Transgenic Animals Research Articles

Inorganic Nanoparticles in Reproduction Biology: Opportunities and Challenges

The development of inorganic metal and metal oxide nanoparticles (MNPs) has attracted significant attention in biomedical and biotechnological fields. An emerging field in this context is the use of MNPs in reproduction biology. Here, we review the development of MNPs in the field of reproduction by focusing on their interactions with highly delicate and specialized germ cells like spermatozoa and oocytes as well as developing embryos. By their unique physicochemical properties, MNPs are good candidates for targeted imaging and delivery of biofunctional molecules to the specific sites of the gametes and reproductive cells. Furthermore, functionalized MNPs can serve as transfection vectors for the generation of transgenic animals by sperm‐mediated gene transfer (SMGT) in artificial insemination or in vitro fertilization. In addition, MNPs have shown great promise in application fields such as semen collection, nano‐purification, cryopreservation, and sex sorting of sperm in the livestock industry. The article includes an in‐depth discussion on the uptake, translocation mechanism, and bioresponse of the MNPs to reproduction‐relevant sites on the clinical, cellular, and molecular levels. Based on these promising achievements, the current challenges and prospects of the development of these functionalized MNPs for clinical research in conjunction with the reproductive system will be discussed.

Exploration of different quantitative polymerase chain reaction-based genotyping methods to distinguish Apcmin/+ mice from wildtype mice.

Various molecular methods have been established for genotyping single-nucleotide variants (SNVs). However, despite the widespread availability of quantitative polymerase chain reaction (qPCR) instruments in biomedical laboratories, the lack of professional analytical tools impedes the application of qPCR in genotyping. Apcmin/+ mice, which harbour a germline Apc mutation (g.2549T>A) associated with multiple intestinal neoplasms, are extensively employed in colorectal cancer research. In this study, we used Apc as a model and assessed the feasibility of different qPCR-based methods for SNV genotyping, considering approaches with and without genotyping analytical tools. We initially employed allele-specific PCR followed by electrophoresis to determine the genotypes of Apc in tail tissues from potential Apcmin/+ mice, and this method served as the benchmark for evaluating the performance of qPCR-based methods. Dye-based qPCR and melting curve assays exhibited distinct dissociation patterns that differentiated between synthesised wildtype (TT) and heterozygous mutant (TA) DNA and between TT and TA genotype mice based on analysis of tissue samples. This discrimination ability of these assays was unaffected by the use of different intercalating dyes (SYBR Green I or EvaGreen). Dual-probe qPCR assays were developed to simultaneously detect mutant and wildtype alleles using differently labelled probes. The genotyping module and delta cycle threshold method were used to facilitate the analysis of results. The qPCR-based methods displayed 100% agreement with the standard genotyping outcomes. When the PCR-electrophoresis method was used, approximately 15% of the samples required re-examination to obtain conclusive results. In contrast, when the qPCR methods were used, success rates exceeding 99% were achieved with a single test. Additionally, all qPCR-based methods determined mouse genotypes by analysis of stool samples, highlighting the applicability of these methods for non-invasive genotyping. Loss of heterozygosity in the Apc gene in intestinal polyps was detected using the dual-probe assay with delta cycle threshold method. In summary, this study successfully implemented intercalator-based and probe-based qPCR methods, with and without professional analytical modules, for characterising Apc in tissue and stool samples. Furthermore, these methods can be extended to allow genotyping of other SNVs and can facilitate non-invasive genotyping of transgenic animals.

Modeling of Parkinson's Disease in Different Models.

Parkinson's Disease (PD) is a progressive disorder worldwide and its etiology remains unidentified. Over the last few decades, animal models of PD have been extensively utilized to explore the development and mechanisms of this neurodegenerative condition. Toxic and transgenic animal models for PD possess unique characteristics and constraints, necessitating careful consideration when selecting the appropriate model for research purposes. Animal models have played a significant role in uncovering the causes and development of PD, including its cellular and molecular processes. These models suggest that the disorder arises from intricate interplays between genetic predispositions and environmental influences. Every model possesses its unique set of strengths and weaknesses. This review provides a critical examination of animal models for PD and compares them with the features observed in the human manifestation of the disease.

Recent advances in immunotherapy targeting amyloid-beta and tauopathies in Alzheimer's disease.

