Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models

The development in cryobiology in animal breeding had revolutionized the field of reproductive medicine. The main objective to preserve animal germplasm stems from variety of reasons such as conservation of endangered animal species, animal diversity, and an increased demand of animal models and/or genetically modified animals for research involving animal and human diseases. Cryopreservation has emerged as promising technique for fertility preservation and assisted reproduction techniques (ART) for production of animal breeds and genetically engineered animal species for research. Slow rate freezing and rapid freezing/vitrification are the two main methods of cryopreservation. Slow freezing is characterized by the phase transition (liquid turning into solid) when reducing the temperature below freezing point. Vitrification, on the other hand, is a phenomenon in which liquid solidifies without the formation of ice crystals, thus the process is referred to as a glass transition or ice-free cryopreservation. The vitrification protocol applies high concentrations of cryoprotective agents (CPA) used to avoid cryoinjury. This chapter provides a brief overview of fundamentals of cryopreservation and established methods adopted in cryopreservation. Strategies involved in cryopreserving germ cells (sperm and egg freezing) are included in this chapter. Last section describes the frontiers and advancement of cryopreservation in some of the important animal models like rodents (mouse and rats) and in few large animals (sheep, cow etc).

The Online Genetically Modified Food Debate: Sociotechnical Imaginaries and Genetically Modified Animals

The aim of this article is to investigate the sociotechnical imaginaries present in UK online news articles and below the line comments in connection with genetically modified animals. This article attempts to provide an answer through a qualitative study using discourse analysis. The findings reveal how sociotechnical imaginaries present in news articles depict genetically modified animals as ‘other’ in comparison to those bred through selective breeding. In the below the line comments, a key feature is of monstrosity. Here, the sociotechnical imaginaries draw on the concept of ‘other’ along with the imagery of Frankenstein. Nature also features in the sociotechnical imaginaries in the news articles. Journalists present genetic modification as overcoming nature, as well as scientists designing nature. The article concludes by discussing how sociotechnical imaginaries can bring invisible nonhuman animals to the fore. Here, difference makes genetically modified animals newsworthy.

Genetically modified animals for use in biopharmacology: from research to production

Introduction: In this review, the analysis of technologies for obtaining biologically active proteins from various sources is carried out, and the comparative analysis of technologies for creating producers of biologically active proteins is presented. Special attention is paid to genetically modified animals as bioreactors for the pharmaceutical industry of a new type. The necessity of improving the technology of development transgenic rabbit producers and creating a platform solution for the production of biological products is substantiated. The advantages of using TrB for the production of recombinant proteins: The main advantages of using TrB are the low cost of obtaining valuable complex therapeutic human proteins in readily accessible fluids, their greater safety relative to proteins isolated directly from human blood, and the greater safety of the activity of the native protein. The advantages of the mammary gland as a system for the expression of recombinant proteins: The mammary gland is the organ of choice for the expression of valuable recombinant proteins because milk is easy to collect in large volumes. Methods for obtaining transgenic animals: The modern understanding of the regulation of gene expression and the discovery of new tools for gene editing can increase the efficiency of creating bioreactors for animals and help to obtain high concentrations of the target protein. The advantages of using rabbits as bioreactors producing recombinant proteins in milk: The rabbit is a relatively small animal with a short duration of gestation, puberty and optimal size, capable of producing up to 5 liters of milk per year per female, receiving up to 300 grams of the target protein.

Transgenes of genetically modified animals detected non-invasively via environmental DNA

We demonstrate that simple, non-invasive environmental DNA (eDNA) methods can detect transgenes of genetically modified (GM) animals from terrestrial and aquatic sources in invertebrate and vertebrate systems. We detected transgenic fragments between 82–234 bp through targeted PCR amplification of environmental DNA extracted from food media of GM fruit flies (Drosophila melanogaster), feces, urine, and saliva of GM laboratory mice (Mus musculus), and aquarium water of GM tetra fish (Gymnocorymbus ternetzi). With rapidly growing accessibility of genome-editing technologies such as CRISPR, the prevalence and diversity of GM animals will increase dramatically. GM animals have already been released into the wild with more releases planned in the future. eDNA methods have the potential to address the critical need for sensitive, accurate, and cost-effective detection and monitoring of GM animals and their transgenes in nature.

Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats

Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future.

Degeneration of Bioprosthetic Heart Valves: Update 2020

The implantation of bioprosthetic heart valves (BHVs) is increasingly becoming the treatment of choice in patients requiring heart valve replacement surgery. Unlike mechanical heart valves, BHVs are less thrombogenic and exhibit superior hemodynamic properties. However, BHVs are prone to structural valve degeneration (SVD), an unavoidable condition limiting graft durability. Mechanisms underlying SVD are incompletely understood, and early concepts suggesting the purely degenerative nature of this process are now considered oversimplified. Recent studies implicate the host immune response as a major modality of SVD pathogenesis, manifested by a combination of processes phenocopying the long‐term transplant rejection, atherosclerosis, and calcification of native aortic valves. In this review, we summarize and critically analyze relevant studies on (1) SVD triggers and pathogenesis, (2) current approaches to protect BHVs from calcification, (3) obtaining low immunogenic BHV tissue from genetically modified animals, and (4) potential strategies for SVD prevention in the clinical setting.

Genetic variation in the social environment affects behavioral phenotypes of oxytocin receptor mutants in zebrafish

Oxytocin-like peptides have been implicated in the regulation of a wide range of social behaviors across taxa. On the other hand, the social environment, which is composed of conspecifics that may vary in their genotypes, also influences social behavior, creating the possibility for indirect genetic effects. Here, we used a zebrafish oxytocin receptor knockout line to investigate how the genotypic composition of the social environment (Gs) interacts with the oxytocin genotype of the focal individual (Gi) in the regulation of its social behavior. For this purpose, we have raised wild-type or knock-out zebrafish in either wild-type or knock-out shoals and tested different components of social behavior in adults. GixGs effects were detected in some behaviors, highlighting the need to control for GixGs effects when interpreting results of experiments using genetically modified animals, since the genotypic composition of the social environment can either rescue or promote phenotypes associated with specific genes.

Genetic variation in the social environment affects behavioral phenotypes of oxytocin receptor mutants in zebrafish

Oxytocin-like peptides have been implicated in the regulation of a wide range of social behaviors across taxa. On the other hand, the social environment, which is composed of conspecifics genotypes, is also known to influence the development of social behavior, creating the possibility for indirect genetic effects. Here we used a knockout line for the oxytocin receptor in zebrafish to investigate how the genotypic composition of the social environment (Es) interacts with the oxytocin genotype (G) of the focal individual in the regulation of its social behavior. For this purpose, we have raised wild-type or knock-out zebrafish in either wild-type or knock-out shoals and tested different components of social behavior in adults. GxEs effects were detected in some behaviors, highlighting the need to control for GxEs effects when interpreting results of experiments using genetically modified animals, since the social environment can either rescue or promote phenotypes associated with specific genes.