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. 2002 Mar 12;99(6):3651–3656. doi: 10.1073/pnas.062032499

Design of an HIV-1 lentiviral-based gene-trap vector to detect developmentally regulated genes in mammalian cells

Zhennan Lai 1,*, Ina Han 1, Misun Park 1, Roscoe O Brady 1
PMCID: PMC122578  PMID: 11891320

Abstract

The recent development of HIV-1 lentiviral vectors is especially useful for gene transfer because they achieve efficient integration into nondividing cell genomes and successful long-term expression of the transgene. These attributes make the vector useful for gene delivery, mutagenesis, and other applications in mammalian systems. Here we describe two HIV-1-based lentiviral vector derivatives, pZR-1 and pZR-2, that can be used in gene-trap experiments in mammalian cells in vitro and in vivo. Each lentiviral gene-trap vector contains a reporter gene, either β-lactamase or enhanced green fluorescent protein (EGFP), that is inserted into the U3 region of the 3′ long terminal repeat. Both of the trap vectors readily integrate into the host genome by using a convenient infection technique. Appropriate insertion of the vector into genes causes EGFP or β-lactamase expression. This technique should facilitate the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of reporter genes. Our findings suggest that the reporter gene is driven by an upstream, cell-specific promoter during cell culture and cell differentiation, which further supports the usefulness of lentivirus-based gene-trap vectors. Lentiviral gene-trap vectors appear to offer a wealth of possibilities for the study of cell differentiation and lineage commitment, as well as for the discovery of new genes.

The vast amount of genetic information in mammalian organisms and the cellular complexity of developing embryos require new experimental approaches that rapidly and effectively identify and analyze genes. Gene-trap vectors mark endogenous genes and enable the detection of changes in gene expression (1–3). Marking a gene enables the study of a specific promoter concurrent with the identification and functional analysis of the corresponding gene (4–6). However, currently available gene-trap vectors, most of which are plasmid or retrovirus-based vectors, are limited by low efficiency, short-term expression or restriction to dividing cells (7–9). Introduction of gene-trap vectors into nondividing cells, including neurons and stem cells, presently necessitates the use of physical and expensive methods such as electroporation to insert the exogenous DNA into the mammalian genome. Recently developed HIV-1-based lentiviral vectors overcome these obstacles and are increasingly being used for gene delivery in vitro (10, 11). They also show promise for permanent gene expression in vivo in cells of the central nervous system (CNS) (12–15), hematopoietic system (16), retina (17), muscle (18), liver (19), and pancreatic islets (20).

Lentiviruses are potentially useful as gene-trap vectors because they incorporate randomly into the host genome in nondividing cells (15, 21). With the advent of human genome sequencing there arises the immense challenge of understanding the function of each gene within the intact organism. We have therefore developed a lentiviral gene-trap vector that offers a wealth of possibilities in the study of genetic function. The insights provided by these studies will be helpful for understanding the genetic programming that underlies embryogenesis and also provide the basis for elucidating normal physiological processes and abnormalities that occur in human diseases. We have constructed an efficient lentiviral gene-trap vector that can be used for identifying reporter genes trapped in mammalian cells in vitro, and assessment of the potential of exogenous embryonic stem (ES) cells to differentiate in the mouse CNS in vivo.

Materials and Methods

Vector Construct and Virus Production.

Plasmid NL-neo is based on the NL 4–3 molecular clone (22) and carries a deletion from the NsiI site to the BglII site. A 1,169-bp fragment carrying the neo gene sequence and SV40 early promoter derived from pBK-CMV (Stratagene) was inserted between the BamHI site and XhoI site (23). To construct the lentivirus-based gene-trap vectors, two different reporter genes, β-lactamase (BLAK, Pluronic, Mount Olive, NJ) and enhanced green fluorescent protein (EGFP; CLONTECH) were inserted into the U3 region of the 3′ long terminal repeat (LTR) between the XhoI and XbaI sites to yield the ZR-1 and ZR-2 vectors, respectively (Fig. 1). A splice acceptor (CLONTECH) site was placed before the reporter gene to allow its expression from an upstream, cell-specific promoter. A polyadenylation [poly(A)] site was placed at the 3′ end of the reporter gene. For the preparation of HIV-1 pseudotypes, helper plasmid DNA (5 μg), Env plasma DNA (5 μg), and vector plasmid DNA (5 μg) were cotransfected into subconfluent 293 T cells by using a transfection kit (Stratagene). Approximately 2 × 106 cells per well were plated into a 6-well plate 24–30 h before transfection. The virus stocks were harvested 60–65 h after transfection and filtered through a 0.45-μm-pore-size filter, aliquoted, and frozen at −80°C (15, 21).

Figure 1.

