Paul Berg and the origins of recombinant DNA.

In fall 1972, Paul Berg's laboratory published articles in PNAS describing two methods for constructing recombinant DNAs in vitro. He received half of the 1980 Nobel Prize in Chemistry for this landmark accomplishment. Here, we describe how this discovery came about, revolutionizing both biological research and the pharmaceutical industry.

Genetically engineering of Saccharomyces cerevisiae for enhanced oral delivery vaccine vehicle.

As a series of our previous studies reported, recombinant yeast can be the oral vaccines to deliver designed protein and DNA, as well as functional shRNA, into dendritic cells (DCs) in mice for specific immune regulation. Here, we report the further optimization of oral yeast-based vaccine from two aspects (yeast characteristics and recombinant DNA constitution) to improve the effect of immune regulation. After screening four genes in negative regulation of glucan synthesis in yeast (MNN9, GUP1, PBS2 and EXG1), this research combined HDR-based genome editing technology with Cre-loxP technology to acquire 15 gene-knockout strains without drug resistance-gene to exclude biosafety risks; afterward, oral feeding experiments were performed on the mice using 15 oral recombinant yeast-based vaccines constructed by the gene-knockout strains harboring pCMV-MSTN plasmid to screen the target strain with more effective inducing mstn-specific antibody which in turn increasing weight gain effect. And subsequently based on the selected gene-knockout strain, the recombinant DNA in the oral recombinant yeast-based vaccine is optimized via a combination of protein fusion expression (OVA-MSTN) and interfering RNA technology (shRNA-IL21), comparison in terms of both weight gain effect and antibody titer revealed that the selected gene-knockout strain (GUP1ΔEXG1Δ) combined with specific recombinant DNA (pCMV-OVA-MSTN-shIL2) had a better effect of the vaccine. This study provides a useful reference to the subsequent construction of a more efficient oral recombinant yeast-based vaccine in the food and pharmaceutical industry.

Genetic constraints in genes exhibiting splicing plasticity in facultative diapause.

Phenotypic plasticity is produced and maintained by processes regulating the transcriptome. While differential gene expression is among the most important of these processes, relatively little is known about other sources of transcriptional variation. Previous work suggests that alternative splicing plays an extensive and functionally unique role in transcriptional plasticity, though plastically spliced genes may be more constrained than the remainder of expressed genes. In this study, we explore the relationship between expression and splicing plasticity, along with the genetic diversity in those genes, in an ecologically consequential polyphenism: facultative diapause. Using 96 samples spread over two tissues and 10 timepoints, we compare the extent of differential splicing and expression between diapausing and direct developing pupae of the butterfly Pieris napi. Splicing differs strongly between diapausing and direct developing trajectories but alters a smaller and functionally unique set of genes compared to differential expression. We further test the hypothesis that among these expressed loci, plastically spliced genes are likely to experience the strongest purifying selection to maintain seasonally plastic phenotypes. Genes with unique transcriptional changes through diapause consistently had the lowest nucleotide diversity, and this effect was consistently stronger among genes that were differentially spliced compared to those with just differential expression through diapause. Further, the strength of negative selection was higher in the population expressing diapause every generation. Our results suggest that maintenance of the molecular mechanisms involved in diapause progression, including post-transcriptional modifications, are highly conserved and likely to experience genetic constraints, especially in northern populations of P. napi.

Investigating the biosynthesis and metabolism of 11ß-hydroxyandrostenedione.

