适于无血清贴壁培养的抗凋亡宿主细胞系CHO-IVB2的构建

应用无血清培养基培养CHO细胞时,由于没有血清提供各种贴壁因子,细胞以悬浮的方式生长。在实际的大规模细胞培养中,CHO细胞往往以贴壁方式培养,要么贴壁于悬浮的微载体中,要么贴壁于固定的聚酯盘状介质或中空纤维中,而很少直接悬浮于培养基中。在无血清培养基中,Vitronectin单一组分可以促使CHO细胞的贴壁和扩增。通过双表达lgf-1和Bcl-2基因,已经构建了可以在无蛋白培养基IMEM中抗凋亡生长的细胞株CHO-IB3。在此基础上,构建了可以同时表达Igf-1、Vitronectin和Bcl-2三个蛋白的三顺反子表达载体pCI—NII—IVB。将该载体转染于CHO—dhfr^-细胞中,构建了一个细胞株CHO—IVB2。该细胞株可以在无蛋白培养基中抗凋亡生长,适于以贴壁的方式大规模培养,用于大量生产外源目的蛋白。     

通过高表达Igf1/Bcl-2或Bcl-2/Cyclin E基因组合使CHO细胞适于在无蛋白培养基中抗凋亡培养

哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中,哺乳动物细胞生长活力差,且容易发生细胞凋亡,因而难以大规模培养。为解决此问题,应用双顺反子表达载体在CHO-dhfr^-细胞中同时表达Igf-1／Bcl-2或Bcl-2／CyclinE基因组合,通过Bcl-2使细胞获得抗凋亡能力;通过1gf-1或CyclinE促进细胞生长分裂,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO-dhfr^-细胞,应用Western blot从G418抗性克隆中分别筛选到Bcl-2高表达克隆若干个,对其中表达Bcl-2最高的CHO-IB3和CHO-Bcl做进一步Western blot和流式细胞分析,确认此两个细胞株分别高表达Igf-1／Bcl-2和Bcl-2／CyclinE基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡,并以流式细胞术和DNA Ladder法检测细胞凋亡,证明CHO-IB3和CHO一BCl均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO-dhfr^-对照细胞。在细胞培养瓶中的连续培养实验表明,CHO-IB3和CHO-BCl在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHO-dhfr^-对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长,适于作为生物工程宿主细胞。     

通过高表达Igf1/Bcl-2或Bcl-2/Cyclin E基因组合使CHO细胞适于在无蛋白培养基中抗凋亡培

哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中,哺乳动物细胞生长活力差,且容易发生细胞凋亡,因而难以大规模培养。为解决此问题,应用双顺反子表达载体在CHO-dhfr-细胞中同时表达Igf-1/Bcl-2或Bcl-2/Cyclin E基因组合,通过Bcl-2使细胞获得抗凋亡能力;通过Igf-1或Cyclin E促进细胞生长分裂,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO-dhfr^-细胞,应用Western blot从G418抗性克隆中分别筛选到Bcl-2高表达克隆若干个,对其中表达Bcl-2最高的CHO-IB3和CHO-BC1做进一步Western blot和流式细胞分析,确认此两个细胞株分别高表达Igf-1/Bcl-2和Bcl^-2/Cyclin E基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡,并以流式细胞术和DNA Ladder法检测细胞凋亡,证明CHO-IB3和CHO-BC1均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO-dhfr-对照细胞。在细胞培养瓶中的连续培养实验表明,CHO-IB3和CHO-BC1在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHOdhfr-对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长,适于作为生物工程宿主细胞。     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因（k2tPA）、扩增基因（dhfr）、重组酶识别序列（FRT）及筛选基因（neo）,且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO／dhfr^-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点（Hotspot）区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO／dhfr-细胞系。随后利用位点特异性重组系统（FLP-FRT）将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17．1μg／10^6cell·24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

