Generation of four gene-edited human induced pluripotent stem cell lines with mutations in the ATM gene to model Ataxia-Telangiectasia.

Ataxia-Telangiectasia (A-T) is an autosomal recessive multi-system disorder caused by mutations in the ataxia-telangiectasia mutated (ATM) gene, resulting, among other symptoms, in neurological dysfunction. ATM is known to be a master controller of signal transduction for DNA damage response, with additional functions that are poorly understood. CRISPR/Cas9 technology was used to introduce biallelic mutations at selected sites of the ATM gene in human induced pluripotent stem cells (hiPSCs). This panel of hiPSCs with nonsense and missense mutations in ATM can help understand the molecular basis of A-T.

In Cerebellar Atrophy of 12-Month-Old ATM-Null Mice, Transcriptome Upregulations Concern Most Neurotransmission and Neuropeptide Pathways, While Downregulations Affect Prominently Itpr1, Usp2 and Non-Coding RNA.

The autosomal recessive disorder Ataxia-Telangiectasia is caused by a dysfunction of the stress response protein, ATM. In the nucleus of proliferating cells, ATM senses DNA double-strand breaks and coordinates their repair. This role explains T-cell dysfunction and tumour risk. However, it remains unclear whether this function is relevant for postmitotic neurons and underlies cerebellar atrophy, since ATM is cytoplasmic in postmitotic neurons. Here, we used ATM-null mice that survived early immune deficits via bone-marrow transplantation, and that reached initial neurodegeneration stages at 12 months of age. Global cerebellar transcriptomics demonstrated that ATM depletion triggered upregulations in most neurotransmission and neuropeptide systems. Downregulated transcripts were found for the ATM interactome component Usp2, many non-coding RNAs, ataxia genes Itpr1, Grid2, immediate early genes and immunity factors. Allelic splice changes affected prominently the neuropeptide machinery, e.g., Oprm1. Validation experiments with stressors were performed in human neuroblastoma cells, where ATM was localised only to cytoplasm, similar to the brain. Effect confirmation in SH-SY5Y cells occurred after ATM depletion and osmotic stress better than nutrient/oxidative stress, but not after ATM kinase inhibition or DNA stressor bleomycin. Overall, we provide pioneer observations from a faithful A-T mouse model, which suggest general changes in synaptic and dense-core vesicle stress adaptation.

Contemporary Transposon Tools: A Review and Guide through Mechanisms and Applications of Sleeping Beauty, piggyBac and Tol2 for Genome Engineering.

Transposons are mobile genetic elements evolved to execute highly efficient integration of their genes into the genomes of their host cells. These natural DNA transfer vehicles have been harnessed as experimental tools for stably introducing a wide variety of foreign DNA sequences, including selectable marker genes, reporters, shRNA expression cassettes, mutagenic gene trap cassettes, and therapeutic gene constructs into the genomes of target cells in a regulated and highly efficient manner. Given that transposon components are typically supplied as naked nucleic acids (DNA and RNA) or recombinant protein, their use is simple, safe, and economically competitive. Thus, transposons enable several avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture comprising the generation of pluripotent stem cells, the production of germline-transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species and therapy of genetic disorders in humans. This review describes the molecular mechanisms involved in transposition reactions of the three most widely used transposon systems currently available (Sleeping Beauty, piggyBac, and Tol2), and discusses the various parameters and considerations pertinent to their experimental use, highlighting the state-of-the-art in transposon technology in diverse genetic applications.