Selectable Marker Gene Research Articles

Overview on Current Selectable Marker Systems and Novel Marker Free Approaches in Fruit Tree Genetic Engineering

Selectable marker genes are useful for recognizing which cells have integrated specific sequences in their genome after genetic transformation processes. They are especially important for fruit trees genetic transformation to individuate putatively genetically modified events, because most of the protocols used to genetic engineer these species are often unsuccessful or with low efficiency. Traditional selectable marker genes, mainly of bacterial origin, confer antibiotics/herbicides-resistance or metabolic advantages to transformed cells. Genes that allow the visual recognition of engineered tissues without using any selective agent, such as morphogenic regulators and reporter genes, are also used as selection tools to in vitro identify genetically modified regenerated lines. As final step, genetic engineered plants should be tested in field conditions, where selectable marker genes are no longer necessary, and strongly unpopular especially for the commercial development of the new products. Thus, different approaches, mainly based on the use of site-specific recombinases and/or editing nucleases, are being now used to recover marker-free fruit crops. This review describes and comments the most used and suitable selection tools of interest, particularly for fruit tree genetic engineering. Lastly, a spotlight highlights the biosafety aspects related to the use of selectable marker genes exploited for fruit species genetic engineering.

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production.

Chlamydomonas incerta, a genetically close relative of the model green alga Chlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta's capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii, C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii. For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta's potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

Use of cardiac cell cultures from salmonids to measure the cardiotoxic effect of environmental pollutants.

Environmental stressors such as micro- and nanosized plastic particles (MNPs) or crude oil have a detrimental effect on aquatic animals; however, the impact upon the cardiovascular system of fish remains relatively under-researched. This study presents a novel approach for investigating the effect of crude oil and MNPs on the cardiac system of fish. We used salmonid larvae and cardiac cell cultures derived from hearts of salmonid fish and exposed them to environmental stressors. Following exposure to plastic particles or crude oil, the larvae exhibited some variation in contraction rate. In contrast, significant alterations in the contraction rate were observed in all cardiac cell cultures. The greatest differences between the control and treatment groups were observed in cardiac cell cultures derived from older brown trout. Following 7 days of exposure to MNPs or crude oil in Atlantic salmon larval hearts or cardiac cell cultures, there were only minor responses noted in mRNA expression of the selected marker genes. These findings show the use of a novel invitro technique contributing to the existing body of knowledge on the impact of MNPs and crude oil on the cardiovascular system of salmonids and the associated risk.

A user-friendly CRISPR/Cas9 system for mutagenesis of Neurosporacrassa

As a widely used eukaryotic model organism, Neurosporacrassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N.crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamineamidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N.crassa compared to traditional methods.

A cyclical marker system enables indefinite series of oligonucleotide-directed gene editing in Chlamydomonas reinhardtii.

CRISPR/Cas9 gene editing in the model green alga Chlamydomonas reinhardtii relies on the use of selective marker genes to enrich for non-selectable target mutations. This becomes challenging when many sequential modifications are required in a single cell line, as useful markers are limited. Here, we demonstrate a cyclical selection process which only requires a single marker gene to identify an almost infinite sequential series of CRISPR-based target gene modifications. We used the NIA1 (Nit1, NR; nitrate reductase) gene as the selectable marker in this study. In the forward stage of the cycle, a stop codon was engineered into the NIA1 gene at the CRISPR target location. Cells retaining the wild-type NIA1 gene were killed by chlorate, while NIA1 knockout mutants survived. In the reverse phase of the cycle, the stop codon engineered into the NIA1 gene during the forward phase was edited back to the wild-type sequence. Using nitrate as the sole nitrogen source, only the reverted wild-type cells survived. By using CRISPR to specifically deactivate and reactivate the NIA1 gene, a marker system was established that flipped back and forth between chlorate- and auxotrophic (nitrate)-based selection. This provided a scarless cyclical marker system that enabled an indefinite series of CRISPR edits in other, non-selectable genes. We demonstrate that this 'Sequential CRISPR via Recycling Endogenous Auxotrophic Markers (SCREAM)' technology enables an essentially limitless series of genetic modifications to be introduced into a single cell lineage of C. reinhardtii in a fast and efficient manner to complete complex genetic engineering.

