Mohri, Zahra; (2004) Recombinant cells and chimeraplasty as tools to study atheroprotective effects of apolipoprotein E. Doctoral thesis (Ph.D), UCL (University College London).

Text Recombinant_cells_and_chimerap.pdf Download (16MB)

Abstract

Apolipoprotein (apo) E is secreted by liver and macrophages and is atheroprotective, in part by contributing to plasma cholesterol homeostasis. Three common isoforms arise from single nucleotide polymorphisms (SNPs): the rarest variant, apoE2, differs from wild-type apoE3 by an R158C substitution, and causes recessive Type III hyperlipidaemia, while apoE4 (C112R) produces a dominant hyperlipidaemia. One aim of this thesis was to determine whether lipidated apoE3 particles, newly-secreted by recombinant Chinese hamster ovary (CHO), were biologically active, using CHO cells engineered to secrete antiatherogenic apoAI as positive controls. An ELISA was established to quantify levels of secreted apoE, while apoAI was measured by immunoblotting and scanning densitometry. Although secreted apoAI was about 55 % better at activating the cholesterol esterifying enzyme, LCAT (lecithin-cholesterol acyltransferase), than apoE after 0.5 (P<0.05) or 1 h (P<0.001) of incubation, both particles were equally effective at promoting cholesterol efflux. These data suggest that these atheroprotective actions may be normal physiological roles for apoE. The second aim was to test the hypothesis that: 'synthetic RNA-DNA oligonucleotides (chimeraplasts) can convert the APOE3 gene to mutant APOE2 and APOE4 in human hepatoblastoma (HepG2) and human monocyte-macrophage (THP-1) cells'. Such new cell lines, secreting dysfunctional apoE2 or apoE4, would allow the atheroprotective properties of the three main human apoE isoforms to be compared in future studies. Initially, CHOE3 and human embryonic kidney (HEK-293; ԑ3/ԑ3) cells, which are both readily transfected, were targeted with apoE3-to-apoE2 chimeraplast complexed with polyethylenimine (PEI); the expected conversions were detected. Dose-dependent conversions using apoE3-to-apoE2 or apoE3-to-apoE4 chimeraplast were also seen in HepG2 cells, as judged by direct sequencing and PCR-RFLP, whereas THP-1 cells proved refractory and the conversion was limited. Trials with end-protected all-DNA molecules proved unsuccessful. Increasing efficiency was subsequently investigated: by using different PEIs (linear vs. branched, and tagging with melittin, galactose or mannose); by centrifugation to enhance cell-complex contact; and by nuclear microinjection. Linear PEI (L-PEI) alone worked best with the apoE3-to-apoE2 chimeraplast, while galactose-4-PEI mixed with L-PEI (1:1) gave good conversion with the apoE3-to-apoE4 reagent. Unexpectedly, the conversion appeared unstable in both THP-1 and HepG2 cells after freeze-thawing and/ or repeated passaging. Attempts to understand this problem were then hampered by difficulties in synthesizing active chimeraplasts. In conclusion, although an emerging technology with enormous potential, chimeraplast-directed gene mutation/repair remains problematical and factors such as chimeraplast quality and design, gene target and cell type all require detailed study in future investigations.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as JSONCSVXML

Archive Staff Only

View Item