Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases

Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.     

Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery

Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13-39%) of editing at the IL-2 receptor common gamma-chain gene (IL2RG) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.     

Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.     

The p53-induced mouse zinc finger protein wig-1 binds double-stranded RNA with high affinity

The p53-induced mouse wig-1 gene encodes a Cys₂His₂-type zinc finger protein of unknown function. The zinc fingers in wig-1 are connected by long (56–75) amino acid linkers. This distribution of zinc finger domains resembles that of the previously described double-stranded (ds)RNA-binding proteins dsRBP-ZFa and JAZ. Ectopically expressed FLAG-tagged mouse wig-1 protein localized to nuclei and in some cells to nucleoli, whereas GFP-tagged mouse wig-1 localized primarily to nucleoli. Electrophoretic mobility shift assay using a recombinant GST–wig-1 fusion protein showed that wig-1 preferentially binds dsRNA rather than single-stranded RNA or dsDNA. A set of deletion/truncation mutants of wig-1 was tested to determine the dsRNA-binding domain(s) or region(s) in wig-1 that is involved in the stabilization of wig-1–dsRNA complexes in vitro. This revealed that the first zinc finger in wig-1 is essential for binding to dsRNA, whereas zinc fingers 2 and 3 are dispensable. wig-1 protein expressed in mammalian cells also showed a high affinity for dsRNA. wig-1 represents the first confirmed p53-induced gene that encodes a dsRNA-binding protein. This suggests that dsRNA binding plays a role in the p53-dependent stress response.     

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.     

Targeted editing of goat genome with modular-assembly zinc finger nucleases based on activity prediction by computational molecular modeling

Zinc finger nuclease (ZFN) technology can mediate targeted genome modification to produce transgenic animals in a high-efficient and biological-safe way. Modular assembly is a rapid, convenient and open-source method for the synthesis of ZFNs. However, this biotechnology is hampered by multistep construction, low-efficiency editing and off-target cleavage. Here we synthesized and tested six pairs of three- or four-finger ZFNs to target one site in goat beta-lactoglobulin (BLG, a dominant allergen in goat milk) gene. Homology modeling was applied to build the structure model of ZFNs to predict their editing activities targeting at goat BLG gene. Goat fibroblast cells were transfected with plasmids that encoded ZFN pairs, and genomic DNA was isolated 72 h later for genome editing efficiency assay. The results of editing efficiency assay demonstrated that ZFNs with optimal interaction modes can edit goat BLG gene more efficiently, whereas ZFNs with unexpected interaction modes showed lower activities in editing BLG gene. We concluded that modular-assembly ZFNs can provide a rapid, public-available, and easy-to-practice platform for transgenic animal research and molecular modeling would help as a useful tool for ZFNs activity prediction.     

Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1.

The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression.     

染色质免疫沉淀技术分析肺腺癌A549细胞中p53蛋白与p21CIP1和Bim基因转录启动子的结合

为了检测肺腺癌A549细胞内抑癌蛋白p53与CDK抑制蛋白p21CIP1和Bim基因转录调控区的结合情况,采用染色质免疫沉淀技术,用p53特异性抗体沉淀DNA,PCR检测p21CIP1和Bim基因5′端特异性序列.结果表明,在抗体免疫沉淀的DNA片段中扩增出p21CIP1和Bim基因5′端的特异性序列.因此证实在A549细胞内,p53蛋白可与p21CIP1和Bim基因转录启动子的特异区域结合,进而参与两基因的表达调控.     

Transactivation by temperature-dependent p53 mutants in yeast and human cells

The p53 protein plays an important role in cancer prevention. In response to stress signals, p53 controls essential cell functions by regulating expression of its target genes. Full or partial loss of the p53 function in cancer cells usually results from mutations of the p53 gene. Some of them are temperature-dependent, allowing reactivation of the p53 function in certain temperature. These mutations can alter general transactivation ability of the p53 protein or they modify its transactivation only towards specific genes. We analyzed transactivation of several target genes by 23 temperature-dependent p53 mutants and stratified them into four functional groups. Seventeen p53 mutants exhibited temperature-dependency and discriminative character in human and yeast cells. Despite the differences of yeast and human cells, they allowed similar transactivation rates to the p53 mutants, thus providing evidence that functional analysis of separated alleles in yeast is valuable tool for assessment of the human p53 status.     

p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200

The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53^K382R failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.     

The p53-activated gene,PAG608, requires a zinc finger domain for nuclear localization and oxidative stress-induced apoptosis

The p53-activated gene PAG608, which encodes a nuclear zinc finger protein, is a p53-inducible gene that contributes to p53-mediated apoptosis. However, the mechanisms by which PAG608 is involved in the apoptosis of neuronal cells are still obscure. In this study, we demonstrated that expression of p53 was induced by 100 microm 6-hydroxydopamine (6-OHDA), accompanied by increased PAG608 expression in PC12 cells. On the other hand, transient or permanent transfection of antisense PAG608 cDNA into PC12 cells significantly prevented apoptotic cell death induced by 100 microm 6-OHDA or 200 microm hydrogen peroxide but not by 250 microm 1-methyl-4-phenylpyridinium ion. The 6-OHDA-induced activation of caspase-3, DNA fragmentation, loss of mitochondrial membrane potential, and induction of p53 and Bax were also prevented in PC12 cells that stably expressed antisense PAG608 cDNA. These results suggest that PAG608 is associated with the apoptotic pathway induced by these oxidative stress-generating reagents, upstream of the collapse in the mitochondrial membrane potential in PC12 cells. Interestingly, transient transfection with PAG608 cDNA increased p53 expression in both PC12 cells and B65 cells, indicating that PAG608 induced by p53 is able to induce p53 expression in these cells inversely. Furthermore, transient transfection of a truncated mutant PAG608 cDNA, lacking the first zinc finger domain, inhibited 6-OHDA-induced cell death and altered the nuclear and nucleolar localization of wild-type PAG608 in PC12 cells. These results suggest that PAG608 may induce or regulate p53 expression and translocate to the nucleus and nucleolus using its first zinc finger domain during oxidative stress-induced apoptosis of catecholamine-containing cells.     

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation

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