Development of a thermostable Cre/lox-based gene disruption system and in vivo manipulations of the megaplasmid pTT27 in Thermus thermophilus HB27

We previously reported the development of a Cre/lox-based gene disruption system for multiple markerless gene disruption in Thermus thermophilus; however, it was a time-consuming method because it functioned at 50 °C, the minimum growth temperature of T. thermophilus HB27. In the present study, we improved this system by introducing random mutations into the cre-expressing plasmid, pSH-Cre. One of the resulting mutant plasmids, pSH-CreFM allowed us to remove selection marker genes by Cre-mediated recombination at temperatures up to 70 °C. By using the thermostable Cre/lox system with pSH-CreFM, we successfully constructed two valuable pTT27 megaplasmid mutant strains, a plasmid-free strain and β-galactosidase gene deletion strain, which were produced by different methods. The thermostable Cre/lox system improved the time-consuming nature of the original Cre/lox system, but it was not suitable for multiple markerless gene disruption in T. thermophilus because of its highly efficient induction of Cre-mediated recombination even at 70 °C. However, in vivo megaplasmid manipulations performed at 65 °C were faster and easier than with the original Cre/lox system. Collectively, these results indicate that this system is a powerful tool for engineering T. thermophilus megaplasmids.

Genoarchitectural Definition of the Adult Mouse Mesocortical Ring: A Contribution to Cortical Ring Theory.

Data mining was performed at the databases of the Allen Institute for Brain Science (RRID:SCR_017001) searching for genes expressed selectively throughout the adult mouse mesocortex (transitional cortex ring predicted within the concentric ring theory of mammalian cortical structure, in contrast with central isocortex [ICx] and peripheral allocortex). We aimed to explore a shared molecular profile selective of all or most mesocortex areas. This approach checks and corroborates the precision of other previous definitory criteria, such as poor myelination and high kainate receptor level. Another aim was to examine which cortical areas properly belong to mesocortex. A total of 34 positive adult selective marker genes of mesocortex were identified, jointly with 12 negative selective markers, making a total of 46 markers. All of them identify the same set of cortical areas surrounding the molecularly different ICx as well as excluding adjacent allocortex. Four representative mesocortex markers-Crym, Lypd1, Cdh13, and Smoc2-are amply illustrated, jointly with complementary material including myelin basic protein, to check myelination, and Rorb, to check layer 4 presence. The retrosplenial (ReSp) area, long held to be mesocortical, does not share any of the 46 markers of mesocortex and instead expresses Nr4a2 and Tshz2, selective parahippocampal allocortex markers. Moreover, it is not hypomyelinic and lacks a Rorb-positive layer 4, aspects generally present in mesocortex. Exclusion of the ReSp area from the mesocortex ring reveals the latter to be closed at this locus instead by two adjacent areas previously thought to be associative visual ICx (reidentified here molecularly as postsplenial and parasplenial mesocortex areas). The concepts of ICx, mesocortex, and parahippocampal allocortex are thus subtly modified by substantial molecular evidence.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

#2485 CellMatchR—RNA-seq based matching of kidney cell lines to kidney reference cell types

