Methods in molecular biology
-
Gene Editing in Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible CRISPR-Cas9 System.
Induced pluripotent stem cells (iPSCs) generated from patients are a valuable tool for disease modelling, drug screening, and studying the functions of cell/tissue-specific genes. However, for this research, isogenic iPSC lines are important for comparison of phenotypes in the wild type and mutant differentiated cells generated from the iPSCs. The advent of gene editing technologies to correct or generate mutations helps in the generation of isogenic iPSC lines with the same genetic background. ⋯ An iPSC line with drug inducible Cas9 expression from the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus offers a controllable expression of Cas9 with robust gene editing. Here, we describe a stepwise protocol for the generation and characterization of such an iPSC line (AAVS1-PDi-Cas9 iPSC) with a doxycycline (dox)-inducible Cas9 expression cassette from the AAVS1 safe harbor site and efficient editing of target genes with lentiviral vectors expressing gRNAs. This approach with a tunable Cas9 expression that allows investigating gene functions in iPSCs or in the differentiated cells can serve as a versatile tool in disease modelling studies.
-
With rapid advances in experimental instruments and protocols, imaging and sequencing data are being generated at an unprecedented rate contributing significantly to the current and coming big biomedical data. Meanwhile, unprecedented advances in computational infrastructure and analysis algorithms are realizing image-based digital diagnosis not only in radiology and cardiology but also oncology and other diseases. Machine learning methods, especially deep learning techniques, are already and broadly implemented in diverse technological and industrial sectors, but their applications in healthcare are just starting. ⋯ Moreover, the applications of genomics data are realizing the potential for personalized medicine, making diagnosis, treatment, monitoring, and prognosis more accurate. In this chapter, we discuss machine learning methods readily available for digital pathology applications, new prospects of integrating spatial genomics data on tissues with tissue morphology, and frontier approaches to combining genomics data with pathological imaging data. We present perspectives on how artificial intelligence can be synergized with molecular genomics and imaging to make breakthroughs in biomedical and translational research for computer-aided applications.
-
Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder affecting many children. The disease is caused by the lack of dystrophin production and characterized by muscle wasting. The most common causes of death are respiratory failure and heart failure. ⋯ Here, we present methodologies to systemically inject PMOs into humanized DMD model mice and determine levels of dystrophin restoration via Western blotting. Using a tris-acetate gradient SDS gel and semi-dry transfer with three buffers, including the Concentrated Anode Buffer, Anode Buffer, and Cathode Buffer, less than 1% normal levels of dystrophin expression are easily detectable. This method is fast, easy, and sensitive enough for the detection of dystrophin from both cultured muscle cells and muscle biopsy samples.
-
The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. ⋯ To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3'-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.
-
The number of studies published in the biomedical literature has dramatically increased over the last few decades. This massive proliferation of literature makes clinical medicine increasingly complex, and information from multiple studies is often needed to inform a particular clinical decision. However, available studies often vary in their design, methodological quality, and population studied, and may define the research question of interest quite differently. ⋯ In addition, since even highly cited trials may be challenged over time, clinical decision-making requires ongoing reconciliation of studies which provide different answers to the same question. Because it is often impractical for readers to track down and review all the primary studies, systematic reviews and meta-analyses are an important source of evidence on the diagnosis, prognosis and treatment of any given disease. This chapter summarizes methods for conducting and reading systematic reviews and meta-analyses, as well as describes potential advantages and disadvantages of these publications.