Genetic Engineering and Competitiveness of Livestock Production

Our ability to modify whole animal genetics has grown considerably in the last two decades. We have seen concerns regarding food safety and protection of breeding rights of genetically modified animals compel redirection of genetic engineering experimentation toward biomedical applications. Indeed, it has been nearly twenty years since the first transgenic livestock appeared in the literature, yet at this time, there are no commercially viable agricultural species. In contrast to commercialization concerns, in a variety of existing transgenic animal models, basic research into the regulation and function of specific genes (including both gain-of-function and ablation of potentially deleterious gene products) has persevered. Pioneering efforts in transgenic animal technology have markedly influenced our appreciation of the factors that govern gene regulation and expression, and have contributed significantly to our understanding of the biology of mammalian development

Klotho pathways, myelination disorders, neurodegenerative diseases, and epigenetic drugs

In this review we outline a rationale for identifying neuroprotectants aimed at inducing endogenous Klotho activity and expression, which is epigenetic action, by definition. Such an approach should promote remyelination and/or stimulate myelin repair by acting on mitochondrial function, thereby heralding a life-saving path forward for patients suffering from neuroinflammatory diseases. Disorders of myelin in the nervous system damage the transmission of signals, resulting in loss of vision, motion, sensation, and other functions depending on the affected nerves, currently with no effective treatment. Klotho genes and their single-pass transmembrane Klotho proteins are powerful governors of the threads of life and death, true to the origin of their name, Fates, in Greek mythology. Among its many important functions, Klotho is an obligatory co-receptor that binds, activates, and/or potentiates critical fibroblast growth factor activity. Since the discovery of Klotho a little over two decades ago, it has become ever more apparent that when Klotho pathways go awry, oxidative stress and mitochondrial dysfunction take over, and age-related chronic disorders are likely to follow. The physiological consequences can be wide ranging, potentially wreaking havoc on the brain, eye, kidney, muscle, and more. Central nervous system disorders, neurodegenerative in nature, and especially those affecting the myelin sheath, represent worthy targets for advancing therapies that act upon Klotho pathways. Current drugs for these diseases, even therapeutics that are disease modifying rather than treating only the symptoms, leave much room for improvement. It is thus no wonder that this topic has caught the attention of biomedical researchers around the world.https://www.liebertpub.com/doi/10.1089/biores.2020.0004Published versio

Pathogenic mitochondrial dysfunction and metabolic abnormalities

Herein we trace links between biochemical pathways, pathogenesis, and metabolic diseases to set the stage for new therapeutic advances. Cellular and acellular microorganisms including bacteria and viruses are primary pathogenic drivers that cause disease. Missing from this statement are subcellular compartments, importantly mitochondria, which can be pathogenic by themselves, also serving as key metabolic disease intermediaries. The breakdown of food molecules provides chemical energy to power cellular processes, with mitochondria as powerhouses and ATP as the principal energy carrying molecule. Most animal cell ATP is produced by mitochondrial synthase; its central role in metabolism has been known for >80 years. Metabolic disorders involving many organ systems are prevalent in all age groups. Progressive pathogenic mitochondrial dysfunction is a hallmark of genetic mitochondrial diseases, the most common phenotypic expression of inherited metabolic disorders. Confluent genetic, metabolic, and mitochondrial axes surface in diabetes, heart failure, neurodegenerative disease, and even in the ongoing coronavirus pandemic.https://doi.org/10.1016/j.bcp.2021.11480

Transgenetic animal technology : a laboratory handbook

xv, 618 p. : il.; 24 cm

Nuclear Expression of a Mitochondrial DNA Gene: Mitochondrial Targeting of Allotopically Expressed Mutant ATP6 in Transgenic Mice

Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE) represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M) or wildtype (A6W) mt-Atp6 transgene. Compared to non-transgenic controls, A6M mice displayed neuromuscular and motor deficiencies (wire hang, pole, and balance beam analyses; P<0.05), no locomotor differences (gait analysis; P<0.05) and enhanced endurance in Rota-Rod evaluations (P<0.05). A6W mice exhibited inferior muscle strength (wire hang test; P<0.05), no difference in balance beam footsteps, accelerating Rota-Rod, pole test and gait analyses; (P<0.05) and superior performance in balance beam time-to-cross and constant velocity Rota-Rod analyses (P<0.05) in comparison to non-transgenic control mice. Mice of both transgenic lines did not differ from non-transgenic controls in a number of bioenergetic and biochemical tests including measurements of serum lactate and mitochondrial MnSOD protein levels, ATP synthesis rate, and oxygen consumption (P>0.05). This study illustrates a mouse model capable of circumventing in vivo mitochondrial mutations. Moreover, it provides evidence supporting AE as a tool for mtDNA disease research with implications in development of DNA-based therapeutics

