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1.
The feasibility of the use of Flp-mediated cassette exchange in the development of a CHO cell line, which produces erythropoietin (EPO) stably and largely, was investigated. A stable, high enhanced green fluorescence protein (EGFP)-producing clone was screened by extensive flow cytometric analysis. An EPO expression unit was targeted into the premarked locus of the stable parental clone by Flp-mediated cassette exchange and a correctly targeted clone (FC28T7) was obtained. The EPO production of FC28T7 was proven to be stable in long-term culture. Furthermore, the Flp-mediated cassette exchange did not alter the stable parental clone's characteristics concerning transgene expression level and stability. Taken together, the data obtained here indicated that the establishment of CHO cell lines stably producing a desired protein is achievable using Flp-mediated cassette exchange.  相似文献   

2.
Site‐specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)‐mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single‐copy integration clones were then co‐transfected with both Flp‐containing plasmid and an EGFP‐containing targeting cassette. Successful cassette exchange was suggested by emergence of G418‐resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re‐usable platform to produce multiple recombinant proteins for fundamental and applied research. Biotechnol. Bioeng. 2012; 109: 2836–2844. © 2012 Wiley Periodicals, Inc.  相似文献   

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4.
Cre recombinase (Cre)-mediated targeted insertion of a transgene is a powerful technique that can be used to tailor genomes. When combined with somatic cell nuclear transfer it could offer an efficient way to generate transgenic livestock with site-specific genetic modifications that are free of antibiotic selection markers. We have engineered primary bovine fibroblasts to contain a chromosomal acceptor site with incompatible loxP/lox2272 sites for Cre-mediated cassette exchange and show for the first time that Cre-mediated targeting can be applied in these acceptor cells. Molecular characterization of the resulting cell clones revealed Cre-mediated transgene insertion efficiencies of up to 98% when antibiotic selection was used to identify transgene containing cell clones. Most clonal lines also contained random insertions of the targeting and Cre expression plasmids with only about 10% of the clones being exclusively modified by the intended targeted insertion. This targeting efficiency was sufficient to enable the isolation of correctly targeted clones without the help of antibiotic selection. Therefore, this recombinase-mediated insertion strategy has the potential to produce transgenic cattle from antibiotic selection marker-free somatic cells with transgenes inserted into proven genomic loci ensuring reliable expression levels.  相似文献   

5.
When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant enhancement in specific TPO productivity (q Tpo) when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly. On the other hand, those clones that showed a significant enhancement inq Tpo at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their cell size increased, which suggested that changes in cell size may be indicative of an enhancedq Tpo. When the enhancement of theq Tpo of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (r 2=0.80,P<0.001, ANOVA), which suggested that an enhancement ofq Tpo after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that the selection of amplified CHO cell clones with enhancedq Tpo can be done based upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.  相似文献   

6.
Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene‐amplified CHO DR1000L‐4N cell line for genome‐wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC‐FISH). Thirteen BAC‐FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR‐deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165‐kb DNA region containing exogenous Dhfr was cloned from the BAC library using high‐density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome. Biotechnol. Bioeng. 2009; 104: 986–994. © 2009 Wiley Periodicals, Inc.  相似文献   

7.
Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.  相似文献   

8.
中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞是生产复杂重组药物蛋白的首选宿主细胞,腺嘌呤磷酸核糖转移酶(adenine phosphoribosyltransferase,APRT)催化腺嘌呤与磷酸核糖缩合形成腺苷一磷酸,是嘌呤生物合成步骤中的关键酶。采用基因编辑技术敲除CHO细胞中aprt基因,验证获得的APRT缺陷型CHO细胞系的生物学特性;构建两种真核表达载体:对照载体(含有目的基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和弱化载体(含有启动子和起始密码子突变的aprt弱化表达盒及EGFP),分别转染APRT缺陷型和野生型CHO细胞并筛选获得稳定转染的细胞池;重组CHO细胞传代培养60代并用流式细胞术检测EGFP表达的平均荧光强度,并比较不同实验组重组蛋白EGFP的表达稳定性。PCR扩增和测序结果表明,CHO细胞aprt基因成功敲除;获得的APRT缺陷型CHO细胞系在细胞形态、生长增殖、倍增时间等生物学特性方面与野生CHO细胞无显著差异。目的蛋白瞬时表达结果表明,与野生型CHO细胞相比,转染对照载体和弱化载体的APRT缺陷型CHO细胞系中EGFP的表达分别提高了42%±6%和56%±9%;特别是长期传代培养时,转染弱化载体的APRT缺陷型细胞中EGFP表达量显著高于野生型CHO细胞(P<0.05);构建的基于APRT缺陷型CHO细胞系能够明显提高重组蛋白的长期表达稳定性。研究结果为建立高效稳定的CHO细胞表达系统提供了一种有效的细胞工程策略。  相似文献   

