Biotechnol Lett DOI 10.1007/s10529-014-1563-9

ORIGINAL RESEARCH PAPER

Distance effect of matrix attachment regions on transgene expression in stably transfected Chinese hamster ovary cells Jun-He Zhang • Xiao-Yin Wang • Tian-Yun Wang • Fang Wang • Wei-Hua Dong • Li Wang • Chun-Peng Zhao Shu-Jie Chai • Rui Yang • Qin Li

•

Received: 2 April 2014 / Accepted: 19 May 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The b-globin matrix attachment regions (MARs) were inserted into the 50 -site of the eukaryotic expression vector cassette and DNA fragments 350 and 750 bp in length were inserted into the site to generate expression vectors with varying distances between the expression cassette and MAR. The vectors containing MARs increased chloramphenicol acetyltransferase (CAT) expression levels compared to the negative control vector lacking the MAR; the highest expression increase was 3.8-fold. A greater MAR-transgene distance (750 bp) correlated with a greater increase in transgene expression when compared to the control vector that lacked separation between the MAR and transgene. CAT gene copy numbers were higher in cells transformed with the J.-H. Zhang  X.-Y. Wang  T.-Y. Wang (&)  F. Wang  W.-H. Dong  L. Wang  C.-P. Zhao Department of Biochemistry and Molecular Biology, Xinxiang Medical University, No. 601 Jinsui Road, Xinxiang 453003, Henan Province, China e-mail: [email protected] S.-J. Chai The Third Affiliated Hospital of Xinxiang Medical University, Xinxiang 453003, China R. Yang Department of Life Science and Technology, Xinxiang Medical University, Xinxiang 453003, China Q. Li Laboratory of Analytical and Testing, Xinxiang Medical University, Xinxiang 453003, China

vector possessing a smaller MAR-transgene distance (350 bp) than in cells belonging to the other three groups. However, MAR-induced transgene expression levels did not exhibit a direct relationship with gene copy number. Keywords Chinese hamster ovary cells  Chloramphenicol acetyltransferase  Distance effect  b-Globin matrix attachment region  Matrix attachment region  Transgene expression

Introduction Transgene silencing and low gene expression levels in mammalian cells are key issues that must be resolved for efficient application of transgenic technology. To obtain stably-expressing transgenic strains, researchers have developed numerous procedures to circumvent transgene silencing, including the use of strong promoters or enhancers in expression vector constructs and demethylation treatments (Finn et al. 2011; Pfaff et al. 2013). The use of matrix attachment regions (MARs) to enhance foreign gene expression is an effective strategy developed to overcome foreign gene silencing. MARs are specific DNA sequences in the eukaryotic chromatin that can interact with the nuclear matrix. The main features of MARs include AT-rich base pairs and characteristic sequence elements such as A-boxes (AATAAAAA/CAA), T-boxes

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(TTTTATTTTT), autonomously replicating sequences (ARS) in yeast, and topoisomerase II recognition sites in Drosophila, which flank non-coding regions of genes. As novel eukaryotic cis-acting elements, MARs reportedly increase transgene expression (Buceta et al. 2011; Girod et al. 2005; Harraghy et al. 2011, 2013; Ley et al. 2013). MARs improve the levels of transgene expression and decrease transgene silencing in transgenic animals and plants, as well as decreasing transgene expression variability in different transformants (Kim et al. 2004; Linnemann et al. 2009; Mock et al. 2012). However, the effects of MARs on transgene expression are not consistent between studies and the underlying mechanism of MAR function remain unclear. Several studies have shown that MARs increase transgene expression levels in stably-transfected Chinese hamster ovary (CHO) cells (Zahn-Zabal et al. 2001; Kim et al. 2004; Wang et al. 2008). In addition, the inclusion of MARs with different sources in the expression cassette can significantly improve transgene expression (Wang et al. 2012) and show significant position effects (Wang et al. 2010). However, studies have yet to be performed to determine whether MAR-regulated transgene expression is related to the distance between the MAR and transgene. In the present study, we have inserted DNA fragments of varying length between the b-globin MAR and transgenes to investigate the mechanism underlying MAR-regulated transgene expression. The constructed expression vectors were transfected into CHO cells and the positional effect of MARs on transgene expression was analyzed.