The "rediscovery" 11ß-hydroxyandrostenedione (11OHA4) placed the spotlight on this unique adrenal-derived hormone with researchers and clinicians once again focusing on the steroid's presence in endocrine pathology. Little was known about the steroid other than its chemical characterisation and that a mitochondrial cytochrome P450 enzyme catalysed the 11ß-hydroxylation of 11OHA4. The fact that neither the biosynthesis nor metabolism of 11OHA4 had been fully characterised presented an ideal opportunity to investigate the metabolic pathways. In addition, methodologies and analytical tools have improved vastly since 11OHA4 was first identified in the 1950s. Cell models, recombinant DNA technology and steroid quantification using liquid chromatography mass spectrometry have greatly facilitated investigations in the field of steroidogenesis. Evident from the structure is that 11OHA4 can be metabolised by hydroxysteroid dehydrogenases and reductases acting on the C4/C5 double bond and on functional moieties at specific carbons on the cyclopentane-perhydro-phenanthrene backbone of the steroid. In this chapter, the biosynthesis and metabolism of 11OHA4 is followed using two strategies that complement each another; (i) human cell models either transiently transfected with recombinant DNA or expressing endogenous steroidogenic enzymes and (ii) steroid identification and quantification using high resolution mass spectrometry. These methodologies have proven invaluable in the determination of 11OHA4's metabolic route. Both strategies are presented with the focus on the accurate identification and quantification of steroids using UHPLC-MS/MS and UPC2-MS/MS. The protocols described in this chapter lay a sound foundation which can aid the researcher and be adapted and implement in future studies.

Poly-Histidine-Tagged Protein Purification Using Immobilized Metal Affinity Chromatography (IMAC).

His-tagging is the most widespread and versatile strategy used to purify recombinant proteins for biochemical and structural studies. Recombinant DNA methods are first used to engineer the addition of a short tract of poly-histidine tag (His-tag) to the N-terminus or C-terminus of a target protein. The His-tag is then exploited to enable purification of the "tagged" protein by immobilized metal affinity chromatography (IMAC). In this chapter, we describe efficient procedures for the isolation of highly purified His-tagged target proteins from an Escherichia coli host using IMAC in a bind-wash-elute strategy that can be performed under both native and denaturing conditions.

Physical characteristics and stability profile of recombinant plasmid DNA within a film matrix.

Plasmids are essential source material for production of biological drugs, vaccines and vectors for gene therapy. They are commonly formulated as frozen solutions. Considering the cost associated with maintenance of cold chain conditions during storage and transport, there is a significant need for alternative methods for stabilization of plasmids at ambient temperature. The objective of these studies was to identify a film-based formulation that preserved transfection efficiency of plasmids at 25 °C. A model plasmid, pAAVlacZ, was used for these studies. Transfection efficiency and agarose gel electrophoresis were utilized to assess bioactivity and changes in physical conformation of plasmid during storage. An amino acid, capable of sustaining a positive charge while supporting an alkaline environment within the film matrix, preserved transfection efficiency for 9 months at 25 °C. Addition of sugar and a plasticizer to the formulation preserved the plasmid in an amorphous state and improved handling properties of the film. The manner in which excipients were incorporated into bulk formulations and environmental humidity in which films were stored significantly impacted transfection efficiency of plasmid in the rehydrated solution. Taken together, these results suggest that plasmids can be stored for extended periods of time without refrigeration within a film matrix.

Differential splicing of neuronal genes in a Trem2*R47H mouse model mimics alterations associated with Alzheimer's disease.

BACKGROUND: Molecular characterization of late-onset Alzheimer's disease (LOAD), the leading cause of age-related dementia, has revealed transcripts, proteins, and pathway alterations associated with disease. Assessing these postmortem signatures of LOAD in experimental model systems can further elucidate their relevance to disease origins and progression. Model organisms engineered with human genetic factors further link these signatures to disease-associated variants, especially when studies are designed to leverage homology across species. Here we assess differential gene splicing patterns in aging mouse models carrying humanized APOE4 and/or the Trem2*R47H variant on a C57BL/6J background. We performed a differential expression of gene (DEG) and differential splicing analyses on whole brain transcriptomes at multiple ages. To better understand the difference between differentially expressed and differentially spliced genes, we evaluated enrichment of KEGG pathways and cell-type specific gene signatures of the adult brain from each alteration type. To determine LOAD relevance, we compared differential splicing results from mouse models with multiple human AD splicing studies. RESULTS: We found that differentially expressed genes in Trem2*R47H mice were significantly enriched in multiple AD-related pathways, including immune response, osteoclast differentiation, and metabolism, whereas differentially spliced genes were enriched for neuronal related functions, including GABAergic synapse and glutamatergic synapse. These results were reinforced by the enrichment of microglial genes in DEGs and neuronal genes in differentially spliced genes in Trem2*R47H mice. We observed significant overlap between differentially spliced genes in Trem2*R47H mice and brains from human AD subjects. These effects were absent in APOE4 mice and suppressed in APOE4.Trem2*R47H double mutant mice relative to Trem2*R47H mice. CONCLUSIONS: The cross-species observation that alternative splicing observed in LOAD are present in Trem2*R47H mouse models suggests a novel link between this candidate risk gene and molecular signatures of LOAD in neurons and demonstrates how deep molecular analysis of new genetic models links molecular disease outcomes to a human candidate gene.