Abstract Background and Aims Cultured kidney cell lines are broadly used to model the physiology and pathophysiology of the kidney. Due to immortalization, passaging and culture conditions, a cell line's gene expression profile might differ greatly from the primary cells it was originally derived from. This might be further enhanced by experimental treatments with compounds or genetic modifications. It is therefore a relevant question if a given cell culture system is still the appropriate model for a certain disease or scientific investigations. Reasoning that RNA sequencing (RNA-seq) based transcriptomes are generated as part of many experimental setups and hence may be used to address this question, we developed and tested an RNA-seq based approach, CellMatchR. This approach matches kidney cell lines, primary cells and even tissue specimen to known kidney reference cell types to determine which primary kidney cell type is the most similar and to what degree global gene expression or the expression of selected marker genes differs. Method CellMatchR uses published murine and human kidney single cell- and tubule-level transcriptomic datasets from healthy mice and human donors as references. Single cell datasets were further processed to pseudobulk references for each of the contained cell types. Then RNA-seq data from cell lines or tissues of interest (test data) was compared to the reference (pseudo)bulk data with Spearman correlations of gene counts-per-million values (CPM) or Euclidean distance using log-transformed CPM values. Both approaches were systematically tested for various combinations of test and reference datasets across different species and using global gene expression (i.e. all genes measured in reference and test datasets) and a set of 315 manually curated, kidney cell type marker genes. Results We sequentially used different kidney tissue types, primary cells and cell lines as positive controls and matched them to our reference datasets. Spearman correlations of gene expression rankings showed the highest correlation coefficients with biologically correct reference cell types (examples in Fig. 1A-C). We found Spearman correlations to be superior to Euclidean distances as method of comparison. Analyses based on global gene expression compared to our curated set of 315 kidney cell type marker genes yielded similar results. Notably, correlation coefficients were higher and showed less variation between reference cell types when using the global gene set. Matchings across species, i.e. using murine test and human reference data or vice versa still yielded mostly correct results. However, correlation coefficients were generally lower (i.e. rho = 0.9 vs. 0.6) and varied more across reference datasets if comparisons were performed across species. Using published RNA-seq data for different cell lines of the proximal tubule (e.g. human HK-2 and opossum OKH cells) we observed low similarities with proximal tubule cells (Fig. 1D), whereas two tested cell lines of the collecting duct (mIMCD-3 and mpkCCD cells) plausibly showed the highest similarity to medullary cell types like collecting duct and Loop of Henle cells. The most similar reference cell types did not change in mIMCD-3 cells when kept in 2-dimensional vs. 3-dimensional culture conditions, and in mpkCCD cells when the osmolality of the culture medium was changed from 300 to 600 mosmol/kg. Also, a Pkd1 knockout in mIMCD-3 cells did not change the most similar reference cell type in our analyses (Fig. 1E and F). Conclusion Our CellMatchR approach uses publicly available kidney single cell and bulk RNA-seq datasets and combines these with simple, computationally fast yet effective statistical methods to determine similarities of kidney cell lines and tissue samples to reference cell types of interest. It can easily be implemented by trained users or integrated into online resources for usage with a visual interface. It relies on RNA-seq data from the cell lines of interest and hence presents a feasible complementary method to check the general similarity of cell culture models to kidney cell types and assess their stability across experimental conditions.

Tandem gene duplication selected by activation of horizontally transferred gene in bacteria

Horizontal gene transfer occurs frequently in bacteria, but the mechanism driving activation and optimization of the expression of horizontally transferred genes (HTGs) in new recipient strains is not clear. Our previous study found that spontaneous tandem DNA duplication resulted in rapid activation of HTGs. Here, we took advantage of this finding to develop a novel technique for tandem gene duplication, named tandem gene duplication selected by activation of horizontally transferred gene in bacteria (TDAH), in which tandem duplication was selected by the activation of horizontally transferred selectable marker gene. TDAH construction does not contain any reported functional elements based on homologous or site-specific recombination and DNA amplification. TDAH only contains an essential selectable marker for copy number selection and 9-bp-microhomology border sequences for precise illegitimate recombination. One transformation and 3 days were enough to produce a high-copy strain, so its procedure is simple and fast. Without subsequent knockout of the endogenous recombination system, TDAH could also generate the relatively stable high-copy tandem duplication for plasmid-carried and genome-integrated DNA. TDAH also showed an excellent capacity for increase gene expression and worked well in different industrial bacteria. We also applied TDAH to select the optimal high copy number of ribA for vitamin B2 production in E. coli; the yield was improved by 3.5 times and remained stable even after 12 subcultures. TDAH is a useful tool for recombinant protein production and expression optimization of biosynthetic pathways.Key points• We develop a novel and efficient technique (TDAH) for tandem gene duplication in bacterium. TDAH is based on the mechanism of HTG rapid activation. TDAH does not contain any reported functional elements based on homologous recombination and DNA amplification. TDAH only contains an essential selectable marker for copy number selection, so its construction and procedure are very simple and fast.• TDAH is the first reported selected and stable tandem-gene-duplication technique in which the selected high-copy plasmid-carried and genome-integrated DNA could remain stable without the subsequent knockout of recombination system.• TDAH showed an excellent capacity for regulating gene expression and worked well in different industrial bacteria, indicating it is a useful tool for recombinant protein production and expression optimization of biosynthetic pathways.• TDAH was applied to select the optimal high copy number of ribA for vitamin B2 production in E. coli; the yield was improved by 3.5-fold and remained stable even after 12 subcultures.