Materials and methods Vector construction Primers (P1, 50 -TTAGTAAGACATCACCTTGCAT TT-30 and P2, 50 -AGCCATAGTTTGAGTTACCC TTT-30 ) were designed according to a previously reported human b-globin MAR sequence (GenBank accession no. L22754). Genomic DNA was extracted from human peripheral blood and used as a template to amplify the human b-globin MAR by using PCR. To directionally clone the b-globin MAR, we introduced NheI and KpnI restriction enzyme sites to the 50 -ends of the primers. PCR was performed as follows: 95 °C for 3 min; 94 °C for 40 s; 60–56 °C for 30 s; 72 °C for 40 s; and 72 °C for 3 min, with four cycles performed

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at each annealing temperature and then 20 cycles at 55 °C. The T-vector (TaKaRa Bio, Inc., Dalian, China) containing MAR fragments and the pCATG plasmid (Wang et al. 2008) were digested using specific restriction enzymes. The resulting fragments were ligated, transformed, and amplified using standard methods. To construct the intermediate vector pCAD, the following primers were designed based on the enhanced green fluorescent protein (EGFP) sequence of the pEGFP-C1 vector (GenBank accession no. U55 763): P3, 50 -CCGGCTAGCGCTACCGGTCGCC A-30 ; P4, 50 -CGCAGATCTCTGAGTGCGGACTTG-30 ; and P5, 50 -AATAGATCTCGGCGCGGGTCTTGTA-30 . P3 and P4 were used to amplify a 750 bp fragment and P3 and P5 were used to amplify a 350 bp fragment. BglII and NheI restriction enzyme sites were also introduced. The pEGFP-C1 plasmid was extracted and used as a template. Two DNA fragments of differing size were amplified using PCR and then directionally inserted between MAR and the pCAD vector expression cassettes to construct pCAC1 and pCAC2 (Fig. 1). The constructed vectors were validated using restriction enzyme digestion and sequencing. Cell culture and transfection CHO cells (Institute of Laboratory Animal Sciences, Beijing, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10 % (v/v) inactivated fetal bovine serum (Sijiqing, Hangzhou, China) at 37 °C and 5 % CO2. These cells were then inoculated into six-well plates with approximately 3 9 106 cells/well. Transfected cells were divided into the following four groups: pCAC1 transfection group; pCAC2 transfection group; pCAD positive transfection control group; and pCATG negative control group. Transfection was performed using a Sunma-sofast gene transfection kit (Sunma Biotechnology Co Ltd, Xiamen, China) according to the manufacturer’s instructions. 24 h after transfection, the cells were screened in DMEM supplemented with 700 lg G418/ml (Calbiochem) for 2 weeks. The culture medium was replaced every 3 days. Cells were digested using 0.25 % trypsin after a stably-transformed cell colony was formed. Each transfected cell group was treated by limiting dilution and was monocloned, and then distributed into 96-well plates for further culture for 7 days. The cells were subsequently transferred into 10 ml flasks, cultured until the

Biotechnol Lett

Fig. 1 Plasmid vectors used in this study. a Control vector pCATG without the MAR element; b expression vector pCAD containing MAR at the 50 site of the CAT reporter gene cassette; c expression vector pCAC1 containing MAR and 750 bp

fragment at the 50 -site of the CAT reporter gene cassette; d expression vector pCAC2 containing MAR and 350 bp fragment at the 50 -site of the CAT reporter gene cassette

cell confluence reached 80–90 %, and collected for analysis.