9.
To meet product quality and cost parameters for therapeutic monoclonal antibody (mAb) production, cell lines are required to have excellent growth, stability, and productivity characteristics. In particular, cell line generation stability is critical to the success of a program, especially where high cell line generation numbers are required for large in‐market supply. However, a typical process for developing such cell lines is laborious, lengthy, and costly. In this study, we applied a FLP/FRT recombinase‐mediated cassette exchange (RMCE) system to build a site‐specific integration (SSI) system for mAb expression in the commercially relevant CHOK1SV cell line. Using a vector with a FRT‐flanked mAb expression cassette, we generated a clonal cell line with good productivity, long‐term production stability, and low mAb gene‐copy number indicating the vector was located in a ‘hot‐spot.’ A SSI host cell line was made by removing the mAb genes from the ‘hot‐spot’ by RMCE, creating a ‘landing pad’ containing two recombination cassettes that allow targeting of one or two copies of recombinant genes. Cell lines made from this host exhibited excellent growth and productivity profiles, and stability for at least 100 generations in the absence of selection agents. Importantly, while clones containing two copies had higher productivity than single copy clones, both were stable over many generations. Taken together, this study suggests the use of FLP‐based RMCE to develop SSI host cells for mAb production in CHOK1SV offers significant savings in both resources and overall cell line development time, leading to a shortened ‘time‐to‐clinic’ for therapeutic mAbs. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1645–1656, 2015  相似文献   

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11.
We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies.  相似文献   

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The mouse whey acidic protein (WAP) gene in mouse embryonic stem (ES) cells has been targeted with a loxP-flanked neomycin phosphotransferase-thymidine kinase (neo-TK) cassette inserted into exon 4. Southern blot revealed that 51 of 199 colonies were correctly targeted (1:4). Next, a Cre-encoding plasmid was electroporated into a targeted cell line to cause the deletion of the neo-TK cassette. Modified ES cell colonies were identified by polymerase chain reaction (PCR); 44 out of 50 colonies (88%) had undergone Cre-mediated deletion. Finally, a loxP-tagged cell line was co-electroporated with a Cre-encoding plasmid and a loxP-containing neo plasmid for site-specific insertion into the WAP locus. The frequency of this event was 23% (11 of 48) of that obtained with random integration. This demonstrates the feasibility of using the Cre-loxP system for site-specific integration in ES cells. Moreover, this is the first report of targeting a loxP-containing transgene into a predetermined location in ES cells. Ultimately, a mouse model derived from these modified ES cells will usher in a second generation of animal “bioreactor” models where the inserted transgene is controlled exclusively by the endogenous locus regulatory elements. In addition, oncogenesis can be explored from single copy oncogene/tumor suppressor gene inserts, which are regulated in a temporal and tissue-specific manner. It is hoped that regulation of transgene expression in this fashion will help elucidate the underlying mechanisms of normal development in the mammary gland. Mol. Reprod. Dev. 48:324–331, 1997. © 1997 Wiley-Liss, Inc.  相似文献   

14.
In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing—new parental cell lines which are inherently more “fit for purpose.” One‐hundred and ninety‐nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell‐specific proliferation rate during extended deep‐well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell‐specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub‐culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N‐glycan processing were identified. Finally, for clonal populations most “evolved” by extended sub‐culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N‐glycosylation. Rapid‐specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone‐specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and isolate CHO cell variants with improved functional properties in vitro. Biotechnol. Bioeng. 2013; 110: 260–274. © 2012 Wiley Periodicals, Inc.  相似文献   