Segregation analysis and floral phenotype of T2 transgenic yellow cosmos (Cosmos sulphureus Cav.) carrying neomycin phosphotransferase II gene in second generation

Abstract. Muchyiddin MGAA, Sawitri WD, Irsyadi MB, Swandari T, Purwantoro A. 2024. Segregation analysis and floral phenotype of T2 transgenic yellow cosmos (Cosmos sulphureus Cav.) carrying neomycin phosphotransferase II gene in second generation. Biodiversitas 25: 1711-1717. The seed of transgenic yellow cosmos (Cosmos sulphureus Cav.) lines harbouring neomycin phosphotranferase II (nptII) gene have been obtained from our previous studies. The nptII selectable marker gene is commonly used for developing a method to estimate the number of targeted gene integrated into the genome. In order to produce successful clones, it is important to ensure that transgenes are inherited sexually as the dominant trait in T2 progeny. This study aimed to obtain the segregation patterns of nptII gene in two populations derived from open- and self-pollinated and evaluated floral phenotypic of T2 transgenic yellow cosmos. The molecular analysis of transgenic yellow cosmos was conducted by PCR and the obtained data was analyzed using a Chi-Square test. The results showed that transgenes were inherited in 1:1 for transgenic and wild-type in both self- and open-pollinated populations. Therefore, it indicated that T2 transgenic plants were still unstable and evolved continuously to segregate independently in the over generation. Surprisingly, the morphological changes were detected in transgenic plants. The mixture of ligulate and tubular types in ray floret was found in some of the flower types in T2 yellow cosmos. In this case, nptII gene may diverge in apparently random genome insertion. This insertion would induce morphological changes in the ray floret type of flower since recombination estimates varied among transformation events.

Enrichment using a selectable CD34t marker enhances TCR/CAR transduced T cell functionality and transgene expression in clinically relevant receptors

Abstract Gene-modified TCR transduced T cells and CAR T cells are used clinically and display a range of efficacy across multiple cancers. Many therapies have successfully controlled patient’s tumors; however, some have shown off-target adverse events which are harmful to patients’ overall outcomes. T cell-based therapies have high variation in patient outcome due to multiple factors, such as transduction efficiency and T cell phenotypes. To reduce product variability, our lab developed a selectable marker gene for use in cellular constructs. This selectable marker is a truncated, non-signaling CD34 molecule (CD34t), which can be used to remove the non-transduced T cells that function as bystanders. Our clinical trials have used two TCRs (TIL 1383I and HERV-E) and one CAR (CD19 CAR). Following transduction with these CD34t constructs, we determined transduction efficiency by expression of CD34t. CD34t expression correlates with transgene, allowing us to select high CD34t expressors, and thus high transgene expressors. The selected population produces higher levels of inflammatory cytokines and CD107a expression compared to the nonselected. This improved functionality is likely due to the selected population having higher levels of transgene across the population, whereas the nonselected has a broader range of transgene expressions with fewer expressors total. This selectable marker contributes to a more functional product, which may in turn improve patient response clinically.

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection

BackgroundMost recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors.ResultsIn micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct.ConclusionsFor the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

The Acute, Short-, and Long-Term Effects of Endurance Exercise on Skeletal Muscle Transcriptome Profiles.

