Expression Pattern of the LacZ Reporter in Secretogranin III Gene-trapped Mice

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping ( SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system:

Inadvertent nucleotide sequence alterations during mutagenesis: highlighting the vulnerabilities in mouse transgenic technology

In the last three decades, researchers have utilized genome engineering to alter the DNA sequence in the living cells of a plethora of organisms, ranging from plants, fishes, mice, to even humans. This has been conventionally achieved by using methodologies such as single nucleotide insertion/deletion in coding sequences, exon(s) deletion, mutations in the promoter region, introducing stop codon for protein truncation, and addition of foreign DNA for functional elucidation of genes. However, recent years have witnessed the advent of novel techniques that use programmable site-specific nucleases like CRISPR/Cas9, TALENs, ZFNs, Cre/loxP system, and gene trapping. These have revolutionized the field of experimental transgenesis as well as contributed to the existing knowledge base of classical genetics and gene mapping. Yet there are certain experimental/technological barriers that we have been unable to cross while creating genetically modified organisms. Firstly, while interfering with coding strands, we inadvertently change introns, antisense strands, and other non-coding elements of the gene and genome that play integral roles in the determination of cellular phenotype. These unintended modifications become critical because introns and other non-coding elements, although traditionally regarded as “junk DNA,” have been found to play a major regulatory role in genetic pathways of several crucial cellular processes, development, homeostasis, and diseases. Secondly, post-insertion of transgene, non-coding RNAs are generated by host organism against the inserted foreign DNA or from the inserted transgene/construct against the host genes. The potential contribution of these non-coding RNAs to the resulting phenotype has not been considered. We aim to draw attention to these inherent flaws in the transgenic technology being employed to generate mutant mice and other model organisms. By overlooking these aspects of the whole gene and genetic makeup, perhaps our current understanding of gene function remains incomplete. Thus, it becomes important that, while using genetic engineering techniques to generate a mutant organism for a particular gene, we should carefully consider all the possible elements that may play a potential role in the resulting phenotype. This perspective highlights the commonly used mouse strains and the most probable associated complexities that have not been considered previously, resulting in possible limitations in the currently utilized transgenic technology. This work also warrants the use of already established mouse lines in further research.

Abstract 234: Identification of a Novel Cardiomyocyte-enriched Long Noncoding Rna Counteracting Against Heart Failure Development

Background: The number of patients with heart failure (HF) continues to increase as the elderly population grows, and mortality remains high, with about 50% of HF patients dying within 5 years of diagnosis. Therefore, defining molecular mechanisms underlying HF pathologies is urgently needed. Methods and Results: Using a gene trapping approach in mouse ES cells, we identified Caren (for car diomyocyte- en riched noncoding transcript), a novel cytoplasmic lncRNA abundantly expressed in cardiomyocytes. Caren expression in mice markedly decreased in heart failure (HF) induced by transverse aortic constriction (TAC), AngiotensinII loaded or aging. Caren ablation accelerated HF-related phenotypes, whereas Caren transgenic (Tg) mice in cardiomyocytes resisted TAC-induced HF. Furthermore, Caren overexpression in cardiomyocytes activated mitochondrial biogenesis and attenuated TAC-induced activation of Ataxia Telangiectasia Mutated (ATM) serine/threonine kinase, a regulator of the DNA damage response (DDR). Next, we generated Tg mice expressing Caren fragments (fragments A-E, from 5’ to 3’) respectively, by inserting that fragment into the corresponding endogenous Caren genomic locus. None of these Tg mice exhibited an anti-HF effect against TAC, suggesting that anti-HF effects of Caren may be more dependent on a structural motif displayed by the full-length lncRNA rather than on a specific Caren sequence. Consistently, restoration of expression of full-length Caren in TAC-induced failing cardiomyocytes slows HF progression in mice injected with AAV6- Caren . Mechanistically, Caren inhibited translation of mRNA encoding ‘ Caren Target Protein 1’ (CTP1), which activates the ATM-DDR pathway and reduces mitochondrial respiratory capacity in cardiomyocytes. CTP1 +/- mice resisted HF development, strongly suggesting that Caren protects against HF by suppressing CTP1 expression. Conclusion: This is the first study reporting that Caren , a novel cytoplasmic lncRNA , counteracts HF development and progression by inactivating the ATM-DDR pathway and activating mitochondrial bioenergetics, in part, by suppressing CTP1 translation in cardiomyocytes. Our findings could encourage development of RNA-based strategies to combat HF development.

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette. This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

We generated new genetic tools to efficiently tag genes in Drosophila. Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on CRE recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette.This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

Reappraisal of putative glyoxalase 1-deficient mouse and dicarbonyl stress on embryonic stem cells in vitro

Glyoxalase 1 (Glo1) is a cytoplasmic enzyme with a cytoprotective function linked to metabolism of the cytotoxic side product of glycolysis, methylglyoxal (MG). It prevents dicarbonyl stress — the abnormal accumulation of reactive dicarbonyl metabolites, increasing protein and DNA damage. Increased Glo1 expression delays ageing and suppresses carcinogenesis, insulin resistance, cardiovascular disease and vascular complications of diabetes and renal failure. Surprisingly, gene trapping by the International Mouse Knockout Consortium (IMKC) to generate putative Glo1 knockout mice produced a mouse line with the phenotype characterised as normal and healthy. Here, we show that gene trapping mutation was successful, but the presence of Glo1 gene duplication, probably in the embryonic stem cells (ESCs) before gene trapping, maintained wild-type levels of Glo1 expression and activity and sustained the healthy phenotype. In further investigation of the consequences of dicarbonyl stress in ESCs, we found that prolonged exposure of mouse ESCs in culture to high concentrations of MG and/or hypoxia led to low-level increase in Glo1 copy number. In clinical translation, we found a high prevalence of low-level GLO1 copy number increase in renal failure where there is severe dicarbonyl stress. In conclusion, the IMKC Glo1 mutant mouse is not deficient in Glo1 expression through duplication of the Glo1 wild-type allele. Dicarbonyl stress and/or hypoxia induces low-level copy number alternation in ESCs. Similar processes may drive rare GLO1 duplication in health and disease.