Aggregation-prone TDP-43 sequesters and drives pathological transitions of free nuclear TDP-43.

Aggregation of the RNA-binding protein, TDP-43, is the unifying hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43-related neurodegeneration involves multiple changes to normal physiological TDP-43, which undergoes nuclear depletion, cytoplasmic mislocalisation, post-translational modification, and aberrant liquid-liquid phase separation, preceding inclusion formation. Along with toxic cytoplasmic aggregation, concurrent depletion and dysfunction of normal nuclear TDP-43 in cells with TDP-43 pathology is likely a key potentiator of neurodegeneration, but is not well understood. To define processes driving TDP-43 dysfunction, we used CRISPR/Cas9-mediated fluorescent tagging to investigate how disease-associated stressors and pathological TDP-43 alter abundance, localisation, self-assembly, aggregation, solubility, and mobility dynamics of normal nuclear TDP-43 over time in live cells. Oxidative stress stimulated liquid-liquid phase separation of endogenous TDP-43 into droplet-like puncta, or spherical shell-like anisosomes. Further, nuclear RNA-binding-ablated or acetylation-mimicking TDP-43 readily sequestered and depleted free normal nuclear TDP-43 into dynamic anisosomes, in which recruited endogenous TDP-43 proteins remained soluble and highly mobile. Large, phosphorylated inclusions formed by nuclear or cytoplasmic aggregation-prone TDP-43 mutants also caused sequestration, but rendered endogenous TDP-43 immobile and insoluble, indicating pathological transition. These findings suggest that RNA-binding deficiency and post-translational modifications including acetylation exacerbate TDP-43 aggregation and dysfunction by driving sequestration, mislocalisation, and depletion of normal nuclear TDP-43 in neurodegenerative diseases.

Identification of reference genes for studies of quantitative gene expression in male and female quail tissues.

In industrial poultry, quail production has gained increasing prominence over the years. It is known that the intensification of genetic studies has contributed greatly to this growth, through techniques, such as analysis of gene expression by PCR, for example. This study aimed to evaluate stability and recommend reference genes for quantitative real-time PCR in different tissues from male and female broiler quails. The stability of 10 housekeeping genes (GAPDH, RPL5, MRPS27, MRPS30, TFRC, HMBS, EEF1, LDHA, B2M, and UBC) by means Bestkeeper, NormFinder, GeNorm softwares with ΔCq method. The tissues analyzed were: heart, thigh muscle, brain, and spleen, considering that they are tissues commonly used in nutrigenomic, immunological, and poultry performance research. As expected, the reference genes tested showed varying stability depending on the tissue evaluated. According to the present study, the most stable housekeeping genes were MRPS30, TFRC, and HMBS in heart; MRPS30, EEF1, and HMBS in thigh muscle; B2M, GAPDH, and UBC in brain; and EEF1, LDHA, and HMBS in spleen. Therefore, it is recommended to be used as reference genes for gene expression studies of male and female quails.

Selection of reference genes for quantitative real-time PCR normalization in European quail tissues.

Coturniculture has been standing out as an industrial poultry activity in several countries around the world because of the several adaptive advantages of quails. Research that considers the analysis of gene expression can enhance this activity. This study aimed to analyze the stability of reference genes (RGs) in different tissues of quails (both males and females) for the recommendation of use in gene expression studies by the quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expression stability of ten RGs (ACTA1, ACTB, B2M, GAPDH, HMBS, SDHA, HPRT1, MRPS27, MRPS30, and RPL5) was analyzed in four tissues (breast muscle, abdominal fat, liver, and intestine), and assessed using the statistical tools geNorm, NormFinder, comparative ΔCq method, and BestKeeper. The HPRT1 gene was the most stable in all quail tissues tested, followed by MRPS27 and MRPS30 in breast muscle, B2M and RPL5 in abdominal fat, HMBS and B2M in the liver, and RPL5 and HMBS in the intestine. These results may help studies using RT-qPCR assays to assess quail tissues from both sexes because they provide data on the most stable genes, which should be tested as candidate RGs for other experimental conditions.

Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum.

Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, we developed a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The HITI strategy was able to facilitate integration of a large DNA fragment, ∼8 kb in length, into the genome of F. oxysporum at the sgRNA cleavage site, and was used to insert a C-terminal 3×sGFP tag to the FoCHS5 gene and a N-terminal mCherry tag to the FoSSO2 gene. The HDRI strategy was used to tag the paralogous gene, FoSSO1, with a C-terminal mCherry marker. FoChs5-3×sGFP localized to conidia, some septa, and fungal tips. A majority of the FoSso1-mCherry was distributed in the conidia, septum, and hyphae that were distal from the fungal tips. While FoSso1-mCherry showed a very weak fluorescent signal at the fungal tips, mCherry-FoSso2 accumulated in the plasma membrane of conidia, germlings, fungal tips, hyphae, and phialides, suggesting FoSSO1 and FoSSO2 are regulated differently during fungal development. These results indicate this EGT system is efficient and can be another molecular tool to resolve the function(s) of proteins and infection strategies of F. oxysporum.