USA) according to the manufacturer’s instructions. Experiments were performed in triplicate. Quantitative PCR

Chloramphenicol acetyltransferase assays Cells were collected, counted, and adjusted to 106 cells/ml before being washed with cold PBS three times. Approximately 1 ml lysis buffer was added (fivefold dilution) and gently mixed with the cells. The resulting mixture was incubated at room temperature for 30 min and centrifuged at *10,0009g for 15 min at 4 °C. The supernatants were then removed and 200 ll was transferred to Eppendorf tubes. Positive and negative controls were also performed. The chloramphenicol acetyltransferase (CAT) content was assayed using a CAT enzyme-linked immunosorbent assay (ELISA) kit (Roche, Indianapolis, IN,

Screened stable cell lines were collected for each group. Genomic DNA of the transgenic cell lines was extracted and used as a template in quantitative PCR (qPCR). CAT primer sequences were as follows: P1, 50 -CATCGCTCTGGAGTGAATACC-30 ; P2, 50 -GGC ATCAGCACCTTGTCG-30 ; and internal control primers P3, 50 -GTCTTTCTTCTGCCGTTCTC-30 and P4, 50 -ACCAGCCTCATTAGGTTTGT-30 . The SYBR Green I fluorescent dye method was used and the target and internal control genes were amplified using an ABI 7500 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). The PCR reactions (20 ll) contained the following components:

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Fig. 2 Restriction endonuclease digestion identification results of the vectors. a pCAC1 vector; b pCAC2 vector; c pCAD vector. Lane 1 digested with KpnI/BglII; lane 2 digested with Bg1II; lane 3 undigested plasmid; M DL5000 DNA ladder

10 ll 29 UltraSYBR Mixture, 1 ll template DNA, 0.5 ll each of the forward and reverse primers, and 10 ll RNase-free water. The following PCR conditions were used: 95 °C denaturation for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The 2DDCt method was used to analyze the relative CAT gene copy numbers. Experiments were performed in triplicate. Statistical analysis Experimental data were statistically analyzed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Measured data were analyzed using analysis of variance at a test level of a = 0.05.

Results

Table 1 Statistical comparison of CAT gene expression of CHO cells transfected with different vectors Items

Vectors pCAC1

pCAC2

pCAD

pCATG

Sample (n)

15

15

15

15

Mean expression

0.3031

0.1725

0.122

0.0807

SD

0.1912

0.1395

0.1298

0.0625

CV

0.6308

0.8087

1.0639

0.7745

Folda

3.8*

2.1*

1.5*

–

Foldb

2.5*

1.4*

–

SD standard deviation, CV coefficient of variation a

Represents pCAC1, pCAC2 and pCAD versus pCATG, *p \ 0.01

b

Represents pCAC1 and pCAC2 versus pCAD, *p \ 0.01

sequencing. Therefore, expression vectors with differing MAR-transgene distances were successfully constructed.

Vector construction CAT expression analysis Constructed vectors were digested using different restriction endonucleases to generate single or double cuts. A 770 bp fragment was obtained from the pCAD vector by KpnI and NheI digestion and DNA was linearized by NheI single digestion, indicating that the MAR vector was successfully constructed (Fig. 2). Fragments with sizes of 1520 and 1120 bp were obtained from pCAC1 and pCAC2 vectors, respectively when digested using KpnI and BglI and DNA was linearized by BglI single digestion (Fig. 2). These fragment sizes were consistent with our predicted results. Vector construction was also confirmed using

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The constructed expression vectors were transformed into CHO cells; the pCATG plasmid lacking the MAR sequence was used as a control. Stably expressing cell lines were obtained using G418 selection and the cells were collected. CAT activity was then analyzed. The MAR-containing expression vectors increased the expression level of the CAT reporter gene to different extents compared to the negative control pCATG vector lacking the MAR sequence. pCAC1 and pCAC2 vectors increased CAT expression by 3.8- and 2.1-fold, respectively. In contrast, the pCAD vector increased