Alzheimer's disease, a devastating neurodegenerative disorder, is characterized by progressive cognitive decline, primarily due to amyloid-beta protein deposition and tau protein phosphorylation. Effectively reducing the cytotoxicity of amyloid-beta42 aggregates and tau oligomers may help slow the progression of Alzheimer's disease. Conventional drugs, such as donepezil, can only alleviate symptoms and are not able to prevent the underlying pathological processes or cognitive decline. Currently, active and passive immunotherapies targeting amyloid-beta and tau have shown some efficacy in mice with asymptomatic Alzheimer's disease and other transgenic animal models, attracting considerable attention. However, the clinical application of these immunotherapies demonstrated only limited efficacy before the discovery of lecanemab and donanemab. This review first discusses the advancements in the pathogenesis of Alzheimer's disease and active and passive immunotherapies targeting amyloid-beta and tau proteins. Furthermore, it reviews the advantages and disadvantages of various immunotherapies and considers their future prospects. Although some antibodies have shown promise in patients with mild Alzheimer's disease, substantial clinical data are still lacking to validate their effectiveness in individuals with moderate Alzheimer's disease.

Transgenic rabbits with genes of human granulocyte colony-stimulating factor and green fluorescent protein

Human granulocyte–colony stimulating factor (GCSF) is one of the pharmacological proteins that can be isolated from the milk of transgenic (TG) animals. The plasmid containing the human GCSF gene under the control of regulatory elements of the bovine β-lactoglobulin gene and the reporter green fluorescent protein (EGFP) gene under the cytomegalovirus (cmv) promoter were obtained. The use of the selected promoters ensures tissue-specific expression of the target protein in the mammary gland of the TG producing animal and a high level of early expression of the reporter protein in eukaryotic cells, which makes it possible to detect TG embryos at the cultivation stage and perform their preimplantation selection. Testing of the gene construct effectiveness was carried out on TG rabbits obtained by microinjection into the male pronucleus of zygotes. It was concluded that GFP is toxic to embryos in the early stages of development due to overexpression of the EGFP gene under a strong cmv promoter. The TG female rabbit (F0) was obtained, in which the level of human GKSF in milk and blood serum was assessed by the ELISA method. Of the 22 baby rabbits obtained from her in four kindling, two were transgenic. Offspring (F1) was obtained from the TG male F0, 56 % of which were males, of which 88 % were TG and did not differ from ordinary rabbits in terms of health. Among females, TG was 10 %, and they died within two weeks after birth.

PET Imaging of a Transgenic Tau Rat Model SHR24 with [18F]AV1451.

Positron Emission Tomography (PET) scans with radioligands targeting tau neurofibrillary tangles (NFT) have accelerated our understanding of the role of misfolded tau in neurodegeneration. While intended for human research, applying these radioligands to small animals establishes a vital translational link. Transgenic animal models of dementia, such as the tau rat SHR24, play a crucial role in enhancing our understanding of these disorders. This study aims to evaluate the utility of SHR24 rat model for PET studies. Dynamic PET scans were conducted in male SHR24 rats and their wild-type SHR (SHRwt) littermates using [18F]AV1451. Rapid blood sampling and metabolite analysis were performed to acquire input curves. Time activity curves were obtained from various brain regions over 60min. Blood-based, 2-Tissue Compartment Model (2-TCM) and Logan graphical analysis were used to obtain kinetic modelling parameters. The ability of reference tissue models to predict the binding potential (BPND) were assessed. Autoradiography studies were performed to corroborate the scan data. Total distribution volume (VT) was the best predicted parameter which revealed significantly higher uptake of [18F]AV1451 in the cortex (5.8 ± 1.1 vs 4.6 ± 0.7, P < 0.05) of SHR24 rats compared to SHRwt rats. Binding potential obtained from 2-TCM was variable, however BPND from reference tissue models detected significantly higher binding in cortex (0.28 ± 0.07 vs 0.20 ± 0.04, P < 0.01 by SRTM) and brainstem (0.14 ± 0.04 vs 0.08 ± 0.02, P < 0.01, by SRTM). With the ability to detect binding of established radioligand [18F]AV1451 in these rats, we have demonstrated the utility of this model for assessing aggregated tau neurobiology by PET, with reference tissue models providing potential for longitudinal studies.

A positive feedback loop between germ cells and gonads induces and maintains sexual reproduction in a cnidarian.