15.
The creation of precise clinical mutations by gene targeting is important in elucidating disease pathogenesis using mouse models. 'Hit and run' gene targeting is an elegant method to achieve this goal. This uses first a positive selection to introduce the targeting vector carrying the required mutation and then a negative selection to identify clones which have removed vector and wild-type sequences by intrachromosomal recombination. However, this approach has only been successfully used in a handful of cases. We used this procedure to introduce precise clinical mutations into the exon 10 region of the cystic fibrosis transmembrane conductance regulator (Cftr) gene. Using a CMV promoter driven hygromycin/thymidine kinase (hyg/tk) fusion gene as both our dominant and negative selectable marker, we targeted the Cftr locus very efficiently but only identified false runs after the negative selection step. This defect in thymidine kinase induced toxicity to gancyclovir correlated with methylation of the transgene. Consequently we devised a stringent screening procedure to select only true 'run' clones. Unfortunately these 'run' clones had lost the mutation so we altered the vector design to bias the run step to retain the mutation and used a different tk selection cassette with a HSVtk promoter sequence. This new vector design allowed both efficient 'hit and run' for two cystic fibrosis (CF) mutations with no false positives and successful germline transmission of the novel G480C missense mutation.  相似文献   

16.
We aim to develop a cultured cell model, to serve as a system with which the altered circadian phenotypes produced by the clock gene variations could be studied in vitro. Tau mutation, which shortens the circadian period of hamsters and mice, was introduced into the CK1ε locus of cultured Rat1-R12 cells by gene targeting mediated by a recombinant adeno-associated virus (rAAV) vector. After transduction of Rat1-R12 cells with rAAV, about 0.14% of the drug-resistant cells underwent gene targeting at CK1ε locus. Of the three clones isolated, only one carried the targeted allele of tau mutation and two carried the targeted wild-type allele. The clone with the targeted tau mutant allele exhibited a significantly shorter circadian period compared to the clone with targeted wild-type allele. rAAV-mediated gene targeting in cultured somatic cells is a convenient and powerful tool for analyzing the phenotypic outcome of clock gene variations, and for elucidating the pathogenesis of the disorders associated with abnormal circadian rhythmicity.  相似文献   

17.
Lentivirus‐derived vectors (LVs) were studied for the generation of stable recombinant Chinese hamster ovary (CHO) cell lines. Stable pools and clones expressing the enhanced green fluorescent protein (eGFP) were selected via fluorescence‐activated cell sorting (FACS). For comparison, cell pools and cell lines were also generated by transfection, using the LV transfer plasmid alone. The level and stability of eGFP expression was greater in LV‐transduced cell lines and pools than in those established by transfection. CHO cells were also infected at two different multiplicities of infection with an LV co‐expressing eGFP and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). At 2‐day post‐infection, clonal cell lines with high eGFP‐specific fluorescence were recovered by FACS. These clones co‐expressed TNFR:Fc with yields of 50–250 mg/L in 4‐day cultures. The recovered cell lines maintained stable expression over 3 months in serum‐free suspension culture without selection. In conclusion, LV‐mediated gene transfer provided an efficient alternative to plasmid transfection for the generation of stable and high‐producing recombinant cell lines. Biotechnol. Bioeng. 2011; 108:600–610. © 2010 Wiley Periodicals, Inc.  相似文献   

18.
Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.  相似文献   

19.
Smads is a new gene family in transforming growth factor-β (TGF- β) signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electroporated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targeting mouse has laid a solid foundation for producing the tissue specific Smad2 gene knockout mice.  相似文献   

20.
Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome. Many of the recombination strategies currently used, however, will introduce a selection marker gene at the modified site. DNA sequences of prokaryotic origin like vector sequences, selection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci. In order to avoid the insertion of excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was devised and used to insert a foreign gene into the β-casein gene in murine embryonic stem cells. The incompatibility of the heterospecific lox sites used for the recombinase-mediated cassette exchange was found to be critical for the success of the strategy. The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach. A mutant lox site carrying two point mutations, however, was highly effective and 90% of the selected cell clones carried the desired modification. This biphasic recombination strategy allows for the efficient and precise modification of gene loci without the concomitant introduction of a selectable marker gene.  相似文献   

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