RGB marking facilitates multicolor clonal cell tracking

We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector-mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.     

Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.     

Tracing cells for tracking cell lineage and clonal behavior

Reconstructing the lineage of cells is central to understanding development and is now also an important issue in stem cell research. Technological advances in genetically engineered permanent cell labeling, together with a multiplicity of fluorescent markers and sophisticated imaging, open new possibilities for prospective and retrospective clonal analysis.     

Effective gene therapy with nonintegrating lentiviral vectors

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.     

Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

Rapid generation of dendritic cell specific transgenic mice by lentiviral vectors

Dendritic cell (DC) specific transgenic mice are a most important model for investigating dendritic cell functions in vivo. Recently, lentivirus mediated gene transfer has become a powerful and convenient method for generation of transgenic mice. We cloned a 1.2 kb CD11c promoter and constructed a lentiviral vector, which efficiently drove DC-specific expression in vitro. After microinjection of purified virus into the perivitelline space of single-cell embryo, more than 80% newborn mice were transgenic and 7 F0 founders were rapidly generated in 2 months. GFP was strictly expressed in CD11c+ cells in spleens, thymus and lymph nodes of the transgenic mice. Importantly, the physiological characteristics and functions of DCs in the transgenic mice were not altered by the specific expression. These results indicate that this vector could be used to rapidly prepare DC-specific transgenic mice. Thus, this lentiviral vector system may provide a convenient and useful tool to study the properties of DCs in vivo.     

Recent advances in lentiviral vectors for gene therapy

Lentiviral vectors (LVs),derived from human immunodeficiency virus,are powerful tools for modifying the genes of eukaryotic cells such as hematopoietic stem cel...     

Functional lentiviral vectors for xeroderma pigmentosum gene therapy

Xeroderma pigmentosum (XP) is a genetic disease characterized by an autosomal-transmitted genodermatosis involving impaired DNA repair activity, where XP patients present severe sensitivity to sunlight (UVB radiation) and are highly victimized by skin cancer. Complementing XP genes by gene therapy is one potential strategy for helping XP patients. However, current viral-based protocols still lack long-term and stable expression, due to limited post-mitotic infection and gene silencing (in the case of retroviruses) or transient expression and activation of immune response (in the case of adenoviruses). Here we demonstrate that the use of third-generation lentiviral vectors can overcome some of these limitations, rescuing the aberrant phenotype in different categories of the disease (XPA, XPC and XPD). Our results show that lentiviruses are efficient tools to transduce XP fibroblasts and correct repair-defective cellular phenotypes by recovering proper gene expression, normal UV survival and unscheduled DNA synthesis after UV radiation. We propose lentiviral vectors as an attractive alternative for gene therapy protocols focusing on DNA repair genetic diseases.     

利用慢病毒载体建立fractalkine基因敲低的HK-2稳定细胞株

目的利用慢病毒载体建立fractalkine(FKN)基因敲低的HK-2稳定细胞株,为探讨FKN基因在肾脏组织肾小管上皮细胞转分化(EMT)重构中的作用奠定实验基础。方法通过双酶切将FKN目的基因连接到GV248载体(hU6-MCS-Ubiquitin-EGFP-IRES-puromycin),经扩增、转化、验证、测序、质粒抽提,将携带目的基因的工具GV248载体质粒及病毒包装辅助质粒Helper1.0、Helper2.0转染293T细胞,完成慢病毒包装及质量检测后获取慢病毒工具载体LV-FKN-RNAi,病毒感染HK-2细胞72h后筛选稳定表达的细胞株HK-2/LV-FKN-RNAi,采用Western blot测定FKN的表达水平,CCK-8检测细胞活力,流式细胞术检测细胞凋亡率。结果经对比,构建LV-FKN-RNAi阳性克隆序列与目的基因FKN相符;HK-2细胞达到80%感染效率的最佳感染条件为:在Enhanced Infection Solution(ENi.S)培养基中进行感染,设定感染复数(MOI)为10,感染时间为72h。Western Blot结果显示,经筛选的HK-2/LV-FKN-RNAi细胞中FKN基因表达水平降低;细胞形态成梭形,多有伪足出现。CCK-8检测显示,FKN基因敲低组细胞活力降低;流式细胞术检测显示,FKN基因敲低组细胞凋亡率增加。结论本研究成功构建一种低表达FKN基因的慢病毒载体;LV-FKN-RNAi感染HK-2细胞后可实现目的基因的低表达,并且FKN基因敲低后HK-2细胞活力下降,凋亡率增加。经筛选的HK-2/LV-FKN-RNAi细胞株可用于后续实验研究。     

The application of lentiviral vectors for tissue-specific gene manipulations

The trophectoderm (TE) ofblastocysts, the first epithelium established in mammalian development, 1) plays signaling, supportive, and patterning functions during pre-implantation development, 2) ensures embryo implantation into the uterine wall, and 3) gives rise to extra-embryonic tissues essential for embryo patterning and growth after implantation. We show that mouse TE, itself permissive to lentiviral (LV) infection, represents a robust non-permeable physical barrier to the virus particles, thereby shielding the cells of the inner cell mass (ICM) from viral infection. This LV feature will allow modulations of gene expression in a lineage-specific manner, thus having significant applications in mouse functional genetics.     

Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectors

Background

Lentiviral vectors allow gene transfer into non‐dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing.

Methods

To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon‐optimized gag‐pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV‐G) under the control of an ponasterone‐inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized.

Results

The RT activity and vector titers of cell clones stably transfected with the inducible gag‐pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone‐inducible VSV‐G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone‐induced producer clones vector titers of more than 1×10⁵ transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production.

Conclusions

The packaging cells described should be suitable for most preclinical applications of SIV‐based vectors. By avoiding regions of high homology between the vector and the packaging constructs, the design of the SIV packaging cell line should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the SIV vector constructs that can be packaged. Copyright © 2002 John Wiley & Sons, Ltd.

     

Multipurpose modular lentiviral vectors for RNA interference and transgene expression

We have created a multipurpose modular lentiviral vector system for expressing both transgenes and miRNA 30-based short hairpins (shRNAmirs) for RNAi. The core of the resulting vector system, pLVmir, allows a simple two step cloning procedure for expressing shRNAmirs under the control of a Pol II promoter in both a constitutive and conditional manner. The adapted cloning method includes a PCR-free method for transferring shRNAmir based RNAi clones from a publicly available library (Open Biosystems). The addition of a Pol II promoter-driven shRNAmir cassette and broadening the choice of Pol III promoters and silencing triggers offers great flexibility to this system. The combination of several preexisting and additional modules created here caters to common needs of researchers. Our modular vector system was validated regarding functionality of promoters, inducibility and reversibility. We successfully applied the system to knockdown Xirp2 mRNA expression in H2kb-tsA58 muscle cells and determined that this had no spurious effect on the expression of a closely related protein. Finally, our set of lentiviral vectors may be used to achieve synergistic effects, for simultaneous knockdown of two genes, as a rescue plasmid and for studying mutant proteins in a physiological context.     

From pathogen to medicine: HIV-1-derived lentiviral vectors as vehicles for dendritic cell based cancer immunotherapy

Over the years, the unique capacity of dendritic cells (DC) for efficient activation of naive T cells has led to their extensive use in cancer immunotherapy protocols. In order to be able to fulfil their role as antigen-presenting cells, the antigen of interest needs to be efficiently introduced and subsequently correctly processed and presented by the DC. For this purpose, a variety of both viral and non-viral antigen-delivery systems have been evaluated. Amongst those, HIV-1-derived lentiviral vectors have been used successfully to transduce DC.This review considers the use of HIV-1-derived lentiviral vectors to transduce human and murine DC for cancer immunotherapy. Lentivirally transduced DC have been shown to present antigenic peptides, prime transgene-specific T cells in vitro and elicit a protective cytotoxic T-lymphocyte (CTL) response in animal models. Different parameters determining the efficacy of transduction are considered. The influence of lentiviral transduction on the DC phenotype and function is described and the induction of immune responses by lentivirally transduced DC in vitro and in vivo is discussed in detail. In addition, direct in vivo administration of lentiviral vectors aiming at the induction of antigen-specific immunity is reviewed. This strategy might overcome the need for ex vivo generation and antigen loading of DC. Finally, future perspectives towards the use of lentiviral vectors in cancer immunotherapy are presented.     

Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator

Transplantation of genetically manipulated cells to the central nervous system holds great promise for the treatment of several severe neurological disorders. The success of this strategy relies on sufficient levels of transgene expression after transplantation. This has been difficult to achieve, however, due to transgene silencing. In this study, we transduced the neural stem cell line RN33B with self-inactivating lentiviral vectors and analyzed transgenic expression of green fluorescent protein (GFP) in several different settings both in vitro and after transplantation to the brain. We found that the transgene was affected of silencing both when transduced cells were proliferating and after differentiation. To prevent silencing, the cHS4 insulator was incorporated into the lentiviral vector. We found that a vector carrying the cHS4 insulator was partially protected against differentiation-dependent downregulation in vitro and in vivo. However, in proliferating cells, we found evidence for variegation and positional effects that were not prevented by the cHS4 insulator, suggesting that the mechanism behind silencing in proliferating cells is not the same mechanism influencing differentiation-dependent silencing. Taken together, these findings favor vector optimization as a strategy for achieving efficient ex vivo gene transfer in the central nervous system.