b-Galactosidase marker genes to tag and track human hematopoietic cells Claude Bagnis, Christian Chabannon, and Patrice Mannoni Centre de The´rapie Ge´nique, Institut Paoli-Calmettes, Centre Re´gional de Lutte contre le Cancer, Marseille, France. Key words: LacZ; human hematopoiesis; stem cell; gene marking; retroviruses; gene therapy. nalysis of the behavior and fate of hematopoietic transmissible, and (c) easily detectable in situ; in addi- Acells in vivo is an effective method of improving our tion, the marker gene or its expression product should understanding of normal hematopoiesis, which relies on not be horizontally transmissible. To achieve these goals a complex interactive network including cytokines, che- with the aim of tagging human hematopoietic cells, we mokines, physical contact, and undefined interaction decided to use the retroviral transfer of the bacterial kinetics, and of establishing the use of hematopoietic b-galactosidase (b-gal) activity encoded by the LacZ cells as therapeutic vehicles in gene therapy.1 gene as a cell-marking activity. Analysis of animal models or patients reinfused with hematopoietic cells is considered important in address- ing these issues. In an autologous context, the most relevant context for this purpose, the tracking of reim- planted cells is impossible to achieve without markers THE LacZ GENE that are able to discriminate between reinfused cells and LacZ and neomycin-resistance (neoR) genes host cells; this emphasizes the need to develop safe, efficient, and easily performed marking strategies. The Thus far, only the neoR gene that induces resistance to injection of nontoxic chemical markers has been widely G418, a neomycin analog, has been used as a genetic used to study the embryonic development of animal marker in clinical trials dealing with hematopoietic cells. models; however, fluorescein-conjugated dodecapep- Several studies have been completed that have provided tides, rhodamine-conjugated dextran, or horseradish interesting information on hematopoietic recovery after peroxidase molecules are diluted during successive divi- stem cell transplantation, or on the origin of relapse in sions of marked cells, thus making long-term studies patients undergoing autologous transplantation. More difficult.2–5 Differences in heterochromatin density in the than 20 gene-marking clinical trials using neoR have quail/chicken chimera model have provided informative been performed or are under investigation and have 6 demonstrated gene transfer and resistance to G418 in a results but cannot be applied to other animal models. 9–12 Defective retroviruses have been found to be very very small percentage of the reinfused cells. How- interesting cell markers in a number of situations, in- ever, the use of neoR is hampered by several problems. cluding preclinical and clinical models, because retrovi- Polymerase chain reaction analysis provides statistical ruses are natural vectors that are able to stably integrate data regarding only integration of the proviral sequence into the genome of the transduced cell; this allows in the studied cell population. This assay does not detection of the provirus in the progeny of genetically necessarily demonstrate transgene expression, nor does modified cells, but in situ detection of a few transduced it give information about individual transduced cells. An cells remains difficult.7,8 analysis of gene expression with the antibiotic resistance A good experimental cell marker must exhibit the assay is limited to clonogenic cells that exhibit different following properties: the expression of the marker must sensitivities to G418 depending upon their nature (i.e., be (a) absent from the cells to be tagged, (b) genetically burst-forming units-E, granulocyte-macrophage colony- forming units (CFU), etc.).13 Finally, some reports sug- gest that the neophosphotransferase encoded by the Received January 3, 1998; accepted May 14, 1998. neoR gene induces modifications of cellular metabolism Address correspondence and reprint requests to Dr. Claude Bagnis, or may act as a transcriptional silencer in eukaryotic 14–16 Centre de The´rapie Ge´nique, Institut Paoli-Calmettes, Centre Re´gional de cells. Therefore, the search for transgenes encoding Lutte contre le Cancer, 232 Boulevard Sainte-Marguerite, 13273 Marseille an activity that is both innocuous and easily recognizable cdx 9, France. E-mail address: [email protected] in situ has led to the use of marker genes such as the © 1999 Stockton Press 0929-1903/99/$12.00/10 LacZ gene. Cancer Gene Therapy, Vol 6, No 1, 1999: pp 3–13 3 4 BAGNIS, CHABANNON, MANNONI: LacZ MARKER GENE AND HEMATOPOIETIC CELLS The LacZ gene product, is the most widely used assay for the detection of transduced cells.28 It is worth noting that cells have to General information. The bacterial LacZ gene which be fixed before addition of the substrate. X-Gal degra- was isolated from the z cistron of the Escherichia coli 7 dation can be inhibited by 4-b-D-galacto-D-glucose. A lactose operon encodes