重组酶介导的定点整合及在构建重组CHO细胞中的应用

中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)是生产治疗性重组蛋白最常用的细胞。随机整合(random integration,RI)工艺是目前构建重组CHO细胞株的主要策略,由于CHO细胞基因组缺乏稳定性,为得到产量高、品质好且适应特定工艺的细胞株,通常需要1~2轮高通量筛选,不仅工作量大、耗时长且批次稳定性差。定点整合(site-specific integration,SSI)基因编辑技术将外源基因整合至细胞基因组的特定位点,经一轮筛选得到稳定、高产且适应特定生产工艺的细胞株,从而缩短细胞株构建周期。近年来,不断有将定点整合策略应用于构建重组CHO细胞株的报道。基因编辑技术的发展是实现细胞外源基因定点整合工艺的基础,常用的基因编辑技术包括核酸酶技术、转座子技术和重组酶技术。比较这三种基因编辑技术在构建流程、整合效率和专利等方面的不同特点,重点讨论重组酶介导的定点整合及其在CHO细胞株构建中的应用。     

CHO细胞表达系统研究进展

CHO细胞表达系统是目前重组糖蛋白生产的首选系统。随着无血清悬浮培养技术、基因工程技术和大规模培养技术的应用和不断发展,CHO细胞表达系统已经成为生物技术药物最重要的表达或生产系统,并被广泛应用于抗体、重组蛋白药物和疫苗等产品的研发和生产中。近年来,针对CHO细胞表达系统在某些重组蛋白的表达和大规模生产中存在的不足,研究者们通过利用基因工程技术手段,结合重组蛋白表达机制的研究成果,为优化和应用CHO细胞表达系统做出了不懈努力。从培养基的优化、高产重组CHO细胞株的构建、大规模培养三个方面综述了CHO细胞表达系统的最近研究进展,以期为CHO细胞表达系统的研究与应用提供参考。     

Bcl-2蛋白的研究进展

Bcl-2基因属于Bcl-2基因家族,其表达的Bcl-2蛋白在细胞凋亡过程中通过抑制线粒体中细胞色素C的释放,可明显地抑制细胞凋亡。由于在大规模的细胞培养过程中,细胞的死亡以凋亡为主,所以可通过建立稳定过表达Bcl-2蛋白的重组细胞株,以抑制细胞的凋亡。同时,Bcl-2蛋白的变化不但可预见肿瘤的发生,而且通过药物降低Bcl-2的含量对于肿瘤的治疗也具有积极的意义。本文综述了Bcl-2蛋白的结构、功能、抗凋亡作用的机理,以及目前在生物制药和临床方面的应用和前景。     

RNA干扰抑制SiHaB2细胞中Bcl-2基因的表达

Bcl-2基因作为细胞凋亡的一个潜在抑制剂调节细胞的死亡,抑制恶性肿瘤细胞中Bcl-2基因的表达可促进肿瘤细胞的凋亡。采用RNA干扰技术,合成了含有21个核苷酸的小双链干扰RNA（siRNA．Bcl-2）,并构建了含有19个核苷酸基因的质粒载体（pSilencer2．1-U6-Bcl-2）,把合成的siRNA．Bcl-2和pSilencer2．1-U．Bcl-2分别转导入Bcl-2高表达的细胞株SiHaB2中,通过Western印迹检测,免疫荧光法检测及DNA梯（ladder）检测,可观察到在导入siRNA．Bcl-2和pSilencer2．1-U．Bcl-2的SiHaB2细胞被培养72h后,可以明显抑制Bcl-2蛋白的表达。     

