A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination

BACKGROUND: Cre-loxP recombination refers to the process of site-specific recombination mediated by two loxP sequences and the Cre recombinase protein. Transgenic experiments exploit integrative recombination, where a donor plasmid carrying a loxP site and DNA of interest integrate into a recipient loxP site in a target genome. Unfortunately, integrative recombination is highly inefficient because the insert is flanked by two loxP sites, which themselves become targets for Cre and lead to subsequent excision of the insert. A small number of mutations have been discovered in parts of the loxP sequence, specifically the spacer and inverted repeat segments, that increase the efficiency of integrative recombination. In this study we introduce a high-throughput in vitro assay to rapidly detect novel loxP spacer mutants and describe the sequence characteristics of successful recombinants. RESULTS: We created synthetic loxP oligonucleotides that contained a combination of inverted repeat mutations (the lox66 and lox71 mutations) and mutant spacer sequences, degenerate at 6 of the 8 positions. After in vitro Cre recombination, 3,124 recombinant clones were identified by sequencing. Included in this set were 31 unique, novel, self-recombining sequences. Using network visualization tools, we recognized 12 spacer sets with restricted promiscuity. We observed that increased guanine content at all spacer positions save for position 8 resulted in increased recombination. Interestingly, recombination between identical spacers was not preferred over non-identical spacers. We also identified a set of 16 pairs of loxP spacers that reacted at least twice with another spacer, but not themselves. Further, neither the wild-type P1 phage loxP sequence nor any of the known loxP spacer mutants appeared to be kinetically favoured by Cre recombinase. CONCLUSION: This study approached loxP spacer mutant screening in an unbiased manner, assuming nothing about candidate loxP sites save for the conserved 4 and 5 spacer positions. Candidate sites were free to recombine with any other sequence in the pool of all possible sites. The subset of loxP sites identified here are candidates for in vivo serial recombination as they have already demonstrated limited promiscuity with other loxP spacer and stability in the presence of Cre

Novel Therapies for Myocardial Irritability following Extreme Hydroxychloroquine Toxicity

Introduction. Hydroxychloroquine (HCQ) overdose is rare and potentially deadly when consumed in large doses. Management of severe HCQ toxicity is limited and infrequently reported. This report presents the case of a massive ingestion of HCQ. Case Report. A 23-year-old female presents following an intentional ingestion of approximately 40 g of HCQ. Within six hours after ingestion, she developed severe hemodynamic instability resulting from myocardial irritability with frequent ventricular ectopic activity leading to runs of polymorphic ventricular tachycardia (PMVT) and ventricular fibrillation (VF) requiring multiple defibrillations. Additional treatments included intravenous diazepam, epinephrine, norepinephrine, sodium bicarbonate, and magnesium sulfate. Despite the ongoing hemodynamic instability, the patient was also treated with Intralipid (ILE) and received hemodialysis. Improvements in her hemodynamics were observed after 18 hours. She survived her massive overdose of HCQ. Conclusion. HCQ poisoning is rare but serious because of its rapid progression to life-threatening symptoms. Hemodynamic support, gastric decontamination, electrolyte monitoring and replacement, and management of arrhythmias are the mainstays of treatment. The combined role of dialysis and ILE in the setting of massive HCQ overdose may improve outcomes

Satellog: A database for the identification and prioritization of satellite repeats in disease association studies

Background. To date, 35 human diseases, some of which also exhibit anticipation, have been associated with unstable repeats. Anticipation has been reported in a number of diseases in which repeat expansion may have a role in etiology. Despite the growing importance of unstable repeats in disease, currently no resource exists for the prioritization of repeats. Here we present Satellog, a database that catalogs all pure 1–16 repeat unit satellite repeats in the human genome along with supplementary data. Satellog analyzes each pure repeat in UniGene clusters for evidence of repeat polymorphism. Results A total of 5,546 such repeats were identified, providing the first indication of many novel polymorphic sites in the genome. Overall, polymorphic repeats were over-represented within 3'-UTR sequence relative to 5'-UTR and coding sequence. Interestingly, we observed that repeat polymorphism within coding sequence is restricted to trinucleotide repeats whereas UTR sequence tolerated a wider range of repeat period polymorphisms. For each pure repeat we also calculate its repeat length percentile rank, its location either within or adjacent to EnsEMBL genes, and its expression profile in normal tissues according to the GeneNote database. Conclusion Satellog provides the ability to dynamically prioritize repeats based on any of their characteristics (i.e. repeat unit, class, period, length, repeat length percentile rank, genomic co-ordinates), polymorphism profile within UniGene, proximity to or presence within gene regions (i.e. cds, UTR, 15 kb upstream etc.), metadata of the genes they are detected within and gene expression profiles within normal human tissues. Unstable repeats associated with 31 diseases were analyzed in Satellog to evaluate their common repeat properties. The utility of Satellog was highlighted by prioritizing repeats for Huntington's disease and schizophrenia. Satellog is available online at http://satellog.bcgsc.ca.Medicine, Faculty ofMolecular Medicine and Therapeutics, Centre forPsychiatry, Department ofScience, Faculty ofNon UBCReviewedFacult