bioRxiv ScienceSearch

Biology subjects

Kahana, C.

Publications and source records attributed to Kahana, C..

2 recordsLinked to original sources

Polyamines support myogenesis by facilitating myoblast migration

The regeneration of the muscle tissue relies on the differentiation of myoblasts into myocytes, to create myotubes and myofibers. Disruption of key events in this process may interfere with the correct formation or repair of muscle tissue. Polyamines, ubiquitous polycations that are essential for fundamental cellular processes, were demonstrated necessary for myogenesis; however, the mechanism by which polyamines contribute to this process has not yet been deciphered. In the present study, we examined the effect of polyamine depletion on the muscle regeneration model of C2C12 myoblasts. Our results reveal a requirement for polyamines at the very beginning of the muscle differentiation process. Myogenesis is accompanied by polyamine synthesis, even though the myoblasts contain high levels of polyamines at the moment of induction. Polyamine depletion at the time of induction, or inability to synthesize more polyamines during the first 24 hours of the process, inhibited myogenesis. Polyamine depletion inhibited the expression of all tested myogenic markers (Pax7, MyoD, Myogenin, Myf5 and Myosin heavy chain), as well as the cells migration and fusion abilities. Real time PCR analysis revealed two key early activation and migration factors, HGF and Annexin A1.

cell biology

Simplifying the combined use of CRISPR-Cas9 and Cre-loxP technologies for the efficient generation of targeted conditional gene knockouts in mammalian cells

Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here we present a simple method that combines the use of CRISPR-Cas9 and Cre-loxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next CRISPR-Cas9 is used to knockout an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate potential mutants of the tested gene.

molecular biology