硫酸化茯苓多糖对人乳腺癌细胞株MCF-7凋亡的影响

为了探讨硫酸化茯苓多糖(SP)对人乳腺癌细胞株MCF-7凋亡的影响,采用MTT法检测不同浓度、作用时间SP对乳腺癌MCF-7细胞的抑制作用,倒置显微镜观察MCF-7细胞的形态学变化,RT-PCR检测SP处理MCF-7细胞凋亡相关基因(Bcl-2,Bax)的表达;Western blotting技术检测SP对乳腺癌细胞凋亡蛋白Bcl-2、Bax表达变化。结果表明,SP对MCF-7细胞增殖有抑制作用,且在一定范围内呈剂量效应;细胞贴壁能力减弱,细胞间隙增大,胞膜褶皱;Bcl-2基因表达水平和蛋白表达水平明显降低(p0.05),Bax基因表达水平和蛋白表达水平明显升高(p0.05)。基于以上研究,SP通过促凋亡基因Bax的表达,抑制抗凋亡基因Bcl-2的表达来下调Bcl-2/Bax比值,激活凋亡途径,诱导MCF-7细胞的凋亡。     

克服位置效应提高外源基因在CHO细胞中稳定高效表达的策略

能够生产有功用的治疗性蛋白的一个重要前提是获得稳定的重组蛋白高表达细胞株,然而筛选一个能够持续稳定表达外源蛋白的重组细胞株是费时费力的过程。有多篇文献报道了重组蛋白细胞株表达的不稳定性。位置效应是高表达细胞株不稳定性的重要因素,克服或利用位置效应是当前获得稳定高表达重组蛋白细胞株的有效途径。为解决外源基因插入的随机性所带来的不可预知的后果,可以事先在CHO细胞基因组中筛选转录热点区域,再通过位点特异性或同源重组的方式,实现外源基因的定点整合。各种调节位置效应的DNA元件陆续被发现,可以利用它们去调控基因表达及增加细胞株的稳定性。     

Bcl-2 overexpression in CHO cells improves polyethylenimine-mediated gene transfection

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI) as a transfection reagent has been considered as an attractive method to produce recombinant proteins rapidly for pre-clinical studies. A high level of transfection efficiency, which is required for high-level TGE in CHO cells, can be achieved by increasing the PEI concentration. However, PEI induces cytotoxicity in a dose-dependent manner. To overcome this problem, Bcl-2 protein, an anti-apoptotic protein, was overexpressed in CHO cells (DG44). At a ratio of PEI to DNA (an N/P ratio) of 10, there were no significant differences in transfection efficiency and cell viability between Bcl-2 overexpressing and non-overexpressing cells. The transfection efficiency and cell viability were 2–11% and 83–92%, respectively. However, there were significant differences (P < 0.05) in the transfection efficiency and cell viability between them at a higher N/P ratio. At an N/P ratio of 40, the transfection efficiency and cell viability of Bcl-2 non-overexpressing cells were 24–38% and 35–40%, respectively, while those of Bcl-2 overexpressing cells were 48–53% and 43–56%, respectively. Furthermore, compared with Bcl-2 non-overexpressing cells, more DNAs entered the Bcl-2 overexpressing cells, resulting in a higher rate of TGE per cell. PE-Annexin V apoptosis revealed that Bcl-2 overexpression suppressed PEI-induced apoptotic cell death at high N/P ratios. Taken together, Bcl-2 overexpression in CHO cells suppresses apoptotic cell death during PEI-mediated transient transfection, resulting in enhanced transfection efficiency and TGE.     

Insulin and IGF-1 mediated inhibition of apoptosis in CHO cells grown in suspension in a protein-free medium

When Chinese hamster ovary (CHO) cells were grown in suspension and deprived of serum, 40% of them became apoptotic after 72 hours, as determined by flow cytometry analysis of TUNEL-labelled cells. Cell viability, assessed by erythrocin B staining, decreased correspondingly. An increase in the total fraction of cells expressing interleukin converting enzyme (ICE; caspase 1), B-cell lymphoma 2 protein (Bcl-2,) and Bcl-2 associated x protein (Bax) was shown by antibody probing and subsequent flow cytometry. The p53 tumour suppressor gene product level remained low within the cell population. Insulin-like growth factor-1 (IGF-1) inhibited cell death in a concentration-dependent manner, and at 20 ng/ml, cell viability was maintained close to 100% and no apoptotic cells were detected. Also, insulin was shown to inhibit cell death - at 1.0 microg/ml, cell viability was 95%, whereas 10% of the cells stained for apoptosis. At the highest concentrations of IGF-1 and insulin, the expression of ICE, Bcl-2 and Bax was fully suppressed, whereas the p53 product level increased, despite still being detectable in a minority of cells. Under these conditions, IGF-1 may increase p53 expression to restrain abnormal cell proliferation. It is concluded that special attention should be paid to exposure and culture conditions that induce acquired susceptibility to a toxic insult, during the development and validation of cell-based assays.     