Figure 1

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Components of the HIV-1 lentiviral gene-trap vector system. (A) Vector construct. (i) The gene-trap vectors contain a reporter gene, either BLAK (ZR-1) or EGFP (ZR-2) preceded by a splice acceptor site. (ii) An HIV-1 lentiviral control vector. BLAK, gene encoding β-lactamase; SA, Splice acceptor site. (B) Helper (Packaging) construct. (C) Envelope construct encoding vesicular stomatitis virus glycoprotein (VSV-G).

Animals.

Mice (57/BL16, 4 days postnatal), obtained from Taconic Farms, were maintained in a BSL2/3 animal facility in a temperature and light-controlled room with sterile food and water available ad libitum. The mice were anesthetized with ketamine/xylazine (77 mg/ml and 4.6 mg/ml) at 1 ml/kg body weight or Avertin solution (Aldrich, 15 μl/g body weight) i.p. before surgery. They were placed in a small-animal stereotactic apparatus fitted to a mouse adaptor with the skull horizontal between lambda and bregma (14, 15). Following the surgery and injection, the animals' scalps were closed and sterilized before return to the recovery cage with their mother. The animal experiment was approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke.

Cell Culture and Immunofluorescence Assay.

Primary human skin fibroblasts and human osteosarcoma (HOS) cells were obtained from American Type Culture Collection and grown in DMEM (GIBCO) containing 10% FBS. The murine ES cells, obtained from StemCell Technologies (Vancouver; refs. 24 and 25), were grown in high glucose DMEM containing 15% FBS (StemCell Technologies), 2 mM glutamine, 10 ng/ml leukemia inhibitory factor (LIF), 100 units/ml penicillin, and 100 g/ml streptomycin.

Primary human skin fibroblasts were transduced with lentiviral gene-trap vector ZR-1 for 4 days. Cells were grown on 12-mm round coverslips coated with poly-L-lysine (Becton Dickinson) in 12-well culture dishes in 2.2 ml of medium. Cells were washed three times with 1× Hanks' balanced salt solution (HSS, GIBCO) before determination of β-lactamase. The Cytoblast substrate (Pluronic) was dissolved in dry DMSO and 100 mg/ml of dry Pluronic F12 to make a 6× stock solution, which was subsequently diluted in a 1× HSS/PBS cell loading buffer. The loading was performed at room temperature for 1 h. After incubation, the cells were washed once with cell loading buffer and were kept in buffer for 10–15 min at room temperature before detection. For the generation of trapped ES cell lines, the murine ES cells were incubated with the lentiviral ZR-2 gene-trap vector at 37°C for 3–5 h as described (21, 23). After G418 selection, drug-resistant colonies were transferred to a 24-well plate and expanded to confluence. In the flow cytometry assay, transduced cells were detached from the plate with 2 mM EDTA PBS for fluorescence-activated cell sorting (FACS) analysis.

Injection of Transduced Cells into Mouse Brain.

The murine ES cells were resuspended in 1× HSS/PBS buffer at a concentration of approximately 8,000 cells per μl after transduction by the ZR-2 lentiviral gene-trap vector in vitro. Using the bregma as a landmark, approximately 24,000 cells were injected slowly over a period of 5 min into the neuroepithelium of the neonatal mouse brains (Fig. 2). Three microliters of the transduced cells were loaded into an internal cannula needle (23 gauge) with cannula tubing connected to a Hamilton syringe mounted on a microinjection pump (Harvard Apparatus). The cells were delivered into the brain at a rate of 1.0 μl/min (14). The needle was allowed to remain in place for an additional 2 min before withdrawal from the brain.

Figure 2.

Figure 2

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Embryonic stem cells were transduced with lentiviral ZR-2 gene-trap vector in vitro and implanted into the neuroepithelium of mouse fetus brain in vivo. Fr, frontal neocortex; fmi, forceps minor corpus callosum; ne, neuroepithelium; ov, olfactory ventricle.

Brain Immunofluorescence Assay.

Animals were decapitated 2.5 weeks after transplantation and the brains were carefully removed. The brains were immediately fixed with 4% paraformaldehyde and 1% glutaraldehyde for 24 h at 4°C, then washed with PBS containing 4% sucrose for 2 days at 4°C. Whole brains were embedded in OCT medium (Tissue-Tek, Torrance, CA) and frozen in a methanol/dry-ice bath for cryosectioning (15 μl per coronal section) at −18°C (14, 15). For immunocytochemical detection of the brain sections, the slides were fixed in 4% paraformaldehyde/1% glutaraldehyde and washed three times with PBT buffer (PBS in 1× HSS, 0.1% BSA, and 0.2% Tween 20), then blocked with 10% goat serum for 15 min. After washing three times with PBT buffer, slides were incubated with primary murine antibodies against the NeuN (neuron-specific nuclear protein, 1:200; Chemicon) at 4°C overnight. The anti-mouse TRITC-conjugated secondary antibody (Sigma) was then added onto slides for 30 min at room temperature (15). The slides were washed with PBT buffer and analyzed by using a Zeiss 510 confocal microscope.