Recombinant Technologies Facilitate Drug Metabolism, Pharmacokinetics, and General Biomedical Research.

The development of safe and effective medications requires a profound understanding of their pharmacokinetic (PK) and pharmacodynamic properties. PK studies have been built through investigation of enzymes and transporters that drive drug absorption, distribution, metabolism, and excretion (ADME). Like many other disciplines, the study of ADME gene products and their functions has been revolutionized through the invention and widespread adoption of recombinant DNA technologies. Recombinant DNA technologies use expression vectors such as plasmids to achieve heterologous expression of a desired transgene in a specified host organism. This has enabled the purification of recombinant ADME gene products for functional and structural characterization, allowing investigators to elucidate their roles in drug metabolism and disposition. This strategy has also been used to offer recombinant or bioengineered RNA (BioRNA) agents to investigate the posttranscriptional regulation of ADME genes. Conventional research with small noncoding RNAs such as microRNAs (miRNAs) and small interfering RNAs has been dependent on synthetic RNA analogs that are known to carry a range of chemical modifications expected to improve stability and PK properties. Indeed, a novel transfer RNA fused pre-miRNA carrier-based bioengineering platform technology has been established to offer consistent and high-yield production of unparalleled BioRNA molecules from Escherichia coli fermentation. These BioRNAs are produced and processed inside living cells to better recapitulate the properties of natural RNAs, representing superior research tools to investigate regulatory mechanisms behind ADME. SIGNIFICANCE STATEMENT: This review article summarizes recombinant DNA technologies that have been an incredible boon in the study of drug metabolism and PK, providing investigators with powerful tools to express nearly any ADME gene products for functional and structural studies. It further overviews novel recombinant RNA technologies and discusses the utilities of bioengineered RNA agents for the investigation of ADME gene regulation and general biomedical research.

Genome- and transcriptome-wide splicing associations with alcohol use disorder.

Genetic mechanisms of alternative mRNA splicing have been shown in the brain for a variety of neuropsychiatric traits, but not substance use disorders. Our study utilized RNA-sequencing data on alcohol use disorder (AUD) in four brain regions (n = 56; ages 40-73; 100% 'Caucasian'; PFC, NAc, BLA and CEA) and genome-wide association data on AUD (n = 435,563, ages 22-90; 100% European-American). Polygenic scores of AUD were associated with AUD-related alternative mRNA splicing in the brain. We identified 714 differentially spliced genes between AUD vs controls, which included both putative addiction genes and novel gene targets. We found 6463 splicing quantitative trait loci (sQTLs) that linked to the AUD differentially spliced genes. sQTLs were enriched in loose chromatin genomic regions and downstream gene targets. Additionally, the heritability of AUD was enriched for DNA variants in and around differentially spliced genes associated with AUD. Our study also performed splicing transcriptome-wide association studies (TWASs) of AUD and other drug use traits that unveiled specific genes for follow-up and splicing correlations across SUDs. Finally, we showed that differentially spliced genes between AUD vs control were also associated with primate models of chronic alcohol consumption in similar brain regions. Our study found substantial genetic contributions of alternative mRNA splicing in AUD.

