91311 (Ефекти, обумовлені введенням у клітини ссавців трансгена аполіпопротеїну А-1 людини), страница 5

The goal of this work was the construction of plasmid vectors, containing genome variant of human apolipoprotein A-1 (APOA1) gene under the transcriptional control of different elements, as well as the study of transient expression of human APOA1 gene in CHO-K1 cells, the examination of the transportation and the distribution of human APOA1 containing plasmid DNA, as a part of complexes of DNA/polyethyleneimine (PEI) in rats and rabbits, the investigation of the time course clearance of target gene and human APOA1 expression in vivo.

The recombinant plasmids containing the genomic human APOA1 gene under the transcriptional control of promoters, namely, the cytomegalovirus immediate early enhancer/chicken β-actin promoter and first intron (CAG promoter) and cytomegalovirus immediate early enhancer/promoter with intron A (hCMV-IA promoter) have been constructed. The transient expression of human APOA1 gene has been studied in CHO-K1 cells using designed and pTRhCMV-IEapo (cytomegalovirus immediate early enhancer/ promoter (hCMV-EI)) vectors. The expression of the transgene for transfected with pTRhCMV-IEapo and pTRhCMV-IAapo CHO-K1 cells have been shown. The expression of human APOA1 on the level of ~ 0,4 and ~ 3 ng of protein per 1 ml of the medium was shown for CHO-K1 cells, transfected correspondingly by pTRhCMV-IEapo and pTRhCMV-IAapo plasmids on the 2 nd day after the transfection.

The recombinant plasmids (pTRhCMV-IEapo-neo^R, pTRhCMV-IAapo neo^R and pTRCAGapo neo^R), containing neomycinphosphotransferase gene (neo^R), have been constructed for obtaining stable transformants. The expression of human APOA1 gene for all pools of stable transformants has been detected. The higher level of expression of the transgene for pools obtained by transfection with pTRhCMV-IAapo neo^R and pTRCAGapo neo^R than with pTRhCMV-IEapo-neo^R has been shown.

The study of the transfer of the plasmid DNA, containing human APOА1 gene, in complex with PEI into rat and rabbit liver parenchyma and time period of transgene clearance in vivo has been performed. The presence of human APOA1 gene was revealed in genetic material, in all organs of experimental animals, the tissues of which had to be analyzed (liver, heart, lungs, kidneys, aorta and lymph nodes). The result obtained may testify in favour of indirect DNA transfer and may be explained by the fact that the part of liver-introduced plasmid DNA gets into the blood stream with intercellular liquid and lymph or with venous blood through capillaries, and is spread in organism. The presence of human APOA1 gene was discovered over the time of 25^th and 85^th days after plasmid injection in total DNA of liver cells rat and rabbit, accordingly.

There are two issues that make the APOA1 human protein analysis more difficult, namely, blood containing animal AроA1 in rather high concentration and molecular weight of APOA1, which does not differ essentially for different species. The selection of system of reagents for immune-chemical detection of human APOA1 protein in blood plasma of rabbits using both ELISA and Western-blot was the part of our work. Therefore, we have optimized the Western-blot method of detection for human APOA1 in blood of rabbits.

Investigating the synthesis of human APOA1 in vivo the rabbits under investigation were divided into two groups. The first group of rabbits was introduced repetitive injection of human APOA1 gene as a part of pTRhCMV-IEapo vector, the second group (control) − repetitive injection of pTR plasmid containing no target gene. All rabbits have been fed a cholesterol-rich diet from day of plasmid injection. On the 6th day after injection, human APOA1 protein in blood plasma of first group of rabbits was detected using the Western-blot method, while in blood plasma of the second group of rabbits it was not detected (sensitivity of Western-blot method was ~ 1 μg of human protein/ml of rabbit blood plasma). Having performed quantitative assessment of transgene expression, the concentration of human APOA1 in blood plasma was 1,6–20,2 μg/ml from rabbit to rabbit. It has been shown that repetitive injection of plasmid DNA, containing human APOA1 gene, in the complex with PEI into the liver parenchyma of cholesterol-rich diet rabbits influenced cholesterol concentration in animal blood and morphology of tissues. Thus, APOA1 injected animals were shown not to reveal the increase in total cholesterol concentration in blood and morphology alteration of liver and vessel tissues, specific for atherosclerosis in cholesterol-fed rabbits.

Key words: apolipoprotein A-1, polyethylenimine, transgene, expression.