Targeted Resequencing of CRISPR Cas9 Mediated ICER Knockout in SK-MEL-24 Cells

Inducible cAMP Early Repressor (ICER) is a small transcription factor that originates from an intronic promoter within the cAMP Response Element Modulator (CREM) gene (Molina et al., 1993). ICER acts as a putative tumor suppressor by mediating cAMP antiproliferative activity by competitively binding to cAMP Response Elements (CRE’s) and repressing transcription of genes involved in cell division (Mémin et al., 2002). ICER has been shown to be effectively absent in cancer and suspected to be targeted for proteasomal degradation via ubiquitination (Healey et al., 2013). ICER is most likely regulated via post-translational modifications as a result of mutations on Ras/Raf oncogenes (Healey et al., 2013). For example, it has been shown that in cancer, mutant activated GTP bound Ras protein continuously activates Braf, which in turn, results in over activation of the Mitogen Activated Protein Kinases, ERK1/ERK2 (Zhang and Liu, 2002). ICER is thought to be phosphorylated by ERK1/ERK2 and subject to proteasomal degradation; a result of ubiquitination in Ras/MAPK-mediated melanoma tumorigenesis (Healey et al., 2013). Melanoma cells rapidly apoptosed when transfected with a mutant form of ICER that did not contain any lysine residues. This was most likely because the transcriptional repressor ICER was unable to be ubiquitinated. While this highlights ICER’s importance in cell division and growth, it is challenging to study the effects of ICER in melanoma if cells are unable to survive. One method to circumvent this issue is to create an inducible cell line, in which a mutant form of ICER with no lysine residues is under a promoter whose expression can be toggled on and off, dependent on the presence of a transactivator. This requires that mutated ICER be knocked-in to an alternative location in the human genome, and that wildtype ICER be efficiently knocked-out in order to solely study the effects of the mutant. In this experiment, CRISPR Cas9 mediated genetic editing was used to knockout ICER, while mitigating off target effects on CREM gene, in an attempt to maintain otherwise normal cell physiology. The target loci of interest in this experiment was the Kozak consensus on an ICER specific promoter and the target of the guide RNA (gRNA) for Cas9 endonuclease activity. Amplicon Libraries of DNA extracted from cells transiently transfected with plasmid containing gRNA and Cas9-GFP cassette and empty vector expressing only EGFP (control) were generated with Nextera index adaptors. Paired-end sequencing on the Illumina Miseq provided sufficient coverage depth to determine how efficient the gRNA was at generating insertions/deletions (indels) or substitutions at the desired loci. Sequencing data between Experimental and Control was first reviewed using CRISPResso, an online bioinformatic tool that analyzes deep sequencing data. An additional bioinformatic analysis was designed and performed to corroborate CRISPResso results and identify any other low-level variants. The goal of this experiment was to develop the workflow to identify possible indels/substitutions that resulted from CRISPR Cas9 induced genetic alteration. This was done with the expectation of identifying a possible knockout of ICER, while minimally affecting CREM. Although no variants identified suggest an ICER knockout, a scalable workflow is now in place to facilitate this stage of the experiment

Continuous Academic Improvement: A Case Study Comparison of Private and Public School

What makes public and private schooling so different in California? This research explores in detail the factors schools pay attention to when it comes down to improving performanc

Mitochondrial genome diversity and evolution in Branchiopoda (Crustacea)

