RESUMO
BACKGROUND: The hCMV promoter is very commonly used for high level expression of transgenes in mammalian cells, but its utility is hindered by transcriptional silencing. Large genomic fragments incorporating the CpG island region of the HNRPA2B1 locus are resistant to transcriptional silencing. RESULTS: In this report we describe studies on the use of a novel series of vectors combining the HNRPA2B1 CpG island with the hCMV promoter for expression of transgenes in CHO-K1 cells. We show that the CpG island gives at least twenty-fold increases in the levels of EGFP and EPO observed in pools of transfectants, and that transgene expression levels remain high in such pools for more than 100 generations. These novel vectors also allow facile isolation of clonal CHO-K1 cell lines showing stable, high-level transgene expression. CONCLUSION: Vectors incorporating the hnRPA2B1 CpG island give major benefits in transgene expression from the hCMV promoter, including substantial improvements in the level and stability of expression. The utility of these vectors for the improved production of recombinant proteins in CHO cells has been demonstrated.
Assuntos
Biotecnologia/métodos , Proteínas Cromossômicas não Histona/genética , Ilhas de CpG , Citomegalovirus/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Inativação Gênica , Técnicas Genéticas , Animais , Células CHO , Mapeamento Cromossômico , Cricetinae , Citometria de Fluxo , Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Modelos Genéticos , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Transcrição Gênica , Transfecção , TransgenesRESUMO
The human TATA binding protein (TBP) locus consists of a functional domain of three closely linkedhousekeeping genes (TBP, PSMB1 (proteasomal C5 subunit), and PDCD2 (programmed cell death-2)) within a 50-kb interval at chromosome position 6q27. Here we demonstrate that a genomic clone spanning the 20-kb TBP gene, with 12 kb 5' and 3' flanking sequences, was fully functional in stable, transfected L-cells harboring a single copy of this transgene, including after long-term (60 day) culture in the absence of drug selective pressure. Furthermore, we were only able to detect DNaseI hypersensitive sites at the TBP and PSMB1 promoters present within this 44-kb fragment. Our data suggest that this 44-kb genomic region possesses genetic regulatory elements that not only drive ubiquitous expression of TBP but also negate chromatin and DNA methylation induced silencing, which is normally associated with transgenes stably integrated into tissue culture cells.
Assuntos
Proteínas de Ligação a DNA/genética , Genoma Humano , Fatores de Transcrição/genética , Transcrição Gênica , Cromossomos Humanos Par 6/genética , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Proteína de Ligação a TATA-Box , TransgenesRESUMO
UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in anintegration independent manner. Efficient expression can bederived from a single copy of an integrated gene site resulting ina higher percentage of cells expressing the marker gene in theselected pool in comparison to standard non-UCOE containingvectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapidproduction of large quantities of protein in a short period oftime. Utilizing this system more than 300 mg of a recombinantantibody has been produced in less than 1 month from transfectionpools in shake flask. Selected subclones have been scaled intosmall bioreactors in less than 2 months, producing significantquantities of monoclonal antibody using a protocol generic for theparent cell line. The increased efficiency obtained with the UCOEvector reduces the number of transfectants which need to bescreened in order to obtain high productivity subclones.Transfection of a standard host cell line, preadapted to grow in alarge-scale setting, allows for rapid cell line developmentdecreasing the transition time from research into development andmanufacturing. Alternatively, the traditional approach of using aparent cell line which requires serum-free and suspensionadaptation after transfection further increases the need forscreening a large number of subclones, because many of thesubclones will not be able to grow under conditions that allowlarge-scale protein production. The use of a preadapted cell linecan reduce the time required to develop a cell line from months toweeks.
RESUMO
The genetic elements that are responsible for establishing a transcriptionally competent, open chromatin structure at a region of the genome that consists only of ubiquitously expressed, housekeeping genes are currently unknown. We demonstrate for the first time through functional analysis in stably transfected tissue culture cells that transgenes containing methylation-free CpG islands spanning the dual divergently transcribed promoters from the human TATA binding protein (TBP)-proteasome component-B1 (PSMB1) and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRPA2B1)-heterochromatin protein 1Hs-gamma (chromobox homolog 3, CBX3) gene loci are sufficient to prevent transcriptional silencing and a variegated expression pattern when integrated within centromeric heterochromatin. In addition, only transgene constructs extending over both the HNRPA2B1 and the CBX3 promoters, and not the HNRPA2B1 promoter alone, were able to confer high and stable long-term EGFP reporter gene expression. These observations suggest that methylation-free CpG islands associated with dual, divergently transcribed promoters possess an independent dominant chromatin opening function and may therefore be major determinants in establishing and maintaining a region of open chromatin at housekeeping gene loci.