CRISPR interference-guided multiplex repression of endogenous competing pathway genes for redirecting metabolic flux in Escherichia coli.

BACKGROUND: Multiplex control of metabolic pathway genes is essential for maximizing product titers and conversion yields of fuels, chemicals, and pharmaceuticals in metabolic engineering. To achieve this goal, artificial transcriptional regulators, such as clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi), have been developed to specifically repress genes of interest. RESULTS: In this study, we deployed a tunable CRISPRi system for multiplex repression of competing pathway genes and, thus, directed carbon flux toward production of molecules of interest in Escherichia coli. The tunable CRISPRi system with an array of sgRNAs successfully repressed four endogenous genes (pta, frdA, ldhA, and adhE) individually and in double, triple, or quadruple combination that are involved in the formation of byproducts (acetate, succinate, lactate, and ethanol) and the consumption of NADH in E. coli. Single-target CRISPRi effectively reduced the amount of each byproduct and, interestingly, pta repression also decreased ethanol production (41%), whereas ldhA repression increased ethanol production (197%). CRISPRi-mediated multiplex repression of competing pathway genes also resulted in simultaneous reductions of acetate, succinate, lactate, and ethanol production in E. coli. Among 15 conditions repressing byproduct-formation genes, we chose the quadruple-target CRISPRi condition to produce n-butanol in E. coli as a case study. When heterologous n-butanol-pathway enzymes were introduced into E. coli simultaneously repressing the expression of the pta, frdA, ldhA, and adhE genes via CRISPRi, n-butanol yield and productivity increased up to 5.4- and 3.2-fold, respectively. CONCLUSIONS: We demonstrated the tunable CRISPRi system to be a robust platform for multiplex modulation of endogenous gene expression that can be used to enhance biosynthetic pathway productivity, with n-butanol as the test case. CRISPRi applications potentially enable the development of microbial "smart cell" factories capable of producing other industrially valuable products.

Repurposing CRISPR System for Transcriptional Activation.

In recent years, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most popular one for genome editing. When the nuclease domains of Cas9 protein are mutated into deactivated form (dCas9), CRISPR/dCas9 still retains the ability to bind the targeted DNA sequence, but loses the endonuclease cleavage activity. Taking advantage of the characteristics of this engineered nuclease inactive Cas9, the CRISPR/dCas system has been repurposed into versatile RNA-guided, DNA-targeting platforms, such as genome imaging, gene regulation, and epigenetic modification. Specifically, fusion of dCas9 with activation domains allows specific and efficient transcriptional activation on a genome-wide scale among diverse organisms. The purpose of this chapter is to review most important the recently published literature on CRISPR/dCas9-based transcriptional activation systems. Compared with the conventional approaches for enhancement of the expression of specific genes of interest, CRISPR/Cas9-based system has emerged as a promising technology for genome regulation, allowing specificity, convenience, robustness, and scalability for endogenous gene activation.

Correlation between in vitro expansion-related cell stiffening and differentiation potential of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are an attractive cell source for tissue regeneration, given their self-renewal and multilineage potential. However, they are present in only small percentages in human bone marrow, and are generally propagated in vitro prior to downstream use. Previous work has shown that hMSC propagation can lead to alterations in cell behavior and differentiation potency, yet optimization of differentiation based on starting cell elastic modulus is an area still under investigation. To further advance the knowledge in this field, hMSCs were cultured and routinely passaged on tissue-culture polystyrene to investigate the correlation between cell stiffening and differentiation potency during in vitro aging. Local cell elastic modulus was measured at every passage using atomic force microscopy indentation. At each passage, cells were induced to differentiate down myogenic and osteogenic paths. Cells induced to differentiate, as well as undifferentiated cells were assessed for gene and protein expression using quantitative polymerase chain reaction and immunofluorescent staining, respectively, for osteogenic and myogenic markers. Myogenic and osteogenic cell potential are highly reliant on the elastic modulus of the starting cell population (of undifferentiated cells), and this potential appears to peak when the innate cell elastic modulus is close to that of differentiated tissue. However, the latent expression of the same markers in undifferentiated cells also appears to undergo a correlative relationship with cell elastic modulus, indicating some endogenous effects of cell elastic modulus and gene/protein expression. Overall, this study correlates age-related changes with regards to innate cell stiffening and gene/protein expression in commercial hMSCs, providing some guidance as to maintenance and future use of hMSCs in future tissue engineering applications.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation.