The fertile gonad includes cells of two distinct developmental origins: the somatic mesoderm and the germ line. How somatic and germ cells interact to develop and maintain fertility is not well understood. Here, using grafting experiments and transgenic reporter animals, we find that a specific part of the gonad-the germinal zone-acts as a sexual organizer to induce and maintain de novo germ cells and somatic gonads in the cnidarian Hydractinia symbiolongicarpus. Germ cells express a member of the transforming growth factor-β family, Gonadless (Gls), that induces gonad morphogenesis. Loss of Gls resulted in animals lacking gonads but having nonproliferative germ cells. We propose that primary germ cells drive gonad development though Gls secretion. The germinal zone in the newly formed gonad provides positive feedback to induce secondary germ cells by activating Tfap2 in resident pluripotent stem cells. The contribution of germ cell signaling to the patterning of somatic gonadal tissue may be a general animal feature.

Advancing Obsessive-Compulsive Disorder Research: Insights from Transgenic Animal Models and Innovative Therapies.

Obsessive-compulsive disorder (OCD) is a prevalent, chronic, and severe neuropsychiatric disorder that leads to illness-related disability. Despite the availability of several treatments, many OCD patients respond inadequately, because the underlying neural mechanisms remain unclear, necessitating the establishment of many animal models, particularly mouse models, to elucidate disease mechanisms and therapeutic strategies better. Although the development of animal models is ongoing, there remain many comprehensive summaries and updates in recent research, hampering efforts to develop novel treatments and enhance existing interventions. This review summarizes the phenotypes of several commonly used models and mechanistic insights from transgenic models of OCD, such as knockout mouse models. In addition, we present the advantages and limitations of these models and discuss their future in helping further understand the pathophysiology and advanced treatment. Here, we highlight current frontline treatment approaches for OCD, including neuromodulation and surgical interventions, and propose potential future directions. By studying gene mutations and observing phenotypes from available OCD animal models, researchers have classified the molecular signatures of each model reminiscent of changes in brain areas and neural pathways, with the hope of guiding the future selection of the most appropriate models for specific research in the OCD field.

Generation of Codon-Optimized Fad3 Gene Transgenic Bovine That Produce More n-3 Polyunsaturated Fatty Acids.

Polyunsaturated fatty acids (PUFAs) such as linoleic acid (18:2, n-6) and α-linolenic acid (18:3, n-3) are essential for the growth, development, and well-being of mammals. However, most mammals, including humans, cannot synthesize n-3 and n-6 PUFAs and these must be obtained through diet. The beneficial effect of converting n-6 polyunsaturated fatty acids (n-6 PUFAs) into n-3 polyunsaturated fatty acids (n-3 PUFAs) has led to extensive research on the flax fatty acid desaturase 3 (Fad3) gene, which encodes fatty acid desaturase. Still, the plant-derived Fad3 gene is used much less in transgenic animals than the Fat-1 gene from Caenorhabditis elegans. To address this problem, we used somatic cell nuclear transfer (SCNT) technology to create codon-optimized Fad3 transgenic cattle. Gas chromatographic analysis showed that the n-3 PUFA content of transgenic cattle increased significantly, and the ratio of n-6 PUFAs to n-3 PUFAs decreased from 3.484 ± 0.46 to about 2.78 ± 0.14 (p < 0.05). In conclusion, Fad3 gene knock-in cattle are expected to improve the nutritional value of beef and can be used as an animal model to study the therapeutic effects of n-3 PUFAs in various diseases.

The current models unravel the molecular mechanisms underlying the intricate pathophysiology of Alzheimer's disease using zebrafish.

The foremost cause of dementia is Alzheimer's disease (AD). The vital pathological hallmarks of AD are amyloid beta (Aβ) peptide and hyperphosphorylated tau (p-tau) protein. The current animal models used in AD research do not precisely replicate disease pathophysiology, making it difficult for researchers to quickly and effectively gather data or screen potential therapy possibilities. Several transgenic animals are used as models for AD; however, they have cost and time concerns. Zebrafish (Danio rerio) has become a suitable model organism for high-throughput pharmacological screening of neuroactive substances and neurodegenerative research. The past few decades have seen a significant increase in research on AD. The fight against amyloidosis has, however, been unexpectedly unsuccessful. It may be due to a need for more relevant in vivo models for high throughput screening, which emphasizes the need to find other anti-AD models. Alternative animal models, including zebrafish, have developed into a potentially useful research tool that must be employed for AD research to be effective. Only a few comprehensive zebrafish models exhibiting AD-like pathogenesis have been reported in the literature, and this book chapter describes these models.

