Study of transient expression of human apoA-1 gene under control of various promoters in CHO-K1 cells

The recombinant plasmids containing the genomic human apoA-1 gene under the transcriptional control of hybrid promoters, namely, the cytomegalovirus immediate early enhancer/chicken ?-actin promoter and first intron (CAG promoter) and cytomegalovirus immediate early enhancer/promoter with intron A (hCMVintronA promoter) have been constructed. The transient expression of human apoA-1 gene has been studied in CHO-K1 cells using designed and pTRapo vectors. The expression of the transgene for transfected with pTRapo and pTRapochCMVintronA CHO-K1 cells have been shown.

Introduction.The way of exogenous DNA delivery into mammalian cells is extremely important to study gene functions as well as to advance gene therapy.At present there are various approaches to the introduction of a heterologous DNA into mammalian cells, among them the usage of vectors, based on mammalian viruses, physical (electroporation in particular) and chemical (usage of liposomes, polycations etc) methods etc.Each of these approaches is aimed at solving definite practical tasks.

Ge nome and Its Regulation
Vectors, designed on the basis of viruses, are widely exploited in gene therapy due to their high specificity and effectiveness of gene transferring in vivo [1].However, the usage of virus vectors in gene therapy is related to such problems as toxicity, increased immunogenicity, and ability to activate protooncogenes and induce the formation of pathogenic variants of viruses due to recombination with endogenous viruses.Therefore, the elaboration of non-virus systems of genetic material introduction is a perspective direction in this area [2].
The plasmid vectors, which may be applied at cells transfection by physical or chemical methods, do not have disadvantages, inherent to virus vectors, though they are characterized by much lower effectiveness of transfection in vivo in comparison with the latter at their usage for genes transfection in vivo [3].On the other hand, the easiness of obtaining and manipulation with recombinant plasmids made them one of the main vectors for genes transfer to obtain stable transformants and transient cultures which may be used while producing recombinant proteins with therapeutic properties in mammalian cells in vitro [4].
It was shown that the expression level of genes, introduced into mammalian cells, depends on many reasons, including the strength of transcription regulatory elements [5].To correct disorder, caused by the deficiency of expression of a specific gene in the organism, high level of expression of a target transgene is often necessary to achieve physiological concentrations of the protein and therapeutic effect correspondingly.One of the perspective strategies of solving the problem, connected with a low level of transgenes expression, is optimization of already known regulatory sequences and search for stronger ones.The advantage of using strong regulatory elements is the possibility to decrease the amount of the vector, which is introduced in vivo to achieve therapeutic effect, or, in case of recombinant proteins production, to increase their production.To some degree it might solve the problem of low effectiveness of cells transfection by the plasmid vectors or decrease possible negative consequences at the usage of vectors, designed on the basis of viruses.
The purpose of this paper was the construction of expression vectors, containing genome variant of human apolipoprotein A-1 (apoA-1) gene under the transcriptional control of different hybrid elements, as well as the study of transient expression of human apoA-1 gene in CHO-K1 cells.
Materials and methods.Plasmids and bacterial strains.Genome variant of human apoA-1 gene was used as a transgene, the source of it was recombinant plasmid pTRapo [6].
The work with bacterial cultures, the transformation of E.coli cells, the restriction and ligation of DNA was performed by standard methods as described [8].The obtained recombinant plasmids were checked by the restriction analysis.
Isolation and purification of plasmid DNA.Plasmid DNA was isolated by the method of alkaline lysis [8].Additional purification of plasmid from RNA and proteins admixtures was conducted by treating the preparation with ribonuclease A (USB, USA), by the method of phenol-chloroform extraction [8] and PEG 8000 precipitation [9].DNA concentration and purity were defined by spectrophotometry according to the ratio A 260 /A 280 [8] on the Specord UV VIS device (CARL ZELSS JENA, Germany) as well as by electrophoresis method in 0.8% agarose gel.
Cells cultures.The cell line of Chinese hamster CHO-K1, obtained from the Russian collection of cell cultures (St.Petersburg) was used in the work.The cells were cultivated at 37°C in the atmosphere, containing 5% CO 2 , in the F10 medium (Sigma, USA) with the addition of 10% embryonic calf serum (Genom, Ukraine), 100 OD/ml penicillin and 100 mg/ml streptomycin (Kyivmedpreparat, Ukraine).
Cells transfection.Two days before transfection the cells were sown in the quantity of 8C10 5 on Petri dishes, 6cm diameter.The transfection was conducted using branched polyethyleneimine (PEI) (25 kDa, Aldrich, USA) by the method, described in the work [10].DNA/PEI preparation was prepared in mass ratio 1:2.For transfection 3.5ìg of plasmid DNA was applied to each dish.The time of cells contact with transforming mixture was 1 hour.