Elucidation of the Landscape of Alternatively Spliced Genes and Features in the Dorsal Striatum of Aggressive/Aggression-Deprived Mice in the Model of Chronic Social Conflicts.

Both aggressive and aggression-deprived (AD) individuals represent pathological cases extensively studied in psychiatry and substance abuse disciplines. We employed the animal model of chronic social conflicts curated in our laboratory for over 30 years. In the study, we pursued the task of evaluation of the key events in the dorsal striatum transcriptomes of aggression-experienced mice and AD species, as compared with the controls, using RNA-seq profiling. We evaluated the alternative splicing-mediated transcriptome dynamics based on the RNA-seq data. We confined our attention to the exon skipping (ES) events as the major AS type for animals. We report the concurrent posttranscriptional and posttranslational regulation of the ES events observed in the phosphorylation cycles (in phosphoproteins and their targets) in the neuron-specific genes of the striatum. Strikingly, we found that major neurospecific splicing factors (Nova1, Ptbp1, 2, Mbnl1, 2, and Sam68) related to the alternative splicing regulation of cAMP genes (Darpp-32, Grin1, Ptpn5, Ppp3ca, Pde10a, Prkaca, Psd95, and Adora1) are upregulated specifically in aggressive individuals as compared with the controls and specifically AD animals, assuming intense switching between isoforms in the cAMP-mediated (de)phosphorylation signaling cascade. We found that the coding alternative splicing events were mostly attributed to synaptic plasticity and neural development-related proteins, while the nonsense-mediated decay-associated splicing events are mostly attributed to the mRNA processing of genes, including the spliceosome and splicing factors. In addition, considering the gene families, the transporter (Slc) gene family manifested most of the ES events. We found out that the major molecular systems employing AS for their plasticity are the 'spliceosome', 'chromatin rearrangement complex', 'synapse', and 'neural development/axonogenesis' GO categories. Finally, we state that approximately 35% of the exon skipping variants in gene coding regions manifest the noncoding variants subject to nonsense-mediated decay, employed as a homeostasis-mediated expression regulation layer and often associated with the corresponding gene expression alteration.

Classical Recombinant DNA Cloning.

Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a gene. The main purpose of cloning is to study either that DNA sequence or the RNA or protein product it encodes. Building on key enzymatic discoveries in the late 1960s, gene cloning was pioneered in the early 1970s. Since then, DNA cloning and manipulation have been used in every area of biological and biomedical research, from molecular genetics, structural biology, and developmental biology to neurobiology, ancient DNA studies, and immunology. It is a versatile technique that can be applied to a variety of starting DNA types and lengths, including cDNAs, genes, gene fragments, chromosomal regions, or shorter fragments such as PCR products and functional control regions such as enhancers or promoters. The starting DNA can originate from any cell, tissue, or organism. In this chapter we will cover traditional ("classic") molecular cloning strategy. This comprises six linked stages in which (1) PCR is used to amplify a DNA region of interest that is then (2) digested with restriction enzymes, alongside a selected vector, to produce complementary ends crucial for the two molecules to be (3) ligated by an ATP-dependent DNA ligase, creating a recombinant DNA molecule. The recombinant DNA is then (4) introduced into competent bacterial cells by transformation and (5) grown on a selective agar media, followed by (6) colony-PCR for screening purposes. We provide a worked example to demonstrate the cloning of an average-size gene (in this case the 2 kb DNA ligase A gene) from E. coli into a common plasmid expression vector. We have included six color figures and two tables to depict the key stages of a classical molecular cloning protocol. If you are cloning a segment of DNA or a gene, remember that each DNA cloning experiment is unique in terms of sequence, length, and experimental purpose. However, the principles of traditional cloning covered in this chapter are the same for any DNA sequence; we have included a detailed notes section, so you should easily be able to transfer them to your own work. Some of the following chapters in this volume will cover other, more recently developed, cloning protocols.

In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1.

Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 µg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.

