced HNSCC cell viability and increased apoptosis, whereas re-expression of short hairpin RNA (shRNA)-resistant SIRPG restored the CLCA2-BAX/BCL2 protein response. CONCLUSION: Together, these findings identify SIRPG as an immune-related prognostic hub and context-dependent tumor-cell regulator associated with apoptosis, immune communication and spatial microenvironmental organization in HNSCC.

Humans↗
PubMed2026

From stress signaling to yield stability: physiological and molecular mechanisms of wheat resilience to heat and drought stress.

Wheat resilience depends on coordinated signaling, reproductive protection, and source-sink regulation, providing a framework to breed robust trait combinations that stabilize yield under combined heat and drought. Climate change is increasing the frequency and severity of heat and drought events, posing a major threat to wheat productivity, yield stability, and food security. Because these stresses often coincide in the field, their combined effects can impair growth, reproductive development, grain filling, and final yield more severely than either stress alone. Wheat resilience under such conditions depends on coordinated physiological adjustment and molecular regulation that sustain cellular homeostasis, protect reproductive tissues, and preserve yield-related traits. This review synthesizes current knowledge on the physiological and molecular bases of wheat resilience to heat and drought, with emphasis on their combined effects. We discuss major physiological responses, including photosynthetic adjustment, stomatal regulation, canopy cooling, osmotic balance, antioxidant defense, membrane stability, and source-sink coordination. We also examine key regulatory pathways involved in stress perception and adaptation, including calcium and reactive oxygen species signaling, mitogen-activated protein kinase cascades, phytohormonal crosstalk, transcriptional regulation, heat shock proteins, late embryogenesis abundant proteins, and osmoprotective and redox-associated pathways. In addition, we highlight the growing contribution of transcriptomics, proteomics, metabolomics, and phenomics to the identification of candidate genes, biomarkers, and adaptive traits. Finally, we consider how mechanistic insights can be translated into wheat improvement through molecular markers, genomic selection, gene editing, and climate-realistic phenotyping. An integrated understanding of stress signaling and adaptive trait deployment will be essential for developing wheat cultivars with improved resilience and yield stability under future climates.

Triticum↗
PubMed2025

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Humans↗