Biotechnol Lett Table 2 Analysis of CAT gene relative copy number from different vectors in stably-transfected CHO cell strains

Fig. 3 Analysis of the amount of CAT from different vectors in the stable transfected CHO cells. pCAC1, pCAC2, pCAD and pCATG were respectively transformed into CHC cells, 15 stably transformants of CHO cells by plasmids pCAC1, pCAC2, pCAD and pCATG were randomly selected, and CAT enzyme amount were analyzed by ELISA method. Each column represents CAT protein of CHO cells transfected by different vectors. 1, pCAC1; 2, pCAC2; 3, pCAD; 4, pCATG

reporter gene expression by 1.5-fold (Table 1). The pCAC1 vector (750 bp MAR-transgene distance) led to an average 2.5-fold increase in CAT expression levels, whereas an average 1.4-fold increase was observed in response to the pCAC2 vector (350 bp MAR-transgene distance) compared to the pCAD vector in which MAR was directly connected to the control expression cassette (Table 1). CAT expression levels differed significantly in cells transfected with the various expression vectors (p \ 0.01; Fig. 3). CAT gene copy number assay The stably-transformed cell lines generated for each vector were randomly selected and genomic DNA was extracted. The relative CAT gene copy number was increased in the three groups of cells stably transformed with MAR-containing vectors. CAT copy number in cells transformed with pCAC2 was higher than that in the cells belonging to the three other groups (p \ 0.01). In cells transformed with pCAC1, the CAT copy number was lower than in those transformed with pCAD, the lowest number among the three MAR-containing groups (Table 2). ELISA analysis indicated that the expression level of the pCAC1 vector was 2.5-fold higher than that of pCAD. Therefore, CAT expression levels were not proportional to the gene copy numbers in different MARcontaining transgenic cell lines, suggesting the effect was copy number-independent.

Groups

Ct mean (vector)

Ct mean (b-actin)

DCt mean

DDCt

Fold

pCAC1

34.89

26.78

8.11

-0.33

1.3

pCAC2

33.28

24.57

8.71

-0.93

2.0

pCAD

35.11

26.87

8.24

-0.46

1.4

pCATG

32.43

24.65

7.78

0

1

Discussion An objective of genetic engineering is to obtain efficient and stable transgenic cell lines. However, in the application of transgenic technology, transgene silencing and low expression levels have impeded the progress of genetic engineering research. At present, several strategies have been developed to overcome transgene silencing at the level of chromatin structure. MARs, which link the chromatin skeleton with genomic DNA sequences, have been widely investigated. Since MAR-regulated transgene expression was first reported (Breyne et al. 1992), several studies have shown that MARs can improve the stability of foreign gene expression levels and overcome gene inactivation (Jin et al. 2012; Sjeklocha et al. 2011). Thus far, MAR-related studies have mainly focused on transgenic plants (Festa et al. 2013; Xu et al. 2011). As a eukaryotic expression system, commonly used CHO cells have many advantages for the production of complex molecules such as monoclonal antibodies (Zahn-Zabal et al. 2001). Therefore, an important research direction is to improve stable and efficient expression of foreign genes in CHO cells. Previous studies showed that MARs improve the stability of foreign gene expression, and different MARs elicit variable effects on transgene expression. To clarify the mechanism of MAR in regulating gene expression, we constructed three expression vectors containing the human b-globin MAR at different positions. The vector containing two MARs flanking the CAT expression cassette led to the highest transgene expression. Expression levels were significantly higher when the MAR was inserted into the 50 site than when it was inserted into the 30 -site. MAR located at different locations in vectors differentially regulate transgene expression, thus exhibit an evident position effect (Wang et al. 2010). Wang et al. (2012)