Results and Discussion

Gene trapping is a powerful tool for identifying genes important in biological phenomena and for the study of cell differentiation and lineage commitment. The number of mutant mouse lines and genes responsible for mutant phenotypes obtained by gene-trap approaches have been increasing (7, 26). For instance, NPC1-trap cells have been suggested to be useful in studying the regulation of cellular cholesterol homeostasis and the pathogenesis of Niemann-Pick disease type C (27). However, most of the gene-trap vectors used today must be introduced into the host genome through labor-intensive technical work combined with the use of expensive equipment such as electroporation. The efficiency of embryonic stem cell transduction by current gene-trap vectors is also limited and needs improvement. We have, therefore, developed an HIV-1 lentiviral gene-trap vector containing a reporter gene that is capable of integrating and transducing mammalian cells such as human skin fibroblasts and murine embryonic stem cells in vitro and in vivo, using a convenient technique. To our knowledge, this is the first report on the use of an HIV-1-based lentiviral gene-trap vector with biological activity in vitro and in vivo.

Generation of HIV-1 Lentiviral Gene-Trap Vector.

The gene-trap vectors (Fig. 1) for pZR-1 and pZR-2 are designed based on the lentiviral vectors (14, 21) that efficiently infect, but do not replicate in mammalian cells. The pZR-1 vector was created by introducing a reporter gene encoding Escherichia coli TEM-1 β-lactamase into the U3 region at the 3′ end LTR of lentiviral vector. The pZR-2 vector uses EGFP to replace the β-lactamase gene in the same region of the 3′ end LTR. To express EGFP or β-lactamase in cells, the proviral form of the vector should insert into an intron of an actively expressed gene in the appropriate region. The coding sequence, in which the 5′ end of the reporter gene is linked to a splice acceptor to allow the expression of reporter gene, is driven by an upstream, cell-specific promoter. The splice donor of the cellular gene would be linked to the splice acceptor in the β-lactamase or EGFP cassette to form a fused transcript. To obtain valid translation of the fusion transcript, a polyadenylation signal in the gene cassette will stop transcription from the fusion gene. Ordinarily, such insertions would disrupt the function of the gene into which the vector inserts. A bacterial neo gene driven by an internal SV40 early promoter was placed between the BamHI and XhoI sites in the lentiviral gene-trap vector, which allows for G418 selection. Furthermore, both pZR-1 and pZR-2 were generated as self-inactivating (SIN) lentiviral gene-trap vectors in which the U3 region of the 3′ LTR was deleted and replaced by either the β-lactamase or EGFP gene. Because the transcriptional inactivation of the long terminal repeat in the SIN provirus should prevent mobilization by replication-competent virus (28–30), these modifications of the lentiviral gene-trap vectors should increase the safety of vector-mediated gene delivery and enhance transduction of genes into nondividing cells.

Immunofluorescence Assay for HIV-1 Lentiviral Gene-Trap Vector in Vitro.

The lentiviral gene-trap vectors were tested in human skin fibroblasts, HOS cells, and ES cells to identify their subcellular distribution in vitro. To understand the regulation of gene expression, it is essential to use an assay of high sensitivity and fidelity that reports expression at the level of single living cells. We constructed a pZR-1 lentiviral gene-trap vector containing a reporter gene, β-lactamase. β-lactamase belongs to a family of bacterial enzymes that efficiently cleave cephalosporins. This enzyme is normally secreted by bacteria into the cytoplasm of host cells, conferring antibiotic resistance by cleavage of cephalosporins (31). Cytoblast substrates such as CCF 2-AM consist of two fluorophores attached to the 7′ and 3′ positions of cephalosporin (31, 32). The uncleaved substrate fluoresces green in cells with excitation at 540 nm. Cleavage by β-lactamase is detected by a shift in fluorescence emission wavelength to 450 nm, fluorescing blue (Fig. 3A). Cells transduced by the lentiviral NL-neo vector did not show blue fluorescence at 450 nm (Fig. 3B). This result demonstrates the use of β-lactamase in our gene-trap vector in combination with a membrane-permeable, fluorogenic substrate to measure the regulation of gene expression in mammalian cells such as human skin fibroblast cells with high sensitivity. We deduced that an upstream promoter caused the expression of β-lactamase in the transduced with the pZR-1 virus (Fig. 3A, blue fluorescence). To generate a direct reporter gene-trap vector, we replaced the β-lactamase with a gene encoding the sequence for EGFP to make our pZR-2 vector. This vector should be very effective in determining genes regulated in vitro and in vivo because the expression of trap vector fused to a cellular gene can be directly observed by following EGFP fluorescence. Fluorescence-activated cell sorting (FACS) analysis indicates that a substantial percentage of HOS cells transduced with pZR-2 virus (Fig. 4 Bottom) show fluorescence above the background level established by the mock-transduced cells (Fig. 4 Top). As expected, up to 100% of the HOS cells transduced with the NL-CMV-EGFP virus are EGFP-positive, indicating that the transduction was highly efficient (Fig. 4 Middle). The expression pattern of EGFP in ES cells is consistent with the previous results from HOS cells transduced by the pZR-2 (data not shown). These findings suggest that the EGFP gene is being driven by an upstream promoter and provide further evidence for the usefulness of lentivirus-based gene-trap vectors.

