Fused in sarcoma (FUS) protein lacking nuclear localization signal (NLS) and major RNA binding motifs triggers proteinopathy and severe motor phenotype in transgenic mice.

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1-359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1-359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5-4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1-359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.

Early lethality and neuronal proteinopathy in mice expressing cytoplasm-targeted FUS that lacks the RNA recognition motif.

Mutations to the RNA binding protein, fused in sarcoma (FUS) occur in ∼5% of familial ALS and FUS-positive cytoplasmic inclusions are commonly observed in these patients. Altered RNA metabolism is increasingly implicated in ALS, yet it is not understood how the specificity with which FUS interacts with RNA in the cytoplasm can affect its aggregation in vivo. To further understand this, we expressed, in mice, a form of FUS (FUS ΔRRMcyt) that lacked the RNA recognition motif (RRM), thought to impart specificity to FUS-RNA interactions, and carried an ALS-associated point mutation, R522G, retaining the protein in the cytoplasm. Here we report the phenotype and results of histological assessment of the brain of transgenic mice expressing this isoform of FUS. Results demonstrated that neuronal expression of FUS ΔRRMcyt caused early lethality often preceded by severe tremor. Large FUS-positive cytoplasmic inclusions were found in many brain neurons; however, neither neuronal loss nor neuroinflammatory response was observed. In conclusion, the extensive FUS proteinopathy and severe phenotype of these mice suggests that affecting the interactions of FUS with RNA in vivo may augment its aggregation in the neuronal cytoplasm and the severity of disease processes.

Transgenic animals in medicine: integration and expression of foreign genes, theoretical and applied aspects.

The production of different pharmaceutically important human proteins in the mammary gland of transgenic animals constitutes an important field of modern biotechnology. In addition, transgenic animals are used to develop suitable models of various human diseases and the possibility of using transgenic technologies to adapt pigs for xenotransplantation of their organs to humans is widely discussed. All these practical applications depend on the availability of reliable techniques to obtain transgenic animals with the necessary spectrum of transgene(s) expression. In the present review we discuss different aspects of producing transgenic animals by microinjection procedure, including both technical aspects and theoretical issues. As far as the technical aspects are concerned, special emphasis is placed on DNA preparation, microinjection timing, vectors permitting delivery of large genomic fragments, and approaches used to increase integration frequency. The molecular basis of the advantage of transgene microinjection into a male pronucleus is discussed. Finally, different strategies used to obtain polytransgenic animals are critically reviewed. In the section devoted to theoretical issues, the mechanisms of integration and molecular systems regulating transgene expression are extensively discussed. In particular, the importance of the domain-level regulatory systems is outlined and experimental approaches permitting the use of the domain-level regulatory systems of the genes encoding milk proteins are described and discussed.

Effect of DNA loop anchorage regions (LARs) and microinjection timing on expression of beta-galactosidase gene injected into one-cell rabbit embryos.

The conditions favoring expression of a reporter gene microinjected into a male pronucleus of naturally ovulated and fertilized rabbit eggs have been studied. Injection of the reporter gene during S phase of the cell-cycle allows the highest level of expression of the gene. Incorporation of DNA loop anchorage regions (LARs) into constructs upstream and/or downstream of the reporter gene significantly increased the efficiency of expression. In all cases the expression of the microinjected gene started after a period of transcriptional quiescence, i.e., together with the expression of the host genome. Correct targeting of microinjected constructs within the nuclei via interaction of LAR elements with receptor sites on the nucleoskeleton may facilitate expression of injected DNA constructs as well as their integration into host cell DNA.