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showed that MARs flanking the expression cassette can more effectively promote transgene expression when they come from different sources than those having the same source, a phenomenon referred to as the ‘‘stacking effect’’. These findings were consistent with the results of Liu and Tabe (1998). Taken together, these data indicate that the effect of MARs on transgene expression has location dependence and species specificity. In this study, DNA fragments with varying lengths were inserted between the b-globin MAR and transgene to construct expression vectors with unequal MAR-transgene distances. The effects of MAR-transgene distance on transgene expression were analyzed in transfected CHO cells. The results showed that the MAR-containing expression vectors increased CAT reporter gene expression to different extents compared to the control vector lacking the MAR sequence (p \ 0.01). This result is consistent with those of previous studies (Grandjean et al. 2011; Kim et al. 2004; Noguchi et al. 2012). As a boundary element, MARs define gene fragments in different chromatin loops. Therefore, the transgene forms an independent structure after gene fragments integrate into a genome receptor loop, leading to enhanced transgene expression (Fukuda and Nishikawa 2003). A long MARtransgene distance improves transgene expression. A MAR-transgene distance of 750 bp induced an average increase in CAT expression of 2.5-fold, whereas a 1.4-fold increase was observed when the distance was 350 bp. MARs elicited different effects on transgenic expression depending where they were inserted relative to the expression cassette, a phenomenon called the ‘‘distance effect’’. This finding suggests that different chromatin packaging modes exist, in which MARs differentially affect chromatin organization based on the distance between the transgene and MAR. Insertion of longer fragments corresponds to higher transcription activities of foreign genes and evident changes in regional chromatin structure. While the expression levels of foreign genes are enhanced, expression level differences exist between different strains (Ji et al. 2013; Johnson and Levy 2005). Real-time PCR analysis of CAT gene copy number suggested that increased transgene expression might be correlated to increased gene copy number. Consistent with our previous findings (Wang et al. 2010), CAT expression levels in different MAR-containing

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transgenic cell lines was not proportional to the gene copy numbers, indicating that the effect was copy number-independent. However, whether the effect of MARs on transgene expression is related to gene copy numbers remains controversial. MARs can also increase gene copy numbers (Buceta et al. 2011; Oh et al. 2005); however, this is in marked contrast to the results of this study. This relationship warrants further investigation. In conclusion, we have observed a distance effect in MAR-regulated transgene expression. Longer MARtransgene distances corresponded to enhanced transgene expression. However, we have not investigated longer regions and only looked at two lengths (350 and 750 bp), whether there is a relationship between length and expression and what is the optimum length remains unclear, and this needs further study. Acknowledgment This work was supported by the National Natural Science Foundation of China (No.31371332). Conflict of interest All authors have no conflict of interest regarding this paper.

References Breyne P, van Montagu M, Depicker N, Gheysen G (1992) Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco. Plant Cell 4:463–471 Buceta M, Galbete JL, Kostic C, Arsenijevic Y, Mermod N (2011) Use of human MAR elements to improve retroviral vector production. Gene Ther 18:7–13 Festa M, Brun P, Piccinini R, Castagliuolo I, Basso B, Zecconi A (2013) Staphylococcus aureus Efb protein expression in Nicotiana tabacum and immune response to oral administration. Res Vet Sci 94:484–489 Finn TE, Wang L, Smolilo D, Smith NA, White R, Chaudhury A, Dennis ES, Wang MB (2011) Transgene expression and transgene-induced silencing in diploid and autotetraploid Arabidopsis. Genetics 187:409–423 Fukuda Y, Nishikawa S (2003) Matrix attachment regions enhance transcription of a downstream transgene and the accessibility of its promoter region to micrococoal nuclease. Plant Mol Biol 51:665–675 Girod PA, Nguyen DQ, Calabrese D, Puttini S, Grandjean M, Martinet D, Regamey A, Saugy D, Beckmann JS, Bucher P, Mermod N (2007) Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nat Methods 4:747–753 Grandjean M, Girod PA, Calabrese D, Kostyrko K, Wicht M, Yerly F, Mazza C, Beckmann JS, Martinet D, Mermod N (2011) High-level transgene expression by homologous