Allotopic Expression of ATP6 in the Mouse as a Model of Targeted Mitochondrial DNA Mutation

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2010.Animal modeling of mitochondrial DNA (mtDNA) mutations has trailed nuclear transgenesis due to a range of cellular and physiological distinctions. mtDNA mutation modeling is of critical importance as mutations in the mitochondrial genome give rise to many pathological conditions. The T to G mutation at nucleotide 8993 of the human mitochondrial genome (ATP6) results in a wide range of clinical manifestations that are broadly grouped into Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa (NARP) or Maternally Inherited Leigh Syndrome (MILS) designations. Nuclear localization and transcription of mtDNA genes followed by cytoplasmic translation and transport into mitochondria provide an opportunity to create in vivo modeling of a targeted mutation in mitochondrial genes. A study was undertaken to develop a mutation model where the mtDNA T8993G mutation was engineered for allotopic expression from the cell’s nucleus. Two murine ATP6 genes were synthesized de novo and cloned into a mammalian expression vector. One ATP6 gene vector encoded the T8993G mutation (A6M); the other coded for the wild-type mouse ATP6 sequence (A6W). Both genes were cloned with nuclear codon substitutions and a Cox VIII N-terminal mitochondrial transport signal. Transgenic mice generated using these constructs allotopically express the recombinant ATP6 protein in mitochondria of brain, heart and skeletal muscle tissues. Moreover, allotopically expressed ATP6 protein is assembled into ATP synthase complexes. Expressed as a percentage, allotopically expressed protein was present in 47-61% of ATP synthase complexes in various tissues. Transgenic mice were subjected to a battery of neuromuscular tasks. Compared to non-transgenic controls, A6M mice display neuromuscular/motor deficiencies in wire hang, pole, and balance beam analyses (p0.05) when compared to non-transgenic control mice. Mice of both transgenic lines did not differ from non-transgenic controls in a number of bioenergetic and biochemical tests including measurements of serum lactate and mitochondrial MnSOD protein levels, ATP synthesis rate, and oxygen consumption (p>0.05). The generalized modest neuromotor impairment seen in A6M transgenic mice suggests a comparison to the NARP phenotype seen in some human patients. Potentially conflicting functional evidence (increased Rota-Rod performance) together with the surprising lack of a biochemical phenotype provide opportunity for further characterizing the pathways leading from mtDNA mutation to functional deficit and eventual disease states. Allotopic expression of a mitochondrial gene in vivo provides a resource for studying mechanisms of pathogenesis in diseases resulting from mitochondrial DNA mutation. This study also provides evidence for the potential utility of allotopic expression as a strategy for gene replacement therapy in patients harboring mitochondrial DNA mutations

NDUFS4 : creation of the first Complex I mouse model mimicking Leigh Syndrome

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2007.Leigh syndrome is an inherited neurometabolic disorder primarily affecting muscle tissue and the nervous system. Disease manifestations are often observed in infants and can be caused by a number of separate mutations involved in energy production and more specifically in the mitochondrial electron transport chain. Mutations in mitochondrial proteins can lead to Leigh syndrome via either pyruvate dehydrogenase deficiency or a bottleneck in the electron transport chain function. The proteins affected by this energetic deficiency have been shown to be involved in support of the electron transport chain or in Complexes I-V directly. Various mutations in the NADH dehydrogenase-ubiquinone-FeS 4 (NDUFS4) gene have been associated with decreased Complex I activity in mitochondrial electron transport and Leigh syndrome. A mouse model harboring a point mutation in the NDUFS4 gene was created to effectively truncate the encoded protein product. The underlying hypothesis was that this truncation would cause decreased Complex I activity which would in turn lead to a Leigh disease phenotype. In initial matings, homozygosity of the point mutation appeared lethal. Therefore, mice heterozygous for the point mutation were characterized. Mitochondria obtained from heterozygotes demonstrated a decreased respiration rate following use of glutamate and malate as substrates with no effect following succinate as a substrate; representing hallmarks of a Complex I disorder. Further, a decreased Complex I activity with steady-state Complex II activity, combined with an increased lactate accumulation, were consistent with Leigh syndrome phenotype. Accordingly, this model will allow for characterization of current and future therapeutic treatments; from dietary mediation to proposed gene therapies. By dissecting the various manifestations of disease at the whole animal, tissue, and cellular levels, coupled with examination of putative alternative endogenous pathways – insights into Leigh syndrome disease progression can now be addressed