Molecular Biology

LncRNAs have been recently implicated in the epigenetic control of muscle differentiation and their functional characterization has traditionally relied upon in vitro models of myogenic differentiation. However, the use of experimental paradigms to specifically target lncRNAs expression in muscle stem cells (MuSCs), also known as satellite cells, represents an important requisite to interrogate their function in more physiological contexts. Since isolation and culture of single myofibers preserves satellite cells within their physiological niche underneath the surrounding basal lamina, this procedure represents the optimal approach to follow satellite cell dynamics ex-vivo, such as activation from quiescence, expansion of committed progenitors, differentiation, and self-renewal. Here, we detail an optimized protocol to isolate viable single myofibers from the extensor digitorum longus (EDL) skeletal muscle of adult mice and to manipulate the expression of lncRNAs by antisense LNA GapmeRs-mediated knock-down (KD). Furthermore, we describe a method of EdU incorporation that, coupled to lncRNA KD and subsequent immunofluorescence analysis of proliferating, differentiating, and satellite cell-specific markers, permits the inference of lncRNAs function on muscle stem cells dynamics.

Graphic abstract:

Graphical representation of the single myofiber isolation method. Experimental workflow showing the main steps of the protocol procedure: EDL muscle harvesting from the mouse hindlimb; EDL digestion into single myofibers; transfection with antisense oligos and culture for 96h; immunofluorescence protocol and image outcome.

Macrophages are immune cells that contribute to host defense through various mechanisms including phagocytosis and antigen presentation. Their antimicrobial capacity is subverted by clinically important intracellular pathogens such as Mycobacterium tuberculosis. The study of host-pathogen interactions using these cells is therefore of considerable interest. Such studies often seek to express tagged proteins to characterize their activities, localizations, and protein-protein interactions. Here, we describe a robust method for transient protein expression in macrophages using mRNA lipoplex transfections.

Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2) to create genetic-modified sheep. Procedure described sgRNA design, construction of gRNA-Cas9 plasmid, efficient detection in fibroblast, embryos and sheep, and some manipulative technologies. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by targeting multiple genes that are in charge of economically significant traits simultaneously.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 activity by sgRNA is to target it with two small RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Both of these small RNAs can be chemically synthesized which makes the production of these RNAs less difficult when compared to sgRNA. Another downside of the CRISPR/Cas9 systems is that off-target effects have been reported. However, modified forms of Cas9 have been developed to minimize off-target effects. For example, nickase-type Cas9 (nCas9) and FokI domain-fused catalytically-inactive Cas9 (FokI-dCas9; fCas9) induce DSBs only when two guide RNAs bind opposite strands within a defined distance. In this protocol, we describe our experimental system for the production of mutant mice using a CRISPR/Cas9 system that combines crRNA, tracrRNA, and modified forms of Cas9. This method not only facilitates the preparation of reagents for the genome editing system but it can also reduce the risk of off-target effects.

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier et al., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.

Aequorin is a Ca²⁺ sensitive photoprotein suitable to measure intracellular Ca²⁺ transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca²⁺ uptake and release of different intracellular organelles. Here, we describe how to use this probe to measure cytosolic Ca²⁺ levels and mitochondrial Ca²⁺ uptake in mammalian cells.

A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).

Small Interfering RNA (siRNA) is a class of double-stranded RNAs of 20-25 nucleotides that play important roles in many biological processes (Hamilton and Baulcombe, 1999). siRNAs act by “neutralizing” the mRNA of the target protein, facilitating degradation of the mRNA and hence altering the biological effect of the protein (reviewed in Hannon and Rossi, 2004). siRNAs may also change the intracellular levels of regulatory RNAs. Use of siRNAs for manipulating the expression of genes of interest in biological research is commonly referred to as RNA interference or knockdown technique (Elbashir et al., 2001). Synthetic siRNAs are an emerging tool that are now widely used in these studies. A variety of algorithms are employed by different companies for the design of siRNA products, which differ in efficacy, specificity and cost among other criteria. An example protocol of siRNA knockdown is explained here using the siGENOME SMARTpool reagents from Dharmacon.