Detection of aberrant locomotor activity in a mouse model of lung cancer via home cage monitoring

IntroductionLung cancer is the first cause of cancer death in the world, due to a delayed diagnosis and the absence of efficacy therapies. KRAS mutation occurs in 25% of all lung cancers and the concomitant mutations in LKB1 determine aggressive subtypes of these tumors. The improvement of therapeutical options for KRASG12C mutations has increased the possibility of treating these tumors, but resistance to these therapies has emerged. Preclinical animal models permit the study of tumors and the development of new therapies. The DVC system was used to measure circadian activity changes indicative of lung cancer progression in KRAS and KRAS-LKB1 transgenic mouse models.Material and methodsKRAS and KRAS-LKB1 conditional transgenic animal models were bred and genotyped. The tumors were inducted using adeno-CRE-recombinase system. The mice were housed in a Digital Ventilated Cage (DVC®) rack measuring the locomotor activity continuously for 24/7. The progression of the tumors was monitored with MRI. The DVC system evaluated a reduction in animal locomotion during the tumor progression.ResultsKRAS and KRAS-LKB1 mutations were induced, and the tumor formation and progression were monitored over time. As expected, the onset of the tumors in the two different breeds occurred at different times. DVC system registered the locomotion activity of the mice during the light and dark phases, reporting a strong reduction, mainly, in the dark phase. In KRAS-LKB1 models, the locomotion reduction appeared more pronounced than in KRAS models.DiscussionsTransgenic animal models represent a fundamental tool to study the biology of cancers and the development of new therapies. The tumors induced in these models harbor the same genotypical and phenotypical characteristics as their human counterparts. DVC methods permit a home cage monitoring system useful for tracking animal behavior continuously 24 hours a day, 7 days a week. DVC system could determine disease progression by monitoring a single animal activity in a cage and also using group-housed animals. For these reasons, the DVC system could play a crucial role in identifying diseases at early stages and in testing new therapeutic approaches with a higher likelihood of efficacy.

Chronic imaging of dendritic spine morphology in 5xFAD-M hybrid line mice of Alzheimer’s disease model

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder of neural structures in different areas of the brain. Loss of synapses is a key stage in the development of AD and it precedes significant loss of neurons. However, the mechanisms of synapse loss are uncertain. Structural and functional changes in synapses are interrelated with the morphology of postsynaptic formations – dendritic spines. This paper describes the implementation of the technology of chronic imaging of dendritic spines in transgenic animals using the methods of multiphoton fluorescence microscopy. Mice of the 5xFAD-M hybrid line were used. 5xFAD-M was derived by crossing transgenic mice with expressions of green fluorescent protein GFP in individual neurons of the brain (M-Line) and a mouse model of AD (5xFAD line). Methodological achievements revealed the multi-day dynamics of the density of dendritic spines in M-Line and 5xFAD-M mice. Transformations of morphological types of spikes were revealed during a long period of observations.

Unveiling the Pathogenic Influence of TDP‐43 on Tau‐Mediated Neurotoxicity in an Alzheimer’s Disease Mouse Model

AbstractBackgroundAlzheimer’s disease (AD) is the leading cause of dementia and one of the most devastating neurodegenerative diseases. In the last decades, a large proportion of AD patients have been described as having aberrant accumulation of TDP‐43 protein, a well‐established driver of neurodegeneration. This TDP‐43 proteinopathy in AD can co‐occur in neurons with the main hallmarks of the disease, toxic amyloid oligomers and neurofibrillary tangles containing hyperphosphorylated Tau, and correlates with rapid progression and worse prognosis. However, the underlying mechanisms of pathogenic TDP‐43 contributing to AD and its possible involvement as a trigger of neurotoxicity remain unclear.MethodWe aimed to explore the role of TDP‐43 in a mouse model of tau‐mediated toxicity. To this end, we created a new mouse model of tau and TDP‐43 co‐pathology that combines the constitutive transgenic expression of wild‐type human tau with adeno‐associated virus (AAV) driven expression of wild‐type human TDP‐43. Using a stereotaxic device, we performed a unilateral intrahippocampal injection of AAV‐9 carrying the human TDP‐43 cDNA (or green fluorescent protein as a control) into 3‐month‐old transgenic mice expressing the most abundant brain isoform (1N4R) of Tau in neurons. Animals were then perfused 2, 4, and 12 weeks after injection, and brains were collected for immunohistochemistry.ResultOur results showed that although TDP 43 overexpression was similar in both transgenic and control mice, the presence of hTDP‐43 promoted an increase in phosphorylated tau species. These data suggest a possible interaction between human tau protein and TDP‐43 proteins leading to the spread of phosphorylated Tau species, thus contributing to the pathology in our mouse model of the disease. Additionally, the co‐expression of both TDP‐43 and Tau proteins induced a transient inflammatory state in transgenic animals, sustained 4 weeks after injury. However, 12 weeks after injury, inflammation was resolved, and the presence of phosphorylated Tau species was significantly reduced.ConclusionThis new mouse model provides a vehicle to explore causes and consequences of co‐pathological tau and TDP‐43. Further studies will be necessary to determine mechanisms underlying TDP‐43 promotion of pathological accumulation of toxic Tau species and neuroinflammation in this experimental model of TDP‐43‐ Tau co‐expression.