Figure 3.

Figure 3

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Detection of β-lactamase activity in human skin fibroblasts (HSF) by fluorescence microscopy. (A′) At 540 nm, all cells that have taken up the Cytoblast substrate show green fluorescence. (A) At 450 nm, β-lactamase-positive cells that were transduced with the ZR-1 gene-trap vector show blue fluorescence while β-lactamase-negative cells remain green. When the HSF cells were transduced by the lentiviral NL-neo vector, all cells show green fluorescence at 540 nm (B′), and there are no cells that show blue fluorescence at 450 nm (B).

Figure 4.

Figure 4

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Analysis of EGFP activity in HOS cells by fluorescence activated cell sorting. (Top) Mock-transduced cells. (Middle) Cells transduced with the positive control NL-CMV-EGFP virus. (Bottom) Cells transduced with the ZR-2 gene-trap vector.

In Vivo Expression of a Reporter Gene Delivered by Gene-Trap Vector in Mouse Brain.

To examine the biological activity of gene-trap vectors in vivo, we transduced murine ES cells in vitro with the pZR-2 trap vector. The target cells were subsequently implanted into the brain of neonatal mice to analyze the expression of the EGFP reporter constructs during brain embryogenesis. The transduced ES cells were implanted into the neuroepithelium (ne) of mice fetus brains in vivo. The mice were killed two and a half weeks after injection. The results showed that some of the transduced stem cells had participated in cell differentiation and tissue formation in vivo. The expression of EGFP was driven by a specific promoter in the CNS during development of the mouse brain (Fig. 5). Interestingly, these differentiated cells derived from these ES cells were found bilaterally, showing that they had migrated into the contralateral cell layer of the ventricle during brain development. This finding suggested that the cellular gene fused to EGFP in the trap vector played an important role on the development of ventricular structure in the mouse brain. Double immunofluorescence and microscopic detection were used to assess the cell types expressing EGFP in the mouse CNS. Most of these differentiated cells were NeuN-immunoreactive neurons (Fig. 6). This finding indicated that stem cells, transduced by the lentiviral vectors, should also be useful as carriers for cell and gene therapy of neurological disorders in the CNS.

Figure 5.

Figure 5

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In vivo differentiation of embryonic stem cells in the mouse brain. ES cells infected with lentiviral ZR-2 gene-trap vector differentiated in the mouse brain. (A) Expression of EGFP was driven by a specific promoter during brain development. (Magnification, ×10.) (B) A differentiated ES cell in the mouse CNS. (Magnification, ×63.)

Figure 6.

Figure 6

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Immunofluorescence assay of mouse CNS cells. (A) Confocal microscopic images of EGFP-expressing cells in mouse CNS (green fluorescence). (B) Antibody staining for neuron-specific marker, NeuN (red fluorescence).

Our results provide evidence that implanting ES cells transduced by a lentiviral gene-trap vector containing a reporter gene is effective in finding mouse genes and investigating their expression patterns during development. This system should ultimately lead to the identification of novel genes involved in the regulation of mammalian development. We also demonstrated that our reporter gene retained biological activity after insertion into the host genome in vivo. In addition, our lentiviral gene trap efficiently transduced target cells and achieved integration through the use of a simple incubation method that saved labor and eliminated the need for electroporation. This system may greatly accelerate the study of the functions of the mammalian genome, as well as other areas of research that require gene targeting techniques.

Acknowledgments

We would like to thank Dr. Jakob Rieser for support during the early phase of this work, Dr. Wahu Choi for help on viral production, and Dr. Judith Davis for providing technical support at the National Institutes of Health BSL 2/3 facility.

Abbreviations

EGFP

enhanced green fluorescent protein

ES

embryonic stem

CNS

central nervous system

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