BACKGROUND: Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and ß-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. AIM: To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. METHODS: We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0), TATA-binding protein (TBP), GAPDH, actin beta (ACTB), tubulin (TUB), aminolevulinic acid synthase 1 (ALAS1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), eukaryotic translational elongation factor 1 alpha (EF1a), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), and beta-2-microglobulin (B2M)] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. RESULTS: All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. CONCLUSION: For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

Employing synthetic biology to expand antibiotic discovery.

Antimicrobial-resistant (AMR) bacterial pathogens are a continually growing threat as our methods for combating these infections continue to be overcome by the evolution of resistance mechanisms. Recent therapeutic methods have not staved off the concern of AMR infections, so continued research focuses on new ways of identifying small molecules to treat AMR pathogens. While chemical modification of existing antibiotics is possible, there has been rapid development of resistance by pathogens that were initially susceptible to these compounds. Synthetic biology is becoming a key strategy in trying to predict and induce novel, natural antibiotics. Advances in cloning and mutagenesis techniques applied through a synthetic biology lens can help characterize the native regulation of antibiotic biosynthetic gene clusters (BGCs) to identify potential modifications leading to more potent antibiotic activity. Additionally, many cryptic antibiotic BGCs are derived from non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) biosynthetic pathways; complex, clustered genetic sequences that give rise to amino acid-derived natural products. Synthetic biology can be applied to modify and metabolically engineer these enzyme-based systems to promote rapid and sustainable production of natural products and their variants. This review will focus on recent advances related to synthetic biology as applied to genetic pathway characterization and identification of antibiotics from naturally occurring BGCs. Specifically, we will summarize recent efforts to characterize BGCs via general genomic mutagenesis, endogenous gene expression, and heterologous gene expression.

Selection and validation of reference genes for RT-qPCR gene expression studies in Candida viswanathii cultivated under different grown conditions.

The properties presented by Candida viswanathii's lipases turn this specie into a promising producer of potentially applicable lipases in several industrial sectors, such as: food, textiles, in the oleochemical and paper industries, and also in different pharmaceutical applications. However, studies for elucidating growth and developmental processes at the molecular level in this species are still incipient. Performing such kinds of studies often rely on the use of the RT-qPCR, which is a highly sensitivity technique, but whose parameters must be carefully planned for achieving reliable data. Among the crucial parameters required for achieving reliable results through this technique, the use of appropriated and validated reference genes is one the most important, constituting a bottleneck, mainly in species where molecular studies are scarce. Thus, the aim of this study was to determine the best reference genes for RT-qPCR gene expression studies in C. viswanathii grown in culture media containing four different carbon sources (Olive oil, Triolein, Tributyrin, and Glucose). Eleven candidate reference genes (ACT, GPH1, AGL9, RPB2, SAP1, PGK1, TAF10, UBC13, TFC1, UBP6, and FBA1) were analyzed for their expression patterns and stability. Analysis of gene expression stability was performed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms, and validation of the results was performed through analyzing the expression of a lipase gene, CvLIP4. Analyzing the four treatments together, CvACT and CvRPB2 constituted the best reference gene pair. When treatments are analyzed individually, CvRPB2/CvACT, CvFBA1/CvAGL9, CvPGK1/CvAGL9 and CvACT/CvRPB2 were the best reference gene pairs for the culture media containing olive oil, triolein, tributyrin, and glucose as carbon sources, respectively. These results are essential and form the basis for the development of relative gene expression studies in C. viswanathii, since adequate reference genes are crucial for the reliability of RT-qPCR data.

CRISPR-Cas systems target endogenous genes to impact bacterial physiology and alter mammalian immune responses.

CRISPR-Cas systems are an immune defense mechanism that is widespread in archaea and bacteria against invasive phages or foreign genetic elements. In the last decade, CRISPR-Cas systems have been a leading gene-editing tool for agriculture (plant engineering), biotechnology, and human health (e.g., diagnosis and treatment of cancers and genetic diseases), benefitted from unprecedented discoveries of basic bacterial research. However, the functional complexity of CRISPR systems is far beyond the original scope of immune defense. CRISPR-Cas systems are implicated in influencing the expression of physiology and virulence genes and subsequently altering the formation of bacterial biofilm, drug resistance, invasive potency as well as bacterial own physiological characteristics. Moreover, increasing evidence supports that bacterial CRISPR-Cas systems might intriguingly influence mammalian immune responses through targeting endogenous genes, especially those relating to virulence; however, unfortunately, their underlying mechanisms are largely unclear. Nevertheless, the interaction between bacterial CRISPR-Cas systems and eukaryotic cells is complex with numerous mysteries that necessitate further investigation efforts. Here, we summarize the non-canonical functions of CRISPR-Cas that potentially impact bacterial physiology, pathogenicity, antimicrobial resistance, and thereby altering the courses of mammalian immune responses.