Distinct associations of excitatory‐inhibitory (E/I) imbalance in aging and in Alzheimer’s disease

AbstractBackgroundNeural circuit hyperexcitability and impaired excitation‐to‐inhibition (E/I) activity is believed to be a key contributor to synaptic and network degeneration in Alzheimer’s disease (AD). Extensive preclinical research on transgenic animal models of AD have demonstrated neuronal and circuit level E/I imbalance mediated by amyloid‐beta (Aβ) and tau proteins. Synaptic and network deficits are also integral changes of aging. It is unknown whether the mechanisms of E/I imbalance in aging are distinct from that in AD. In this study we leveraged the aperiodic spectral slope measure, which represent the spontaneous neuronal firing, and which has been shown to indicate higher E/I as reduced slope values.MethodsWe used source reconstructed, high spatiotemporal resolution signals, from magnetoencephalography (MEG) for the spectral analysis and used the neural mass model (NMM) to estimate excitatory and inhibitory neuronal parameters, in patients with AD (n = 85) and age‐matched elderly‐controls (n = 45). The NMM estimates the time constants of excitatory (te) and inhibitory (ti) neuronal subpopulations and depicts E/I as 1/inhibitory‐neural‐gain (gii). We estimated the aperiodic slope from the spectral analysis (15‐50 Hz) and examined the correlations with NMM parameters. In a subset of AD patients, we recorded the presence or absence of epileptiform activity (EPI) and examined the associations of neuronal‐time‐contents and inhibitory‐neural‐gain in AD‐EPI+ (n = 20) vs. AD‐EPI− (n = 30).ResultsAD patients showed reduced inhibitory‐neural‐gains compared to elderly‐controls indicating increased E/I (Fig. 1.A). AD patients also showed increased neuronal‐time‐constants than controls (Fig. 1.B‐C). The spatial patterns of reduced inhibitory‐neural‐gains overlapped with AD vulnerable anatomic regions showing a posterior temporo‐parietal‐occipital distribution (Fig. 1.D). Greater reductions of aperiodic‐slope distinctly correlated with inhibitory‐neural‐gains in AD and with higher neuronal‐time‐constants in elderly‐controls (Fig. 1.G‐L). Moreover, AD‐EPI+ showed greater reductions in inhibitory‐neural‐gains than AD‐EPI−, but no differences in neuronal‐time‐constants (Fig. 1.M‐O).ConclusionsOur findings identified impaired inhibitory synaptic activity as the strongest correlate of neural circuit hyperexcitability in AD. In contrast, impaired timing of both excitatory and inhibitory neurons were the best predictors of neural circuit hyperexcitability in aging. These results suggest different mechanisms of neural circuit hyperexcitability in AD vs. aging and have important implications to translational as well as clinical studies probing early network changes.

XBP‐1s transcriptional targets in the endoplasmic reticulum unfolded protein response ameliorate tauopathy