Tip60's Novel RNA-Binding Function Modulates Alternative Splicing of Pre-mRNA Targets Implicated in Alzheimer's Disease.

The severity of Alzheimer's disease (AD) progression involves a complex interplay of genetics, age, and environmental factors orchestrated by histone acetyltransferase (HAT)-mediated neuroepigenetic mechanisms. While disruption of Tip60 HAT action in neural gene control is implicated in AD, alternative mechanisms underlying Tip60 function remain unexplored. Here, we report a novel RNA binding function for Tip60 in addition to its HAT function. We show that Tip60 preferentially interacts with pre-mRNAs emanating from its chromatin neural gene targets in the Drosophila brain and this RNA binding function is conserved in human hippocampus and disrupted in Drosophila brains that model AD pathology and in AD patient hippocampus of either sex. Since RNA splicing occurs co-transcriptionally and alternative splicing (AS) defects are implicated in AD, we investigated whether Tip60-RNA targeting modulates splicing decisions and whether this function is altered in AD. Replicate multivariate analysis of transcript splicing (rMATS) analysis of RNA-Seq datasets from wild-type and AD fly brains revealed a multitude of mammalian-like AS defects. Strikingly, over half of these altered RNAs are identified as bona-fide Tip60-RNA targets that are enriched for in the AD-gene curated database, with some of these AS alterations prevented against by increasing Tip60 in the fly brain. Further, human orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brains, implicating disruption of Tip60's splicing function in AD pathogenesis. Our results support a novel RNA interaction and splicing regulatory function for Tip60 that may underly AS impairments that hallmark AD etiology.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) has recently emerged as a hotbed for RNA alternative splicing (AS) defects that alter protein function in the brain yet causes remain unclear. Although recent findings suggest convergence of epigenetics with co-transcriptional AS, whether epigenetic dysregulation in AD pathology underlies AS defects remains unknown. Here, we identify a novel RNA interaction and splicing regulatory function for Tip60 histone acetyltransferase (HAT) that is disrupted in Drosophila brains modeling AD pathology and in human AD hippocampus. Importantly, mammalian orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brain. We propose that Tip60-mediated AS modulation is a conserved critical posttranscriptional step that may underlie AS defects now characterized as hallmarks of AD.

Specific expression of alternatively spliced genes in the turkey (Meleagris gallopavo) reproductive tract revealed their function in spermatogenesis and post-testicular sperm maturation.

The tissue-specific profile of alternatively spliced genes (ASGs) and their involvement in reproduction processes characteristic of turkey testis, epididymis, and ductus deferens were investigated for the first time in birds. Deep sequencing of male turkey reproductive tissue RNA samples (n = 6) was performed using Illumina RNA-Seq with 2 independent methods, rMATs and SUPPA2, for differential alternative splicing (DAS) event prediction. The expression of selected ASGs was validated using quantitative real-time reverse transcriptase-polymerase chain reaction. The testis was found to be the site of the highest number of posttranscriptional splicing events within the reproductive tract, and skipping exons were the most frequently occurring class of alternative splicing (AS) among the reproductive tract. Statistical analysis revealed 86, 229, and 6 DAS events in the testis/epididymis, testis/ductus deferens, and epididymis/ductus deferens comparison, respectively. Alternative splicing was found to be a mechanism of gene expression regulation within the turkey reproduction tract. In testis, modification was observed for spermatogenesis specific genes; the changes in 5' UTR could act as regulator of MEIG1 expression (a player during spermatocytes meiosis), and modification of 3' UTR led to diversification of CREM mRNA (modulator of gene expression related to the structuring of mature spermatozoa). Sperm tail formation can be regulated by changes in the 5' UTR of testicular SLC9A3R1 and gene silencing by producing dysfunctional variants of ODF2 in the testis and ATP1B3 in the epididymis. Predicted differentially ASGs in the turkey reproductive tract seem to be involved in the regulation of spermatogenesis, including acrosome formation and sperm tail formation and binding of sperm to the zona pellucida. Several ASGs were classified as cilia by actin and microtubule cytoskeleton organization. Such genes may play a role in the organization of sperm flagellum and post-testicular motility development. To our knowledge, this is the first functional investigation of alternatively spliced genes associated with tissue-specific processes in the turkey reproductive tract.