A better understanding of the cellular and molecular mechanisms that are involved in skeletal muscle adaptation to exercise is fundamentally important to take full advantage of the enormous benefits that exercise training offers in disease prevention and therapy. The aim of this study was to elucidate the transcriptional signatures that distinguish the endurance-trained and untrained muscles in young adult males (24 ± 3.5 years). We characterized baseline differences as well as acute exercise-induced transcriptome responses in vastus lateralis biopsy specimens of endurance-trained athletes (ET; n = 8; VO2max, 67.2 ± 8.9 mL/min/kg) and sedentary healthy volunteers (SED; n = 8; VO2max, 40.3 ± 7.6 mL/min/kg) using microarray technology. A second cohort of SED volunteers (SED-T; n = 10) followed an 8-week endurance training program to assess expression changes of selected marker genes in the course of skeletal muscle adaptation. We deciphered differential baseline signatures that reflected major differences in the oxidative and metabolic capacity of the endurance-trained and untrained muscles. SED-T individuals in the training group displayed an up-regulation of nodal regulators of oxidative adaptation after 3 weeks of training and a significant shift toward the ET signature after 8 weeks. Transcriptome changes provoked by 1 h of intense cycling exercise only poorly overlapped with the genes that constituted the differential baseline signature of ETs and SEDs. Overall, acute exercise-induced transcriptional responses were connected to pathways of contractile, oxidative, and inflammatory stress and revealed a complex and highly regulated framework of interwoven signaling cascades to cope with exercise-provoked homeostatic challenges. While temporal transcriptional programs that were activated in SEDs and ETs were quite similar, the quantitative divergence in the acute response transcriptomes implicated divergent kinetics of gene induction and repression following an acute bout of exercise. Together, our results provide an extensive examination of the transcriptional framework that underlies skeletal muscle plasticity.

A comparison of marker gene selection methods for single-cell RNA sequencing data

BackgroundThe development of single-cell RNA sequencing (scRNA-seq) has enabled scientists to catalog and probe the transcriptional heterogeneity of individual cells in unprecedented detail. A common step in the analysis of scRNA-seq data is the selection of so-called marker genes, most commonly to enable annotation of the biological cell types present in the sample. In this paper, we benchmark 59 computational methods for selecting marker genes in scRNA-seq data.ResultsWe compare the performance of the methods using 14 real scRNA-seq datasets and over 170 additional simulated datasets. Methods are compared on their ability to recover simulated and expert-annotated marker genes, the predictive performance and characteristics of the gene sets they select, their memory usage and speed, and their implementation quality. In addition, various case studies are used to scrutinize the most commonly used methods, highlighting issues and inconsistencies.ConclusionsOverall, we present a comprehensive evaluation of methods for selecting marker genes in scRNA-seq data. Our results highlight the efficacy of simple methods, especially the Wilcoxon rank-sum test, Student’s t-test, and logistic regression.

Increasing biosensor-based cell selection pressure improves microbial biosynthesis of 4-hydroxybenzoate

Biosensors are powerful tools for improving microbial biosynthesis. For example, biosensors can be used to establish feedback control mechanisms to inhibit the growth of low performing cells in a microbial population. In this study, a previously developed biosensor-assisted cell selection system was further engineered to increase the cell selection pressure for improving the bioproduction of 4-hydroxybenzoate (4HB) in E. coli. First, additional sensor protein PobR binding sites were introduced to reduce the binding of PobR controlling the expression of the selection marker gene (antibiotic tetracycline resistance tetA), which stimulated the engineered cells to produce more 4HB to compensate for the tetA expression reduction due to the loss of the PobR binding. Second, the degradation of the PobR sensor protein was promoted using an E. coli ssrA-SspB system to press the cells to make more 4HB. Third, the dosage of antibiotic tetracycline controlling the selection strengths was dynamically increased during the cultivation to align the selection pressure elevation with the cell growth. Our findings show these strategies all effectively improved the 4HB biosynthesis in E. coli and thus demonstrate their potential of wider applications in microbial biosynthesis.

Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

Divergent effects of the antiretroviral drugs, dolutegravir, tenofovir alafenamide, and tenofovir disoproxil fumarate, on human adipocyte function