虫草素对肺癌细胞生长及迁移的影响

探讨虫草素对非小细胞肺癌细胞株H1781细胞凋亡及迁移的影响及作用机制。培养H1781细胞并分组,对照组用不含药物的培养基处理,虫草素组用含有10、20、30和40 μmol/L虫草素处理,处理24 h后测定细胞活力,通过显微镜观察细胞形态学,HE染色观察虫草素对细胞整体的影响,细胞免疫荧光技术检测细胞中MMP-9和DAPI核染色情况观察细胞凋亡,Western blotting检测凋亡等相关蛋白表达。与对照组相比,虫草素处理24 h后,H1781细胞系活力显著降低;细胞数量明显减少;HE染色观察发现随着虫草素浓度增加,细胞数量及细胞集团明显变少,免疫荧光技术检测发现药物处理后细胞凋亡明显促进;划痕实验发现虫草素明显降低细胞迁移能力;Western blotting实验中Bax、cleaved caspase-3蛋白表达明显上调,MMP-9、Bcl-2蛋白表达明显下调。虫草素对肺癌H1781细胞迁移有抑制作用、对凋亡有促进作用,推测其作用机制为上调促凋亡蛋白表达、下调抗凋亡蛋白表达。     

Regulation of cell proliferation and apoptosis in CHO-K1 cells by the coexpression of c-Myc and Bcl-2

Proliferation and cell death are regarded as key targets for the optimization of animal cell culture processes and for the maximization of product yield. Although chemical and physical factors are vitally important, of primary interest is the utilization of genetic engineering to regulate cellular processes. CHO cells were first genetically modified to enhance proliferation rate in both suspension and monolayer cultures. Under the constitutive control of c-myc overexpression the CHO cultures showed an increase in growth rate and maximum cell number accompanied by a similar decrease in specific glucose consumption rate. Although the c-myc transfected cell line exhibited apoptosis at much lower rates than is widely reported and associated with the overexpression of c-Myc, it was nevertheless apparent that c-Myc was responsible for the induction of higher apoptotic rates when compared with the control cell line. Hence, the anti-apoptotic gene bcl-2 was also used to transfect the c-Myc CHO cell line, to reduce cell death. Overexpression of both oncoproteins resulted in a cell line that exhibited higher proliferation rates and maximum cell numbers, with a decrease in apoptosis when compared to the parental cell line. In conclusion, it was shown that Bcl-2 protein overexpression specifically abrogates c-Myc-induced apoptosis without affecting the c-Myc mitogenic function.     

FAS/ActD通过线粒体途径引起细胞阻滞诱导人宫颈癌HeLa细胞凋亡

为了探讨FAS抗体与放线菌素D(actinomycin D,ActD)联合作用诱导人宫颈癌HeLa细胞凋亡的分子机制,通过MTT法检测细胞活力,利用流式细胞仪检测细胞凋亡和细胞周期,从而研究FAS/ActD抑制细胞增殖的作用. 结果表明,FAS/ActD能明显降低HeLa细胞的活力,并且通过G1/G0期阻滞和S期阻滞诱导HeLa细胞凋亡. 此外,Western印迹分析进一步显示,FAS/ActD还能引起Bcl-2蛋白表达降低, Bax蛋白表达增加,Bid蛋白发生断裂激活,导致细胞质中Cyto-c释放的增加,并激活在细胞凋亡的执行过程中起着关键作用的caspase 9和caspase 3. 以上结果提示,FAS抗体与ActD的联合作用可能经线粒体途径引起细胞周期阻滞,从而诱导HeLa细胞凋亡. 该研究为宫颈癌的免疫治疗提供了新的思路.     