BackgroundThe crustacean class Branchiopoda includes fairy shrimps, clam shrimps, tadpole shrimps, and water fleas. Branchiopods, which are well known for their great variety of reproductive strategies, date back to the Cambrian and extant taxa can be mainly found in freshwater habitats, also including ephemeral ponds. Mitochondrial genomes of the notostracan taxa Lepidurus apus lubbocki (Italy), L. arcticus (Iceland) and Triops cancriformis (an Italian and a Spanish population) are here characterized for the first time and analyzed together with available branchiopod mitogenomes.ResultsOverall, branchiopod mitogenomes share the basic structure congruent with the ancestral Pancrustacea model. On the other hand, rearrangements involving tRNAs and the control region are observed among analyzed taxa. Remarkably, an unassigned region in the L. apus lubbocki mitogenome showed a chimeric structure, likely resulting from a non-homologous recombination event between the two flanking trnC and trnY genes. Notably, Anostraca and Onychocaudata mitogenomes showed increased GC content compared to both Notostraca and the common ancestor, and a significantly higher substitution rate, which does not correlate with selective pressures, as suggested by dN/dS values.ConclusionsBranchiopod mitogenomes appear rather well-conserved, although gene rearrangements have occurred. For the first time, it is reported a putative non-homologous recombination event involving a mitogenome, which produced a pseudogenic tRNA sequence. In addition, in line with data in the literature, we explain the higher substitution rate of Anostraca and Onychocaudata with the inferred GC substitution bias that occurred during their evolution

Software educativo y aprendizaje basado en problemas - ABP : estrategia didáctica para el fortalecimiento de habilidades en Ciencias Naturales

1 recurso en línea ( 158 páginas) : ilustraciones, tablas, gráficas.El presente trabajo incorpora una estrategia didáctica que se apoya en un instrumento guía que orienta el proceso de enseñanza y aprendizaje en ciencias naturales, específicamente en el tópico “ordenamiento de los seres vivos” siguiendo los pasos del aprendizaje basado en problemas – ABP, además este proceso genera una consulta o búsqueda de información en esta temática, la cual es proporcionada por un software educativo, pensado y diseñado para puntualizar conceptos, que permiten al estudiante mejorar las habilidades de indagación y explicación de fenómenos para esta área.Bibliografía y webgrafía: páginas 151-158.MaestríaMagíster en Ambientes Educativos mediados por TI

An introduction to scripting in Ruby for biologists

<p>Abstract</p> <p>The Ruby programming language has a lot to offer to any scientist with electronic data to process. Not only is the initial learning curve very shallow, but its reflection and meta-programming capabilities allow for the rapid creation of relatively complex applications while still keeping the code short and readable. This paper provides a gentle introduction to this scripting language for researchers without formal informatics training such as many wet-lab scientists. We hope this will provide such researchers an idea of how powerful a tool Ruby can be for their data management tasks and encourage them to learn more about it.</p

Effective detection of rare variants in pooled DNA samples using Cross-pool tailcurve analysis

Sequencing targeted DNA regions in large samples is necessary to discover the full spectrum of rare variants. We report an effective Illumina sequencing strategy utilizing pooled samples with novel quality (Srfim) and filtering (SERVIC4E) algorithms. We sequenced 24 exons in two cohorts of 480 samples each, identifying 47 coding variants, including 30 present once per cohort. Validation by Sanger sequencing revealed an excellent combination of sensitivity and specificity for variant detection in pooled samples of both cohorts as compared to publicly available algorithms

A primary breast cancer with distinct foci of estrogen receptor-alpha positive and negative cells derived from the same clonal origin as revealed by whole exome sequencing

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Background/purpose: Tumor heterogeneity is a now well-recognized phenomenon that can affect the classification, prognosis and treatment of human cancers. Heterogeneity is often described in primary breast cancers based upon histologic subtypes, hormone- and HER2-receptor status, and immunolabeling for various markers, which can be seen within a single tumor as mixed cellular populations, or as separate discrete foci. Experimental design/methods: Here, we present a case report of a patient’s primary breast cancer that had two separate but adjacent histologic components, one that was estrogen receptor (ER) positive, and the other ER negative. Each component was subjected to whole exome sequencing and compared for gene identity to determine clonal origin. Results: Using prior bioinformatic tools, we demonstrated that both the ER positive and negative components shared many variants, including passenger and driver alterations. Copy number variations also supported the two components were derived from a single common clone. Conclusions: These analyses strongly suggest that the two ER components of this patient’s breast cancer were derived from the same clonal origin. Our results have implications for the evolution of breast cancers with mixed histologies, and how they might be best managed for optimal therapy