Splicing factor mutations in the myelodysplastic syndromes: Role of key aberrantly spliced genes in disease pathophysiology and treatment.

Mutations of splicing factor genes (including SF3B1, SRSF2, U2AF1 and ZRSR2) occur in more than half of all patients with myelodysplastic syndromes (MDS), a heterogeneous group of myeloid neoplasms. Splicing factor mutations lead to aberrant pre-mRNA splicing of many genes, some of which have been shown in functional studies to impact on hematopoiesis and to contribute to the MDS phenotype. This clearly demonstrates that impaired spliceosome function plays an important role in MDS pathophysiology. Recent studies that harnessed the power of induced pluripotent stem cell (iPSC) and CRISPR/Cas9 gene editing technologies to generate new iPSC-based models of splicing factor mutant MDS, have further illuminated the role of key downstream target genes. The aberrantly spliced genes and the dysregulated pathways associated with splicing factor mutations in MDS represent potential new therapeutic targets. Emerging data has shown that IRAK4 is aberrantly spliced in SF3B1 and U2AF1 mutant MDS, leading to hyperactivation of NF-κB signaling. Pharmacological inhibition of IRAK4 has shown efficacy in pre-clinical studies and in MDS clinical trials, with higher response rates in patients with splicing factor mutations. Our increasing knowledge of the effects of splicing factor mutations in MDS is leading to the development of new treatments that may benefit patients harboring these mutations.

Development of a compartmentalised self-replication protocol for selection of superior blunt-end DNA ligases.

DNA ligases are widely used in molecular biology to generate recombinant DNA. However, having evolved for nick-sealing, they are inefficient at catalysing the blunt-ended ligations that are critical to many biotechnological applications, including next-generation sequencing. To facilitate engineering of superior blunt-ended DNA ligases, we have developed and validated a compartmentalised self-replication protocol that can select for the most effective ligases from a library of variants. Parallel cultures of Escherichia coli cells expressing different plasmid-encoded variants act as both a source of template DNA for discrete whole-plasmid PCR reactions, and a source of expressed ligase to circularise the corresponding PCR amplicons. The most efficient ligases generate the greatest number of self-encoding plasmids, and are thereby selected over successive rounds of transformation, amplification and ligation. By individually optimising critical steps, we arrived at a coherent protocol that, over five rounds of selection, consistently enriched for cells expressing the more efficient of two recombinant DNA ligases.

Blood transcriptome analysis and identification of genes associated with supernumerary teats in Chinese Holstein cows.

In dairy cows, supernumerary teats (SNT) are not desired as they are considered a repository for bacteria; thus, SNT are a risk factor for mastitis. Supernumerary teats are a heritable oligo- or polygenic trait. The incidence of SNT in offspring must be reduced by genomic selection. However, in modern dairy farming, farmers often ignore the effects of SNT on cows. The study aimed to elucidate the effects of SNT on dairy cows from the blood transcriptome level and identify genes associated with SNT in Chinese Holstein cows. We selected 6 SNT cows (Yes) and 6 non-SNT cows (No). In the 6 SNT cows, 3 cows had 1 SNT (One) and 3 cows had 2 SNT (Two). They were divided into 3 comparison groups (One vs. No; Two vs. No; and Yes vs. No). RNA was extracted from blood white membrane cells of 12 cows, and RNA sequencing was performed. Differential gene expression analysis based on the negative binomial distribution was used to detect differentially expressed genes in the One versus No and Two versus No comparison groups. Genes that were significantly upregulated or downregulated both in the One versus No and Two versus No groups (shared genes, SG) were obtained for further analysis. We also performed gene set enrichment analysis for all genes expressed in the Yes versus No group, correlation analysis between SG and the hematological parameters, protein-protein interaction network analysis of SG to select hub genes, and alternative splicing analysis for Yes versus No group to explore the functions of differentially spliced genes. We detected 289 SG. Gene set enrichment analysis, gene ontology, and the Kyoto Encyclopedia of Genes and Genomes enrichment analysis results showed that SNT affect immunity, inflammation, and lactation-related pathways in dairy cows. Correlation analysis showed that LOC104968484, SLC25A6, GADD45G, BAX, APAF1, ATM, XIAP, MDM4, BDP1, CEP350, MED13, TAOK1, SMG1, and RIF1 are associated with white blood cell count and absolute value of lymphocytes in SNT cows only, so they might be genes associated with SNT in Chinese Holstein cows. We found 2 genes (BAX and MDM4) were also differentially spliced genes. However, the causal relationship between these genes and the SNT phenotype needs to be further studied. This study is the first to reveal the adverse effects of SNT on dairy cows at a transcriptional level, and the genes we found can be used as a reference for further searching for candidate genes for the SNT phenotype.