The transfection effectiveness was defined using the transfer of pTR-EGFP vector into CHO-K1 cells, which contains a marker gene of green fluorescent protein (egfp) [6].
The anal y sis of apoA-1 syn the sis.The level of human apoA-1 ac cu mu la tion in a cul tural me dium was deter mined by the meth ods of West ern-blot and dot-blot-anal y sis.The pro teins of cul tural me dium were con cen trated pre vi ously us ing trichloroacetic acid [11], the pre cip i tate was sep a rated from the supernatant by centrifugation, and dis solved in the buffer, con tain ing 4M urine and 0.1M Tris.The sam ples for pro teins sep ara tion in Ds-Na-PAAG were pre pared ac cord ing to the method [8].The pro teins were sep a rated in 12% Ds-Na-PAAG.For immunodetection the pro teins from gel were trans ferred to PVDF mem brane (Amersham Bio sci ences, Swe den).Free sites on mem brane were blocked by 3% so lu tion of non-fat dry milk.The membrane was in cu bated for 1 hour with monoclonal an tibod ies against hu man apoA-1 (MGHLaa 1/500, IMTEK, Rus sia).In the fol low ing ex per i ment the mem brane was in cu bated in the course of 1 hour with an ti bod ies against mouse G immunoglobulins, con ju gated with horse rad ish peroxidase (P-GAM 1/1000, IMTEK).Immune com plexes were visualized using the ECL method [12], and luminol (Sigma) as a substrate.
Chemiluminescence was registered using the X-ray film Retina XBM (Lizophorm, Ukraine).The obtained images were scanned and the quantitative estimation of signals intensity was performed using Total Lab program (Amersham Biosciences).Human apoA-1 protein (Sigma) was a standard of molecular weight and concentration.
The ApoA-1 protein content in cultural medium was also determined by the method of indirect immune-enzyme analysis (ELISA).
Results and discussion.The promoter of immediate early gene for human cytomegalovirus -hCMV-IE is most frequently used as an element of transcription regulation of heterologous genes in mammalian cells.First of all, it is conditioned by such characteristics as high transcription rate, absence of 212

Study of transient expression of human apoA-1
Fig ure 1.The scheme of ob tain ing the re com bi nant con struc tion pTRapohCMVâ-actin: apoA-1 -hu man gene of apolipoprotein A-1; hCMV IEcytomegalovirus im me di ate early gene enhancer/pro moter; CAG -hu man cytomegalovirus im me di ate early gne enhancer, chicken â-actin gene promoter, chicken first intron â-actin gene; ITR -in verted ter mi nal re peats of adenoassociated vi rus; pA polyadenilation site tissue specificity and, at the same time, size compactness (700 n.p.) [13,14].
It was shown that the including of some genome elements, introns and 3'-and 5'non-translated sequences, in particular, into cDNA of target genes increases the level of transgene expression both in vitro and in transgene animals [15].The mechanism of such sequences influence on the level of transgenes expression is not defined in all the cases, but it is thought to be connected with the phenomena of increasing transcription rate, the rise of stability, transport and/or the effectiveness of 3'-end mRNA formation at its maturing [16].
At pres ent a large quan tity of hy brid el ements of the tran scrip tion reg u la tion is cre ated [16][17][18].It was de ter mined in the pre vi ous works [5,19] us ing marker genes, that hy brid se quences -hu man cytomegalovirus im me di ate early enhancer/chicken 213 Y.M. Gilchuk, O.M. Sukhorada, T.A. Ruban, O.K. Toporova, V.A. Kordyum Fig ure 2. The scheme of ob tain ing the re com bi nant con struc tion pTRapohCMVintronA: apoA-1 -hu man gene of apolipoprotein A-1; hCMV IE -cytomegalovirus im me di ate early gene enhancer/pro moter; hCMVintronA -hu man cytomegalovirus im me di ate early gene enhancer and human cytomegalovirus im me di ate early gene enhancer with intron A; ITR -in verted ter mi nal re peats of adenoassociated vi rus; pApolyadenilation site â-actin pro moter with chicken first intron of â-actin gene (CAG se quence) and enhancer/pro moter of cytomegalovirus im me di ate early gene with intron A of the same gene (hCMV-intronA se quence) pro vide the high est level of transgene ex pres sion in vi tro and in vivo.There fore, the us age of these reg u la tory el e ments for the in crease of ex pres sion level of a tar get gene in mammalian cells is perspective.
To estimate the influence of abovementioned hybrid promoters on the expression of human apoA-1 gene in vitro we designed several plasmid vectors.
While designing the expression vector pTRapo-hCMVâ-actin in pTRapo plasmid on sites of restrictase Eco1051-XbaI there was a substitution of hCMV-IE promoter for hybrid CAG sequence (Fig. 1).
