Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.

Abstract

We have inactivated the endogenous apolipoprotein E (apoE) gene by using gene targeting in mouse embryonic stem (ES) cells. Two targeting plasmids were used, pJPB63 and pNMC109, both containing a neomycin-resistance gene that replaces a part of the apoE gene and disrupts its structure. ES cell colonies targeted after electroporation with plasmid pJPB63 were identified by the polymerase chain reaction (PCR) followed by genomic Southern analysis. Of 648 G418-resistant colonies analyzed, 9 gave a positive signal after PCR amplification, and 5 of them were confirmed as targeted by Southern blot analysis. The second plasmid, pNMC109, contains the negatively selectable thymidine kinase gene in addition to the neomycin-resistance gene. After electroporation with this plasmid, 177 colonies resistant both to G418 and ganciclovir were analyzed; 39 contained a disrupted apoE gene as determined by Southern blotting. Chimeric mice were generated by blastocyst injection with 6 of the targeted lines. One of the lines gave strong chimeras, three of which transmitted the disrupted apoE gene to their progeny. Mice homozygous for the disrupted gene were produced from the heterozygotes; they appear healthy, even though they have no apolipoprotein E in their plasma.

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA

“…22 Animals and intramuscular injections C57BL/6J apoE-deficient mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were maintained on a normal chow diet. 46 ApoE-deficient mice received injections of plasmid DNA dissolved in 225 mm NaCl. DNA injection was performed into the rectus femoris muscles using a disposable insulin syringe with a sterile 29-gauge needle.…”

Role of H3-Receptor-Mediated Signaling in Anxiety and Cognition in Wild-Type and Apoe–/– Mice

“…Mice deficient in apoE (Apoe -/-) (Piedrahita et al, 1992;Plump et al, 1992) have been used to define the role of apoE in brain function. Cardiac and serum histamine levels are higher in Apoe -/-than wild-type mice and Apoe -/-mice have 37% more cardiac mast cells (Huang et al, 2002).…”

Inhibition of atherosclerosis in apolipoprotein-E-deficient mice following muscle transduction with adeno-associated virus vectors encoding human apolipoprotein-E

“…16,17 ApoE is normally expressed predominantly by hepatocytes and macrophage cells of haematopoietic lineage, and transplantation of normal bone marrow cells into apoE -/-mice results in increased lipoprotein clearance, reversal of hypercholesterolaemia, and protection against development of atherosclerosis. 18,19 Liver-directed somatic gene transfer with recombinant adenovirus vectors (rAds) containing the apoE3 cDNA has also been shown to result in transient normalisation of lipoprotein and cholesterol plasma profiles and inhibition of atherosclerotic lesion development.…”

“…16 The animals were maintained on a normal chow diet and blood samples were taken after a 4 h fast from the tail vein. The rAAV vector stocks were diluted to 6.25 × 10 11 viral particles/ml using PBS-MK and 40 l (2.5 × 10 10 virus particles) of either rAAV/apoE2 or rAAV/apoE3 were injected into both TA muscles of mice anaesthetised by intra-peritoneal administration of a 25% solution of hypnorm and hypnovel (3 l per gram body weight).…”

“…Each muscle was thawed out in 0.5 ml of RNAlater (Ambion, Abingdon, UK) and sliced into small pieces, followed by isolation of total RNA with Trizol Reagent (Life Technologies), according to the manufacturer's instructions. DNase treated RNA (1 g) was reverse transcribed with Superscript II RNase H -Reverse Transcriptase (Life Technologies) and an oligo (dT) [12][13][14][15][16][17][18] primer at 42°C, following the manufacturer's instructions. The derived cDNA (1 l from a 20 l reaction) was used to amplify a 227 bp product using a primer set specific for the human apoE sequence.…”