[Consideration on the Research and Development of Anti-tumor Bispecific Antibody Drugs].

The drugs of programmed cell death 1 and its ligand 1 immune checkpoint inhibitors have ushered in a new era of anti-tumor immunotherapy, which has shown outstanding efficacy in some tumors, such as Hodgkin lymphoma, but there is still low response rate in some kinds of tumors. In recent years, bispecific antibodies prepared by cell fusion, recombinant DNA, protein engineering and other technologies can specifically bind two antigens or epitopes at the same time or successively, play a synergistic role in tumor treatment, can effectively inhibit tumor immune escape, and improve the effect of anti-tumor treatment has become a hot spot in tumor research. This paper will summarize the clinical research and development of bispecific antibodies, to provide reference for the industry.
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Modern biotechnological strategies for vaccine development in aquaculture - Prospects and challenges.

Advances in genomics and the gradual reduction of cost for technologies like whole-genome sequencing have provided exciting opportunities for developing modern biotechnological-based vaccines in aquaculture. This systemic review describes the prospects and challenges of implementing these high-tech vaccines in fish species. The majority of the commercial vaccines in aquaculture utilize conventional procedures for which cost of administration, protective immunity and safety issues are the major challenges. In recent years, more efficient vaccines are being developed by adopting the advances in vaccine technology. Vaccines based on surface antigens, protein/peptide/polysaccharide subunits, recombinant DNA/mRNA/plasmids, novel antigen expression and delivery systems (bacteriophage particles, virus like particles/VLPs, recombinant yeast, mucosal vaccines), novel molecular adjuvants (IL-8, IL-12, HSPs), and encapsulation polymers and polysaccharides like chitosan nanoparticles and PLGA microcapsule were successfully developed. These biotechnology-based vaccines have proved to be very efficient in field trials, but are always in the research pipeline or as patents. Only very few of them are licensed for use, that too, in high-valued fishes like salmonids. Currently, commercial aquaculture vaccines are available for Aeromonas salmonicida, Vibrio salmonicida, Yersinia ruckeri, Vibrio anguillarum, Edwardsiella ictalurid, and for certain Betanodaviruses. Nevertheless, no registered vaccines are available for other major infectious diseases/pathogens such as viral hemorrhagic septicemia virus (VHSV), viral nervous necrosis virus (VNN) and certain other betanodaviruses, channel catfish virus (CCV), gill disease bacteria, mycobacteria, flavobacterium, Edwardsiella tarda, and certain streptococci. Despite the important economic losses that the pathogens cause to aquaculture worldwide, the commercialization of vaccines remains limited due to immunological pitfalls in aquatic species, large-scale vaccination issues, unregulated use of antibiotics and chemicals, gene-based vaccine regulations and commercial viability. If attempts are to be made to develop novel delivery methods, cost-effective procedures, and relaxations in DNA vaccine regulations, biotechnology-based vaccination could circumvent the emerging disease challenges in aquaculture.