Three strategies of transgenic manipulation for crop improvement.

Heterologous expression of exogenous genes, overexpression of endogenous genes, and suppressed expression of undesirable genes are the three strategies of transgenic manipulation for crop improvement. Up to 2020, most (227) of the singular transgenic events (265) of crops approved for commercial release worldwide have been developed by the first strategy. Thirty-eight of them have been transformed by synthetic sequences transcribing antisense or double-stranded RNAs and three by mutated copies for suppressed expression of undesirable genes (the third strategy). By the first and the third strategies, hundreds of transgenic events and thousands of varieties with significant improvement of resistance to herbicides and pesticides, as well as nutritional quality, have been developed and approved for commercial release. Their application has significantly decreased the use of synthetic pesticides and the cost of crop production and increased the yield of crops and the benefits to farmers. However, almost all the events overexpressing endogenous genes remain at the testing stage, except one for fertility restoration and another for pyramiding herbicide tolerance. The novel functions conferred by the heterologously expressing exogenous genes under the control of constitutive promoters are usually absent in the recipient crops themselves or perform in different pathways. However, the endogenous proteins encoded by the overexpressing endogenous genes are regulated in complex networks with functionally redundant and replaceable pathways and are difficult to confer the desirable phenotypes significantly. It is concluded that heterologous expression of exogenous genes and suppressed expression by RNA interference and clustered regularly interspaced short palindromic repeats-cas (CRISPR/Cas) of undesirable genes are superior to the overexpression of endogenous genes for transgenic improvement of crops.

Construction of a human hTERT RPE-1 cell line with inducible Cre for editing of endogenous genes.

The human retinal pigment epithelial RPE-1 cell line immortalized with hTERT retains a stable karyotype with a modal chromosome number of 46 and has been widely used to study physiological events in human cell culture systems. To facilitate inducible knock-out or knock-in experiments in this cell line, we have modified the AAVS1 locus to harbour a DNA fragment encoding ERT2-Cre-ERT2 fusion protein under regulation of a Tet-On expression system. In the generated cell line, active Cre recombinase was induced by simple addition of doxycycline and tamoxifen to the culture medium. As proof of concept, we successfully introduced an oncogenic point mutation to the endogenous KRAS gene locus of this cell line. The cell line will serve as a powerful tool to conduct functional analyses of human genes.

Tailoring Cardiac Synthetic Transcriptional Modulation Towards Precision Medicine.

Molecular and genetic differences between individual cells within tissues underlie cellular heterogeneities defining organ physiology and function in homeostasis as well as in disease states. Transcriptional control of endogenous gene expression has been intensively studied for decades. Thanks to a fast-developing field of single cell genomics, we are facing an unprecedented leap in information available pertaining organ biology offering a comprehensive overview. The single-cell technologies that arose aided in resolving the precise cellular composition of many organ systems in the past years. Importantly, when applied to diseased tissues, the novel approaches have been immensely improving our understanding of the underlying pathophysiology of common human diseases. With this information, precise prediction of regulatory elements controlling gene expression upon perturbations in a given cell type or a specific context will be realistic. Simultaneously, the technological advances in CRISPR-mediated regulation of gene transcription as well as their application in the context of epigenome modulation, have opened up novel avenues for targeted therapy and personalized medicine. Here, we discuss the fast-paced advancements during the recent years and the applications thereof in the context of cardiac biology and common cardiac disease. The combination of single cell technologies and the deep knowledge of fundamental biology of the diseased heart together with the CRISPR-mediated modulation of gene regulatory networks will be instrumental in tailoring the right strategies for personalized and precision medicine in the near future. In this review, we provide a brief overview of how single cell transcriptomics has advanced our knowledge and paved the way for emerging CRISPR/Cas9-technologies in clinical applications in cardiac biomedicine.

A Direct Comparison of Thyroid Hormone Receptor Protein Levels in Mice Provides Unexpected Insights into Thyroid Hormone Action.