The pTRapohCMVintronA plasmid was designed through the intermediate construction pBluSKM-hCMVintronA.To obtain this plasmid the hybrid sequence hCMVintronA was cloned on the sites of restrictase EcoRI-PstI from the plasmid pTR-GT60 into a pBluSKM vector, which was hydrolized by restriction enzymes Eco-RI-PstI.Then promoter hCMV IE was substituted in plasmid pTRapo on the sites of EcoRI-XbaI restriction for hybrid sequence hCMVintronA (Fig. 2).
In the investigated vectors human apoA-1 gene was located under the control of different hybrid sequences: hCMVintronA, CAG and hCMV IE promoter.These elements have a common enhancerhuman cytomegalovirus immediate early gene enhancer and different promoters: human cytomegalovirus immediate early promoter in case of hCMV IE and sequence hCMVintronA, and chicken â-actin promoter for CAG element.Besides, hybrid sequences hCMVintronA and CAG include first introns: of human cytomegalovirus immediate early gene and chicken â-actin gene (Fig. 3).
The peculiarities of transgene expression under the control of mentioned elements were studied in vitro at transient synthesis of apoA-1 in CHO-K1 cells.The advantages of using transient transformers for such an analysis is the fact that the expression level does not depend on the "position effect" i.e. regulatory chromosome elements do not influence it, as transgene expression occurs from non-integrated vectors.Moreover, much less time is needed to conduct such investigations than to obtain and analyze stable transformants [18].
It is worth mentioning that human apoA-1 protein is secretory.ApoA-1 gene contains a sequence, coding its own signal peptide, which provides its secretion into the medium.In this regard apoA-1 was detected directly in the cultural medium according to the following scheme: in 24 hours after the transfection the medium was changed for non-serum one, next day it was collected completely and frozen, while the fresh one was added to the cells.Thus, the samples of cultural medium were taken on the 2 nd -5 th days after the cells transfection.
On the second day after transfection the presence of target protein was detected by the method of immune-chemical analysis in the cultural medium of cells, transfected by the expression vectors pTRapo and pTR-apohCMVintronA.
The example of experiment is presented at Fig. 4. The ELISA testing of cultural media using did not give positive results (the data are not shown), which is connected with insufficient sensitivity of the method for the detection of the quantity of apoA-1 protein, which 214 Study of transient expression of human apoA-1 Fig ure 3. The gen eral scheme of reg u la tory el e ments lo ca tion: ITR -in verted ter mi nal re peats of adenoassociated vi rus; pA -polyadenilation site; enh hCMV IE -hu man cytomegalovirus im me di ate gene early enhancer; P hCMV IE -hu man cytomegalovirus im me di ate early pro moter; intron A -hu man cytomegalovirus im me di ate early gene enhancer with intron A; P â-actin -chicken â-actin gene pro moter; â-actin intron -first intron of chicken â-actin gene; BGH pA -polyadenilation site of ox hor mone growth gene; apoA-1 -hu man gene of apolipoprotein A-1 was synthesized by CHO-K1 cells.The comparison of plasmids with different elements of transcription regulation showed that pTRapo-hCMVintronA construction provides a higher level of transgene expression than pTRapo plasmid.
The immunodetection showed that the level of apoA-1 synthesis by cells, transfected by pTRapohCMVintronA plasmid, amounts to about ~3nm of protein per 1ml of medium a day, while at cells transfection by pTRapo palsmid it is ~0.4nm/ml.Not high level of apoA-1 synthesis at the conditions of this experiment may be explained by insufficient effectiveness of cells transfection by a gene vector.
The dynamics analysis of protein accumulation in the samples of cultural medium using dot-blotting did not reveal apoA-1 protein starting from the 3 rd day.The transgene expression from plasmid vectors in vitro during a week was described for transient cultures, longer expression is impossible due to the loss of a transgene (at the elimination of plasmid DNA, division or death of cells).The level of transgene expression, controlled by hybrid CAG sequence, was lower than the sensitivity of the used analysis method (<0.2 ng/ml).The designed genetic constructions will be further employed for the obtaining and analysis of human apoA-1 gene expression by stable transformants of CHO-K1.
Conclusions.The recombinant plasmids were designed, containing genome variant of human qpoA-1 gene under the transcriptional control of different regulatory elements.The expression of human apoA-1 gene on the level of ~0.4 and ~3 ng of protein per 1ml of the medium was shown for CHO-K1 cells, transfected correspondingly by pTRapo and pTRapohCMVintronA plasmids on the 2 nd day after the transfection.The obtained data on the increase in human apoA-1 gene expression under the control of hybrid hCMVintronA sequence in comparison with the level of transgene expression under the hCMV IE promoter in general are in good agreement with the results of other researches.Êëþ÷åâûå ñëîâà: òðàíçèòîðíàÿ ýêñïðåññèÿ, àïîëèïîïðîòåèí À-1, ÑÍÎ-Ê1.