B-RAF基因特异的siRNA干扰对胃癌BGC823细胞的影响

为探讨B-RAF基因特异的siRNA干扰对胃癌BGC823细胞的增殖和凋亡的影响, 设计并合成B-RAF小分子干扰RNA(B-RAF-siRNA)和阴性对照siRNA, 用TransMessenger介导转染胃癌BGC823细胞, RT-PCR分析检测胃癌BGC823细胞中B-RAF基因以及Bcl-2基因的表达; MTT检测胃癌BGC823细胞增殖情况; 流式细胞仪检测细胞凋亡情况, 并与对照组进行比较。TransMessenger能够有效介导B-RAF-siRNA和阴性对照siRNA转染胃癌BGC823细胞, TransMessenger介导的B-RAF-siRNA有效地抑制胃癌BGC823细胞B-RAF以及Bcl-2基因的表达, 与对照组相比, 抑制率达90.0%以上, 最高达100%; 同时明显抑制胃癌BGC823细胞增殖; 促进胃癌BGC823细胞的凋亡(P < 0.01)。B-RAF基因特异的siRNA干扰能有效地抑制胃癌BGC823细胞中B-RAF基因以及Bcl-2基因的表达, 同时促进胃癌细胞凋亡和抑制胃癌细胞增殖。     

Baicalein induces the apoptosis of U251 glioblastoma cell lines via the NF-kB-p65-mediated mechanism

Glioblastoma (GBM) is the most aggressive cerebral gliomas. Moreover, the overall prognosis of GBM is still little. Baicalein (BA) is a flavonoid derived from the Scutellaria baicalensis root, and has historically been used in anticancer therapies. However, its apoptosis role and related mechanisms in GBM has not yet been researched clearly. Thus, this study aimed to investigate the effects of BA on human GBM U251 cell line. The effects of BA on proliferation of U251 cells were measured by Cell Counting Kit-8 assay. Cellular apoptosis was detected by flow cytometry with annexin V-FITC/propidium iodide staining. The expression of apoptosis-related protein Bcl-2, Bax and cleaved-caspase3 was detected by quantitative real-time PCR and western blot. The expression of nuclear p65 protein, the active subunit of nuclear factor-kappa B (NF-κB), was determined by immunofluorescence and western blot. Our results showed that the viability of U251 cells significantly decreased in a time- and dose-dependent manner after treated with BA, and the apoptotic ratio of BA-treated groups was significantly higher than that of control groups. Furthermore, the expression of NF-kB-p65 in the nucleus was remarkably reduced, and the activity of NF-kB-p65 was remarkably inhibited after BA treatment. Combined treatment with a NF-kB-P65 inhibitor (QNZ) and BA resulted in the synergistic reduction of Bcl-2 expression and then increase of Bax and cleaved-caspase3 expression; and the viability of U251 cells was also inhibited. In conclusion, BA inhibits GBM cells viability and induces apoptosis via inhibit the activity of NF-kB-p65, suggesting that BA is a potential therapeutic agent for GBM.     

CHO细胞基因组NW-003614092.1内稳定表达位点的发现*

目的:获得中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)内稳定表达位点信息,为构建重组蛋白CHO稳定表达株、缩短研发时间线提供信息明确的稳定位点。方法:对慢病毒随机整合Zsgreen1基因的具有潜在稳定表达位点的CHO-K1-1d2细胞株进行连续传代培养,验证表达稳定性;通过染色体步移分析慢病毒载体整合位点,并利用CRISPR/Cas9技术验证位点的可编辑性。结果:CHO-K1-1d2细胞在连续贴壁培养20代、悬浮培养50代过程中,能够100 %发绿色荧光,且荧光强度稳定,能够稳定表达Zsgreen1蛋白;染色体步移分析测序结果表明,CHO-K1-1d2细胞中慢病毒载体整合于CHO细胞基因组NW-003614092.1上第1 159 463与1 159 467碱基间;共转染sgRNA与Cas9质粒后,测序结果表明,该位点可被CRISPR/Cas9技术编辑。结论:CHO细胞基因组NW-003614092.1内存在一个信息明确的、能够被CRISPR/Cas9技术编辑的稳定表达位点。     

Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody

The influence of Bcl-2 expression on the robustness of a CHO cell line (22H11) developed for the industrial production of a chimeric antibody was evaluated. Western blot analysis following transfection with the expression vector unexpectedly revealed upregulation of endogenous Bcl-2 expression in the control (Neo) cell line in response to exposure to the selection drug G418. This indicated that geneticin may function by inducing apoptosis in cells not carrying the control plasmid or expressing very low levels of survival genes. Thus, exposure to the drug enriched the culture for a population of cells which expressed enhanced levels of endogenous Bcl-2. In batch cultures, ectopic bcl-2 expression resulted in a 75% increase in maximum viable cell density over control cultures. Moreover, the rate of decrease in viability in the Bcl-2 cultures was significantly lower than that in the control cultures. After 18 days, the Bcl-2 viability was around 90%, compared to 20% in the control cultures. Evaluation of the mechanism of cell death revealed very few cells with classical apoptotic morphology. Around 10% were clearly necrotic, but the majority of dead cells were seen as chromatin free but otherwise relatively intact structures. Because of the relatively low rate of cell death in both cell lines, few cells were observed in the transitional, easily identifiable early stages of apoptosis. However, DNA gel electrophoresis revealed a clear ladder-pattern, but only in the control cultures, thus confirming high levels of apoptotic death. Antibody concentrations during both sets of cultures were very similar, both during the growth and death phases, with a maximum titer of around 40 microgram/ml. Analysis of Bcl-2 expression by flow cytometry revealed that the cultures contained two populations of cells: a large population which expressed high levels of Bcl-2 and a relatively smaller low-expressing population. During the course of the batch, the smaller, low-expressing population declined in frequency, suggesting that these cells were more sensitive to cell death. In addition, the mean level of Bcl-2 expression in the overexpressing population also declined significantly, presumably reflecting the exhaustion of precursors for protein synthesis following nutrient depletion. Importantly, when cells were taken from day 40 of the significantly extended Bcl-2 batch cultures, they immediately proliferated, confirming that they had retained their replicative potential. Cultivation of the cells in basal medium lacking (individually) serum, all amino acids, glutamate/asparagine, and, finally, glucose, resulted in relatively lower viable cell numbers and viability in the control cell line compared to the Bcl-2 cell line. Exposure of cells to ammonia toxicity also revealed the relative robustness of the bcl-2 transfected cells. When growth was arrested by treatment with 4 mM thymidine, Bcl-2 overexpressing cells exhibit a viability of over 80% after 5 days in culture, compared to only 40% in the control cell line. However, under growth-arrested conditions, there was no major difference in antibody titer between the two cell lines.     

Identification of regulatory motifs in the CHO genome for stable monoclonal antibody production

Chinese hamster ovary (CHO) cell lines are widely used for therapeutic protein production. When a transgene is integrated into the genome of a CHO cell, the expression level is highly dependent on the site of integration because of positional effects such as gene silencing. To overcome negative positional effects and establish stable CHO cell lines with high productivity, several regulatory DNA elements are used in vector construction. Previously, we established the CHO DR1000L-4N cell line, a stable and high copy number Dhfr gene-amplified cell line. It was hypothesized that the chromosomal location of the exogenous gene-amplified region in the CHO DR1000L-4N genome contains regulatory motifs for stable protein production. Therefore, we isolated DNA regulatory motifs from the CHO DR1000L-4N cell line and determined whether these motifs act as an insulator. Our results suggest that stable expression of a transgene can be promoted by the CHO genome sequence, and it would be a powerful tool for therapeutic protein manufacturing.