AbstractBackgroundProtein homeostasis (proteostasis) mechanisms fail with aging and disease, promoting toxic protein accumulation. Neurons are particularly vulnerable to proteostatic disruption leading to aging related neurodegeneration. Abnormal activation of the endoplasmic reticulum unfolded protein response (UPRER) is implicated in tauopathies, a group of neurodegenerative diseases characterized by pathological accumulation of the microtubule‐associated protein tau. Previous work showed neuronal overexpression of IRE1a branch UPRER transcription factor XBP‐1s suppresses tauopathy in C. elegans.MethodsWe conducted RNA sequencing of mRNAs to analyze the transcriptome of xbp‐1s and tau transgenic animals to identify genes required for XBP‐1s mediate protection from tauopathy.ResultsTranscriptomic analysis of XBP‐1s animals showed upregulation of the following genes with human homologs: csp‐1, F42G8.7, F41E7.6, Y19D10A.16, C01B4.6, dnj‐28, hsp‐4, ckb‐2, mct‐2, lipl‐3, and eol‐1. Surprisingly, each one of these genes is required for xbp‐1s‐mediated suppression of tauopathy, suggesting that XBP‐1s activates a broad and non‐redundant network of cellular mechanisms to reduce tau pathology. Of these, we examined the critical UPRER regulator BiP/hsp‐4, the ER resident HSP70 homolog. Hsp‐4 loss of function, but not loss of the cognate ER resident DNAJ protein dnj‐28, eliminates xbp‐1s‐mediated suppression of tauopathy. While hsp‐4 loss of function exacerbates tau‐induced behavioral deficits, tau protein level and phosphorylation are unaffected. High level overexpression of hsp‐4 exacerbates tau‐induced behavioral deficits and protein accumulation, while moderated hsp‐4 overexpression ameliorates this phenotype. Furthermore, caspases also appear to play an important role downstream of XBP‐1s: both the XBP‐1s target csp‐1 and its non‐XBP‐1s target gene family member ced‐3 are required for xbp‐1s‐mediated suppression of tauopathy.ConclusionsWe present a dataset illuminating the mechanism of xbp‐1s‐mediated suppression of tauopathy involving a suite of diverse transcriptional targets.

Transgenic animals: catalysts in drug discovery

Transgenic technologies have fundamentally transformed scientific research by enabling precise genetic modifications with wide-ranging applications across various fields. This review examines the application of transgenic mice across various research domains including cancer, diabetes, cardiovascular science, neurology, gastrointestinal research, and reproductive science. We explored how these models are important in enhancing the knowledge of complex biological methods, from elucidating pathways of the disease to enhancing the quality of research outcomes in these fields. Key methodologies such as CRISPR-Cas9, TALENs, knock-out, knock-in, microinjection, embryonic stem (ES) cell transfection are highlighted, with a focus on their impact on experimental results. Additionally, the review addresses the use of various transgenic strains in drug discovery and the challenges associated with transgenic research. By integrated findings from multiple research areas, this review underscores the transformative potential of transgenic technologies and their pivotal role in driving innovation and addressing global challenges.

Substitution of Glutamic Acid at Position 71 of DRβ1*04:01 and Collagen-Specific Tolerance Without Alloreactivity.

The DRB1 locus is strongly associated with both susceptibility and resistance to rheumatoid arthritis (RA). DRB1 alleles encoding the VKA or VRA epitope in positions 11, 71, and 74 confer the highest risk of developing RA, whereas the allele encoding VEA is protective. We therefore investigated the feasibility of creating antigen-specific tolerance without inducing alloreactivity by replacing lysine with glutamic acid at position 71 in DRβ1*04:01. Individual DRB1 alleles and the DRB1*04:01K71E allele were cloned into T2 cell lines to measure binding of biotinylated peptides. Transgenic animals expressing DRB1*04:01, DRB1*01:01, or DRB1*04:01K71E were injected with collagen to measure T cell proliferation. Skin and bone marrow transplants between DRB1*04:01K71E and DRB1*04:01 mice were performed to determine if the single amino acid change at position 71 would be recognized as foreign. DRB1*04:01 mice transplanted with DRB1*04:01K71E bone marrow were injected with collagen to test if resistance to collagen sensitization could be transferred. Replacing lysine (K) at position 71 in DRβ1*04:01 with glutamic acid (E) blocked collagen peptide binding and rendered the DRB1*04:01K71E mice resistant to collagen sensitization. Skin and bone marrow transplants from DRB1*04:01K71E mice were not rejected by DRB1*04:01 mice, suggesting the single E71 difference was not recognized as allogeneic. Bone marrow from DRB1*04:01K71E mice adoptively transferred antigen-specific tolerance to collagen to DRB1*04:01 mice. These studies demonstrate that editing a single amino acid in DRβ1*04:01 blocks collagen peptide binding without inducing alloreactivity and could therefore represent a gene therapy approach to induce antigen-specific passive tolerance.