Biotechnol Lett recombination-mediated gene transfer. Nucleic Acids Res 39:e104 Harraghy N, Regamey A, Girod PA, Mermod N (2011) Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections. J Biotechnol 154:11–20 Harraghy N, Buceta M, Regamey A, Girod PA, Mermod N (2012) Using matrix attachment regions to improve recombinant protein production. Methods Mol Biol 801:93–110 Ji L, Xu R, Lu L, Zhang J, Yang G, Huang J, Wu C, Zheng C (2013) TM6, a novel nuclear matrix attachment region, enhances its flanking gene expression through influencing their chromatin structure. Mol Cell 36:127–137 Jin Y, Liu Z, Cao W, Ma X, Fan Y, Yu Y, Bai J, Chen F, Rosales J, Lee KY, Fu S (2012) Novel functional MAR elements of double minute chromosomes in human ovarian cells capable of enhancing gene expression. PLoS ONE 7:e30419 Johnson CN, Levy LS (2005) Matrix attachment regions as targets for retroviral integration. Virol J 2:68 Kim JM, Kim JS, Park DH, Kang HS (2004) Improved recombinant gene expression in CHO cells using matrix attachment regions. J Biotechnol 107:95–105 Ley D, Harraghy N, Le Fourn V, Bire S, Girod PA, Regamey A, Rouleux-Bonnin F, Bigot Y, Mermod N (2013) MAR elements and transposons for improved transgene integration and expression. PLoS ONE 8:e62784 Linnemann AK, Platts AE, Krawetz SA (2009) Differential nuclear scaffold/matrix attachment marks expressed genes. Hum Mol Genet 18:645–654 Liu JW, Tabe LM (1998) The influences of two plant nuclear matrix attachment regions (MARs) on gene expression in transgene plants. Plant Cell Physiol 39:115–123 Mock U, Thiele R, Uhde A, Fehse B, Horn S (2012) Efficient lentiviral transduction and transgene expression in primary human B cells. Hum Gene Ther Methods 23:408–415

Noguchi C, Araki Y, Miki D, Shimizu N (2012) Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production. PLoS ONE 7:e52990 Oh SJ, Jeong JS, Kim EH, Yi NR, Yi SI, Jang IC, Kim YS, Suh SC, Nahm BH, Kim JK (2005) Matrix attachment region from the chicken lysozyme locus reduces variability in transgene expression and confers copy number-dependence in transgenic rice plants. Plant Cell Rep 24:145–154 Pfaff N, Lachmann N, Ackermann M, Kohlscheen S, Brendel C, Maetzig T, Niemann H, Antoniou MN, Grez M, Schambach A, Cantz T, Moritz T (2013) A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny. Stem Cells 31:488–499 Sjeklocha L, Chen Y, Daly MC, Steer CJ, Kren BT (2011) bglobin matrix attachment region improves stable genomic expression of the sleeping beauty transposon. J Cell Biochem 112:2361–2375 Wang TY, Yang R, Qin C, Wang L, Yang XJ (2008) Enhanced expression of transgene in CHO cells using matrix attachment region. Cell Biol Int 32:1279–1283 Wang TY, Zhang JH, Jing CQ, Yang XJ, Lin JT (2010) Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells. Cell Biol Int 34:141–145 Wang F, Wang TY, Tang YY, Zhang JH, Yang XJ (2012) Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells. Gene 500:59–62 Xu MY, Zhang X, Zhang L, Luo YZ, Fan YL, Wang L (2011) Functional analysis of BnMAR element in transgenic tobacco plants. Mol Biol Rep 38:3285–3291 Zahn-Zabal M, Kobr M, Girod PA, Imhof M, Chatellard P, de Jesus M, Wurm F, Mermod N (2001) Development of stable cell lines for production or regulated expression using matrix attachment regions. J Biotechnol 87:29–42

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