b-gal, a glycoside hydrolase. The recent report suggests that the use of this detection 3081-base pair coding sequence encodes a nonglycosy- strategy in vivo may underestimate the extent of gene lated protein of 1027 amino acids with a molecular mass expression compared with the immunostaining per- of ;11 kDa17 The active enzyme is a tetramer that is formed with monoclonal or polyclonal antibodies.29 The made up of four identical monomers encoded by the use of fluorescein digalactose (FDG) combined with LacZ sequence. fluorescence-activated cell-sorting (FACS) analysis of- Optimal pH and endogenous activity. Mammalian cells fers an interesting alternative. FDG is a galactose com- possess endogenous b-gal involved in lactose metabo- plex that traps and inactivates a fluorescein molecule lism and glycolipid and mucosubstance catabolism. which can be released as an active form by b-gal activity. These enzymes are found mainly in lysosomes. Bacterial The FDG-FACS assay is the most sensitive b-gal detec- b-gal works optimally at a neutral pH, whereas mamma- tion assay, because 1–10 enzyme molecules per cell are lian b-gal requires an acidic pH (pH 4–5).18 Thus, by enough to detect a b-gal activity; in contrast, 100-1000 selecting appropriate reaction buffers, it is possible to molecules per cell are necessary to achieve a positive preferentially observe the enzymatic activity attributable X-Gal staining. In addition, the staining of cells with to the exogenous bacterial b-gal.19 In addition, chloro- FDG does not require the fixation of cells and allows the quine treatment of the cells can increase the internal pH sorting and subsequent culture of viable cells.27,30–33 of lysosomes and consequently prevent detection of the However, because FDG is a large molecule, the staining endogenous b-gal activity.20,21 procedure requires a gentle permeation of cell mem- branes using a mild osmotic shock, which makes this Subcellular localization. In mammalian cells, the LacZ practical approach rather difficult when assaying primary gene product is found mainly in the cytoplasm.22 When hematopoietic cells. Lipophilic alternatives to FDG have chromogenic substrates are used for in situ detection, been developed, but the interest of these molecules still cells expressing LacZ can be visualized by their cytoplas- remains to be demonstrated.33 mic staining surrounding a pale lighter area (the un- stained nucleus). In CV-1 cells, it has been shown that LacZ and cell behavior. Most of the results published b-gal has no membrane component and cannot be found thus far concern nonhematopoietic cell or tissue types; exposed at the cell surface.23 Moreover, b-gal is not able several examples are reported below. Sanes et al7 have to cross intracellular or synaptic junctions; consequently, shown that the growth of murine NIH-3T3 fibroblasts marking activity does not leak from LacZ-expressing infected with a retrovirus coding for the LacZ gene is cells to neighbor cells that do not express the LacZ identical with that of noninfected NIH-3T3 cells. Fur- gene.24 thermore, the same experiments have also shown that Alternatively, the development of a fusion protein the percentage of LacZ-expressing cells remains stable composed of the nuclear localization sequence of the over 50 cell divisions. The LacZ gene was also used as a simian virus 40 T antigen (Ag) at the 59 end of the LacZ reporter gene to analyze the transcription of specific or coding sequence results in a b-gal enzyme that is located ubiquitous promoters in different cell and tissue types. primarily in the nucleus of eukaryotic cells.22,25 Expres- LacZ was transferred to the cotton rat or to nonhuman sion of the fusion protein nls-LacZ during mitosis sug- primate airway epithelium using adenoviral vectors in gests that the location of the enzyme is mostly perinu- preparation for a clinical trial of cystic fibrosis.34–36 The clear. Using photon microscopy, b-gal staining is not gene encoding b-gal was also transferred to neuro- diffuse throughout the nucleus, but rather appears as a nal37–40 and retinal cells,41–43 keratinocytes,44,45 blad- dark rim on the external face of the nuclear membrane. der46 and gallbladder cells,47 mesangial and tubular Galileo et al reported that the nuclear localization of cells,48,49 endothelial and other vascular cells,50–54 skel- nls-b-gal is not totally stable, with a cytoplasmic staining 55–59 60–63 1 etal and heart muscle cells, hepatocytes, sali- in 1% of postmitotic nls-b-gal embryonic cells in vary gland cells,64 and even thyroid follicular cells65 vitro.26 using different, mostly adenoviral vectors in different Detection of expression of the LacZ gene. Expression of species. In these experiments, limited side effects were the LacZ gene or its derivatives can be detected using observed and were believed to be related to the vector various substrates.27 Cellular extracts can be analyzed itself,34,51,57,65,66 rather than to the LacZ gene or its using O-nitrophenol-b-D-galactoside or 4-methyl-umbel- product.