Background: Thyroid hormone (TH) action is mediated by three major thyroid hormone receptor (THR) isoforms α1, ß1, and ß2 (THRA1, THRB1, and THRB2). These THRs and a fourth major but non-TH binding isoform, THRA2, are encoded by two genes Thra and Thrb. Reliable antibodies against all THR isoforms are not available, and THR isoform protein levels in mammalian tissues are often inferred from messenger RNA (mRNA) levels. Methods: We generated knock-in mouse models expressing endogenously and identically 2X hemagglutenin epitope (HA)-tagged THRs (THRA1/2, THRB1, and THRB2), which could then be detected by commercially available anti-HA antibodies. Using nuclear enrichment, immunoprecipitation, and Western blotting, we determined relative THR protein expression in 16 mouse organs. Results: In all peripheral organs tested except the liver, the predominant THR isoform was THRA1. Surprisingly, in metabolically active organs such as fat and muscle, THRB1 protein levels were up to 10 times lower than that of THRA1, while their mRNA levels appeared similar. In contrast to peripheral organs, the central nervous system (CNS) had a unique pattern with relatively low levels of both THRB1 and THRA1, and high levels of THRA2 expression. As expected, THRB2 was highly expressed in the pituitary, but a previously unknown sex-specific difference in THRB2 expression was found (female mice having higher pituitary expression than male mice). Higher THRB2 expression appears to make the central axis more sensitive to TH as both serum thyrotropin and Tshb mRNA levels were lower in female mice. Conclusions: Direct comparison of THR protein abundance in different organs using endogenously tagged HA-THR mouse lines shows that expression of THR isoforms is regulated at transcriptional and posttranscriptional levels, and in organ-specific manner. The prevalence of THRA1 and low abundance of THRB1 in majority of peripheral tissues suggest that peripheral actions of these isoforms should be revisited. A unique pattern of high THRA2 in CNS warrants further exploration of this non-TH binding isoform in brain development. Finally, THRB2, in addition to cell-specific control, is also regulated in a sex-specific manner, which may change the hypothalamus-pituitary-thyroid axis set point and perhaps metabolism in males and females.

Endogenous p21-Dependent Transgene Control for CHO Cell Engineering.

Numerous engineering efforts have been made in Chinese hamster ovary (CHO) cells for high level production of therapeutic proteins. However, the dynamic regulation of transgene expression is limited in current systems. Here, we investigated the effective regulation of transgene expression in CHO cells via targeted integration-based endogenous gene tagging with engineering target genes. Targeted integration of EGFP-human Bcl-2 into the p21 locus effectively reduced the apoptosis, compared with random populations in which Bcl-2 expression was driven by cytomegalovirus (CMV) promoter. Endogenous p21 and EGFP-human Bcl-2 displayed similar expression dynamics in batch cultures, and the antiapoptotic effect altered the expression pattern of endogenous p21 showing the mutual influences between expression of p21 and Bcl-2. We further demonstrated the inducible transgene expression by adding low concentrations of hydroxyurea. The present engineering strategy will provide a valuable CHO cell engineering tool that can be used to control dynamic transgene expression in accordance with cellular states.

CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi.

Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca2+ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca2+ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP3R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.

Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi.

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have generated homogeneous populations of genetically modified parasites in 4-5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the gene. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines generated by CRISPR/Cas9, and C-terminal tagging of endogenous genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.

Endogenous gene selection for relative quantification PCR and IL6 transcript levels in the PBMC's of severe and non-severe dengue cases.

OBJECTIVES: Dengue viral infection ranges from dengue fever to dengue haemorrhagic fever and lethal dengue shock syndrome. Currently no means are available to monitor the progression of disease. Real time PCR based gene expression analyses are used to find potential molecular markers for effective prediction of dengue clinical outcome. The accuracy of qPCR analysis is strongly dependent on transcript normalization using stably expressed endogenous genes, which if selected imprecisely can lead to misinterpreted results. We aimed to determine the best fit for endogenous gene among six genes namely COX, ACTB, GAPDH, HMBS, HPRT and B2M for dengue viral infection cases. Gene stability was inferred from qPCR data by normalizing with two algorithms geNorm and Normfinder and the rankings generated were validated by gene expression analysis against target gene IL-6. RESULTS: Both the algorithms showed ACTB, HPRT, GAPDH as most stable genes. Normalizing with the stable genes revealed a significant fold change (p < .05) in IL-6 levels of .32, .52, .69, and .62 in non-dengue febrile illness, non severe, severe and All Dengue groups respectively compared to healthy controls. based on our study, we suggest ACTB with HPRT/GAPDH combination for normalization in qPCR for precise quantification of transcripts in dengue infected studies.