engleski

Establishment of recombinant mouse embryonic stem cells for use in DNA flexibility assays

Using quantitative measurement of DNA flexibility in HEK293 cells by methods of site-specific recombination and Southern blot, it has been observed that in vivo DNA flexibility is double that of in vitro DNA, which has been ascribed to nucleosomal DNA wrapping and chromatin remodeling. Silencing of development-specific genes by local chromatin remodeling could also cause a change in DNA flexibility. The aim of this Master's thesis was to prepare the mouse embryonic stem cells (mESCs) for the comparison of DNA flexibility with the neural stem cells (NSCs). In this work, gene cloning was used to prepare a series of ten plasmids containing the loxP sites of different distances (from 70 bp to 2359 bp), 5' and 3' homology to Nanog gene and blasticidin resistance gene. β-galactosidase test was used to assess the inducilibity of mESCs containing tamoxifen- inducible CreERT2 recombinase and loxP-lacZ reporter gene, which is utilized for inducibility test and as a reference in flexibility measurement. Also, probes for radioactive Southern blotting were produced by gene cloning. Using gene targeting assisted by CRISPR-Cas9, all 10 targeting constructs were successfully inserted into the mNanog gene, which was verified by Southern blotting. Lastly, a method for additional gene targeting validation based on the polymerase chain reaction was developed.

DNA flexibility ; chromatin remodeling ; mouse embryonic stem cells ; neural stem cells ; Cre-loxP ; CRISPR-Cas9 ; Southern blot

nije evidentirano