Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos

Abstract

E mbryonic stem (ES) cells are derived from early mammalian embryos and display characteristics of totipotency, i.e., after transfer to a suitable in vivo environment they contribute to the primary germ layers (ectoderm, endoderm, and mesoderm) and populate the germline of mice (1, 2). ES cells can be propagated in an undifferentiated state and genetically manipulated in vitro. Thus, transgenic animals can be generated by introducing foreign genes into ES cells, followed by transplantation of the ES cells into embryos and germ-line transmission.The first reports of genetic manipulation of ES cells demonstrated that vectors derived from retroviruses can infect ES cells and that the integrated virus (provirus) is transmitted through the germline (3, 4). Furthermore, it was shown that retroviral vectors are able to infect preimplantation embryos, giving rise to transgenic animals that transmit the proviral DNA to offspring (5-8). However, further analysis revealed that both infected ES cells and preimplantation embryos lack significant provirus transcription. Two major mechanisms have been identified for retrovirus silencing (see references in ref. 9): trans-acting factors that bind to the viral promoters in the long terminal repeats (LTRs) and methylation of the integrated retroviral genome and flanking host DNA sequences. According to the organization of their genome (for review see ref. 10), one can distinguish simple retroviruses, such as the prototypic murine leukemia virus, from complex retroviruses like the lentiviruses. HIV type 1 (HIV-1) is one of the best-studied complex retroviruses and has the ability to infect nondividing cells presumably by import of the viral DNA through the nuclear pore and subsequent integration into the host genome (references in ref. 11). Vectors derived from lentiviruses can transduce a broad spectrum of terminally differentiated, nondividing cells, as well as, hematopoietic stem cells of multiple mammalian species (references in ref. 11).In this communication, we show that (i) unlike traditional oncoretroviral vectors, expression of transgenes introduced by lentiviral vectors into murine or human ES cells is not silenced.(ii) Transgene expression is not ''shut off'' during differentiation, and the transgene is expressed in multiple tissues of chimeric animals generated by transfer of lentivector-transduced ES cells in blastocysts. (iii) Germ-line transmission of transgenes introduced into ES cells by lentiviral vectors and (iv) preimplantation embryos at morula stage can be successfully transduced with lentiviral vectors, and the resulting progeny express the transgene. We therefore conclude that lentiviral vectors will be excellent tools for generating transgenic animals. Materials and MethodsVirus Production. LV-green fluorescent protein (GFP) was constructed by cloning the CAG promoter into the ClaI and BamHI sites of the vector LV-pGFP (12), thereby replacing the phosphoglycerate kinase (PGK) promoter. LV-Lac was cloned by introducing the LacZ-woodchuck hepatitis virus frag...

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

“…35 The lentiviral vector used in this report contain several elements previously shown to enhance vector function, including a central polypurine tract (cPPT) for improved replication and nuclear import, 36 a promoter from the murine stem cell virus (MSCV), which has been shown to lessen vector silencing in some cell types, 37,38 a woodchuck hepatitis virus posttranscriptional responsive element (WPRE) for improved transcriptional termination, 39 and the backbone was a deleted 3 0 -LTR self-inactivating (SIN) vector design that may have improved safety, sustained gene expression and antisilencing properties. 5,[40][41][42] The ability of lentiviral vector to transduce minimally stimulated T cells 43 may have significant advantages. In vitro analysis of human T cells transduced with g-retroviral vectors following T-cell activation indicated that both TCR Vb usage and global gene Figure 5 Alternative furin site vectors.…”

“…In two constructs we dissected the V5 into two parts and accordingly made the two constructs as depicted in Figure 3e (second and third images). Next, we replaced the V5 peptide with a synthetic hinge region commonly used in chimeric TCR constructs 30 ((G4S)2, construct 4) or naturally occurring hinge regions from primate immunoglobulins 31 (constructs [5][6][7][8]. The original vector with the furin site plus the complete V5 peptide (construct 9) consistently displayed the highest level of TCR expression, whereas other vectors showed a performance similar to the vector where furin was linked directly to F2A (construct 10).…”

“…In T-cells, the level of transgene expression is modulated by the activation state of the cell, 3 and g-retroviral based vectors have been shown to be subject to gene silencing in certain cell types, 4,5 and they have a preference for integration near transcription start sites, 6 which may increase the potential for insertional mutagenesis. Furthermore, the current g-retroviral vector based protocols require T cells to be fully activated for efficient transduction and this may be deleterious to their function.…”

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

“…7 Furthermore, lentiviral vectors possess a higher stability of expression in vivo than oncoretroviral ones, supporting their use for long-term corrections of genetic disorders. 8,9 Chromatin insulator is a DNA sequence that serves as a boundary element between differentially regulated genes. Various insulators have been found from Drosophila and vertebrates, including chicken, mouse and human.…”

Status of transgenic chicken models for developmental biology

“…A common problem with some retroviral vectors used in transgenic technology has been gene silencing. A new approach that does not appear to be affected by gene silencing has been to use lentiviral vectors, which are a type of retrovirus that can infect both dividing and quiescent cells (Pfeifer et al, 2002). The preintegration complex of lentiviral vectors can penetrate the intact membrane of the nucleus of the target cell, and lentiviral vectors have been used to develop transgenic mice (Lois et al, 2002).…”