In-vitro Cell Culture: A Promising Technology in the Advancement of Science and Research as a Solution to Human and Animal Health Issues

From ancient Romans till late nineties, conceptualized first by Aristotle, it was believed that life evolved from non-living matter, the spontaneous generation theory. With the invention of microscopy, invisible life was for the first time visible as “animalcules”, that were later described as cells and the cell theory and theory of tissue formation came into limelight. In late nineties cell culture methods were discussed and in-vivo as well as in-vitro cell culture was achieved. In-vitro cell culture techniques were defined, improved, augmented and refined with the advancement in biological sciences. The applications of cell culture technology spanned through various fields, in pathology, microbiology, reproduction, physiology and production of biologicals, monoclonal antibodies, vaccines. In-vitro fertilization, embryo splitting and transgene delivery for augmenting fertility in human and animals, production of animal products, key biomolecules, proteins are some of the endowed applications of in-vitro cell culture technique. Cell culture technology has taken leaps and has become a key modelling system for large class of biotechnological interventions, clinical and industrial applications. Production of important hormones, growth factors, biomolecules and therapeutic agents are all blessings of in-vitro cell culture technology. In-vitro cell culture technology has entered into new era of development of organoids, organ chips, RNA/ DNA / recombinant vaccines, transgenic animals, tissue reconstructs etc. in pursuit of achieving better health care and food security. In-vitro cell culture technology has been playing a significant role in the global economy with huge market size in stem cell therapy, human infertility and production of therapeutic biomolecules. The in-vitro cell culture technology is promising, fast evolving, robust and matured technology with huge potential to revolutionize the fields of medicine, biological sciences and biomedical-engineering.

Percutaneous microwave ablation of a transgenic large animal porcine liver tumor model after intra-arterial embolization

Objectives: Percutaneous ablation with microwave ablation (MWA) successfully treats hepatic tumors (HTs) up to 3 cm in size when appropriate margins are achieved. MWA is limited when treating larger HT due to the disbursement of heat from adjacent tissue and vasculature. Embolization before MWA can achieve a larger ablation zone (AZ); however, no evaluation has been performed to assess the influence of proximal or distal embolization on AZ. Material and Methods: Using a transgenic porcine liver tumor model, angiography and embolization of HT were performed with lipiodol or different-sized particles, ranging from 40 to 1200 µm to complete vascular occlusion followed by MWA for 4 min at 65 watts with subsequent ex vivo assessment of AZ. Results: AZ volume using 40 µm, 100 µm, and 300–500 µm microparticles were significantly larger than for the control, non-embolization group (mean ± standard deviation: 40 µm: 17.48 cm3 ± 1.22, P ≤ 0.001; 100 µm: 14.81 cm3 ± 0.43, P ≤ 0.001; and 300–500 µm: 12.16 cm3 ± 0.8, P ≤ 0.001 compared to 6.06 cm3 ± 2.02 in the control group. Conclusion: Distal embolization with smaller particles produced significantly larger AZ in an in vivo liver tumor when compared to no embolization control, lipiodol, or proximal large particle embolization.

Studying C9orf72 dipeptide repeat polypeptide aggregation using an analytical ultracentrifuge equipped with fluorescence detection

Sedimentation velocity, using an analytical ultracentrifuge equipped with fluorescence detection, and electrophoresis methods are used to study aggregation of proteins in transgenic animal model systems. Our previous work validated the power of this approach in an analysis of mutant huntingtin aggregation. We demonstrate that this method can be applied to another neurodegenerative disease studying the aggregation of three dipeptide repeats (DPRs) produced by aberrant translation of mutant c9orf72 containing large G4C2 hexanucleotide repeats. These repeat expansions are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We analyzed the aggregation patterns of (Gly-Pro)47, (Gly-Ala)50, and (Gly-Arg)50 fused to fluorescent proteins in samples prepared from D. melanogaster, and (Gly-Ala)50 in C. elegans, using AU-FDS and SDD-AGE. Results suggest that (GP)47 is largely monomeric. In contrast, (GA)50 forms both intermediate and large-scale aggregates. (GR)50 is partially monomeric with some aggregation noted in SDD-AGE analysis. The aggregation of this DPR is likely to represent co-aggregated states with DNA and/or RNA. The power of these methods is the ability to gather data on aggregation patterns and characteristics in animal model systems, which may then be used to interpret the mitigation of aggregation through genetic or molecular therapeutic interventions.