Temperature-sensitive yeast mutants have been isolated that block each of the enzymatic steps in the synthesis of the dolichol-l
inked oligosaccharide precursor for n-linked glycosylation. why do mutations that block synthesis of the intermediate with the structure dolichol-pp-(glcnac)2man5 completely prevent addition of n-linked oligosaccharide chains to secretory proteins, whereas mutations that block conversion of this intermediate into the completed precursor—dolichol-pp-(glcnac)2man9glc3—allow the addition of n-linked oligosaccharide chains to secretory glycoproteins?
This is because the seven-sugar intermediate is synthesized by sugar addition to cytosolic-facing dolichol phosphate. The intermediate is flipped from the cytosol face of the ER membrane to the the luminal face. Additionally, the sugar additions then occur within the lumen of the ER. The short forms of the intermediate are on the wrong side of the membrane to add to nascent polypeptides within the ER lumen. Incomplete adductants within the ER lumen are located appropriately to N-glycosylate nascent polypeptide.
CRISPR can be used to reintroduce dystrophin back into the KO mouse
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and is used to for gene editing
CRISPR/Cas-mediated genome editing has been shown to permanently correct DMD mutations and restore dystrophin function in mouse models
Germline editing by injecting zygotes with CRISPR/Cas9 editing component was first done in mdx mice by correcting the mutated exon 23
Postnatal editing of mdx mice was then achieved using recombinant adeno-associated virus to deliver CRISPR/Cas9 genome editing components and correct the dystrophin gene by skipping or deleting the mutated exon 23 in vivo
Germline and postnatal CRISPR/Cas9 editing approaches both successfully restored dystrophin function in the mice and same technique can be used for KO mouse model
