Generation of an Endogenous MAGP2 Mutant via CRISPR-Cas9 to Investigate the Role of Enzymatic Cleavage
Başlık:
Generation of an Endogenous MAGP2 Mutant via CRISPR-Cas9 to Investigate the Role of Enzymatic Cleavage
Yazar:
Song, Ann, author.
ISBN:
9780438053540
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (62 pages)
Genel Not:
Source: Masters Abstracts International, Volume: 57-06M(E).
Advisors: Alison Miyamoto Committee members: Esther Chen; Math Cuajungco.
Özet:
Microfibril-associated glycoprotein 2 (MAGP2) is a small secreted protein associated with elastic fibers in the extracellular matrix. MAGP2 function may be regulated by cleavage of the last 20 amino acids by proprotein convertases (PC); for instance, PC cleavage promoted the binding of exogenously expressed MAGP2 to microfibrils in immunofluorescence studies. In order to test endogenous MAGP2, a cell culture model carrying a mutation in the PC cleavage site of MAGP2 was generated using the homology directed repair (HDR)-mediated CRISPR-Cas9 system to study MAGP2 expressed at natural levels. Specifically, a MAGP2 gRNA, Cas9 plasmid, and PC mutation donor plasmid were co-transfected into immortal human dermal fibroblasts to study the role of PC cleavage in MAGP2 microfibril binding. However, only random integration of the mutation was achieved, possibly due to low transfection efficiency. Thus, CRISPR plasmids were transfected into HEK293 cells that have high transfection efficiency, and three heterozygous mutants were successfully generated. This finding is consistent with reported CRISPR studies using HDR; homozygosity is much more difficult to obtain than heterozygosity. Future studies could include using a more efficient gene delivery system for site-specific integration. Additionally, HDR could be promoted using inhibitors to repress the nonhomologous end joining pathway.
Notlar:
School code: 6060
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(689840.1) | 689840-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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