Combined antiretroviral therapy (cART) has been associated with increased body weight accompanied by metabolic alterations in people living with human immunodeficiency virus (PLWH). To gain insight into the combined effects of cART components on adipocyte dysfunction, we assessed whether and how treatment of human adipocytes with dolutegravir (DTG) and the nucleotide-analog reverse-transcriptase inhibitors (NRTIs), tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF), alone and in combination, altered biological processes related to adipose tissue dysfunction. DTG, TAF, and TDF were applied to human Simpson-Golabi-Behmel syndrome (SGBS) adipose cells during differentiation (day 10) and ensuing differentiation (day 14). Expression of selected marker genes was determined by qPCR, the release of adipokines and inflammatory cytokines to the culture media was assessed, and cell respiration was measured. Adipogenesis was not altered by the combined treatment of human adipocytes. However, DTG at the highest dose repressed adipogenesis marker genes expression, and TAF and TDF appeared to mitigate this effect. DTG repressed the expression of adiponectin and the release of adiponectin and leptin in differentiating adipocytes, and these effects were mantained in combination with TAF and TDF. DTG plus TAF or TDF on human adipocytes enhanced inflammation and stress and increased the release of proinflammatory cytokines to the culture media. Together, our results show that combined therapy with these drugs can alter inflammation, cellular stress, and fibrosis in human adipocytes. These findings may improve our understanding and management of the effects of cART on body adiposity and metabolic dysregulation in PLWH.

A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas.

Gene knockout is a widely used technique for engineering bacterial genomes, investigating the roles of genes in metabolism, and conferring biological characteristics. Herein, we developed a rapid, efficient, and simple method for the knockout of long gene cassettes in Pseudomonas spp., based on a traditional allelic exchange strategy. The upstream and downstream sequences of the target gene cluster to be deleted were amplified using primers with 5'-end sequences identical to the multiple cloning sites of a suicide plasmid (mutant allele insert vector). The sequences were then fused with the linearized suicide plasmid in one step via seamless cloning. The resulting allelic exchange vector (recombinant plasmid) was introduced from the donor strain (Escherichia coli SM 10) into recipient cells (Pseudomonas putida, P. composti, and P. khazarica) via conjugation. Single-crossover merodiploids (integrates the vector into host chromosome by homologous recombination) were screened based on antibiotic resistance conferred by the plasmid, and double-crossover haploids (deleting the target gene clusters and inserted alien plasmid backbone) were selected using sucrose-mediated counterselection. Unlike other approaches, the method described herein introduces no selective marker genes into the genomes of the knockout mutants. Using our method, we successfully deleted polysaccharide-encoding gene clusters in P. putida, P. composti, and P. khazarica and generated four mutants with single-gene cassette deletions up to 18 kbp and one mutant with double-gene cassette deletion of approximately 34 kbp. Collectively, our results indicate that this method is ideal for the deletion of long genetic sequences, yielding seamless mutants of various Pseudomonas spp.

Identification of novel associations of candidate marker genes with resistance to onion-fusarium basal rot interaction pathosystem

We evaluated the selected populations of eighteen open-pollinated short-day onion genotypes for FBR-Fusarium basal rot (Fusarium oxysporum f. sp. cepae-FOC) susceptibility; population genetic variation via ISSR marker; and transcriptome analysis using qRT-PCR with six novel selected marker genes: R1, R5, RGA29, lectin, LOX, and Osmotin, at tree time post inoculation (wpi). Screening for resistance showed the average severity between 4.7 and 88.9%; of which, the lowest one was in ‘Saba’ and ‘Saba – HS (6.7 and 4.7%, respectively). ISSR analysis recorded 226 amplified bands, of which 160 bands showed polymorphism, of which ISSR1 and ISSR10 primers showed the best performance. We also found that following inoculation with FOC could regulate defense-related marker genes; R1, PR5, Lectin, LOX, Osmotin, and RGA29 in resistant onion “Saba” and ‘Saba’-HS in comparison to susceptible and controls, non-inoculated ones ranging from 1.23 to 6.99 -fold significantly. Surprisingly, marker genes; Lectin, LOX, and Osmotin were also expressed to FOC simultaneous, though basically are resistance to other biotic and abiotic stress: Lectin to Rhizoctonia solani, Aphid, and major sap-sucking pests; LOX to root-knot and cyst nematode, Heterodera glycines; Osmotin to drought stredd, and oxidative burst in plants. This indicates the double, and or multiple roles of our selected marker genes covering two or more functions at a time. The findings introduce newly resistant onion genotypes, and also can be used in management programs to reduce damages caused by FOC disease. Cumulatively, the transcriptome-data provide novel-insights into the response of onions for improving onion-breeding to FOC.