Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg,
Hamburg, Federal Republic of Germany
1 Communicating author

Summary

A protocol has been established which allows obtaining growth-factor-independent cell mutants by retroviral insertion mutagenesis in vitro. In order to identify gene alterations possibly leading to growth-factor independency, two techniques were established for the cloning of retroviral insertion sites. One technique makes use of the amplification of retroviral flanking fragments by inverse polymerase chain reaction (IPCR). The other strategy involves complementation of a truncated kanamycin gene present in abacterial plasmid vector by a neomycin gene fragment originating from the retroviral vector, which allows direct selection for kanamycin resistant bacterial cell clones. Using these techniques flanking fragments with several putative genes have been obtained. One flanking fragment shows high homology to the rat ionotrophic glutamate receptor, i.e. GluR5.

Introduction

Omission of growth factor from proliferating cells leads to cell death unless oncogenes such as those encoding tyrosine kinase activity abrogate the factor requirement of factor-dependent cells (Pierce et al., 1985; Mathey et al., 1986; Wheeler et al., 1987; Cleveland et al., 1989; Katzav et al., 1989; Mecckling et al., 1992). In fact, in hematopoietic stem cells, the primary function of colony-stimulating factors appears to be suppression of programmed cell death (apoptosis, Williams et al., 1993), and this effect allows an intrinsically determined pathway of differentiation to be followed (Fairbairn et al., 1993). Several different messenger systems have been associated with induction of apoptosis, and the final response varies with the cell type and the other signals being received. Consequently, in addition to genes exclusively involved in cell death, there are likely to be some that can influence other aspects of cell behavior as well. In fact, genes mediating or modulating apoptosis such as myc, ras, p53, bcl-2, bcl-x, bax etc. are critically involved in control of proliferation and differentiation (reviewed by Freeman et. al., 1993). Since the molecular control of proliferation and differentiation has been studied far more extensively than control of apoptosis, some proteins with long established roles in these areas could also be important in apoptosis. Retroviral insertion mutagenesis has proved useful for identifying oncogenes in provirally induced tumors (Habets et al., 1994), and for identifying genes or genetic loci involved in growth factor independence (Stocking et al., 1988, Dorsers et al., 1993). We asked whether it is applicable to obtain growth-factor independent mutants of hematopoietic stem cells in vitro. We anticipated that retroviruses integrate preferentially into open chromosomal domains (reviewed by Breindl et. al. 1989), which may lead to: a) gene inactivation, b) gene truncation yielding an altered phenotype or c) constitutive gene expression due to an exchange of transcription control regions.

Results and discussion

Establishment of growth-factor independent mutants

The human, growth factor-dependent, hematopoietic precursor TF -1 cell line derived from a patient with erythroleukemia (Kitamura et al., 1989) was used for the retroviral insertion mutagenesis. TF-l cells require IL-3 or GM-CSF for long term growth. Other factors such as stem cell factor (SCF) and erythropoietin (Epo) induce short term proliferative signals. TP A induces differentiation into macrophage like cells. Hemin and Epo induce hemoglobin synthesis. TF-I mutants grow stroma cell dependent (Itoh, et al., 1994). For infection with the retroviral vector TF-l cells were cocultivated with irradiated pM3neo-supF producing fibroblasts for 8 or 16 rounds of cocultivation. Subsequent growth factor removal allowed the detection of growth-factor independent cell mutants. This regime (Fig. 1) led to the generation of 241 growth- factor independent cell lines (Stocking et at., 1993). The construction of the retroviral vector pM3neo-sup containing a gag-neoR fusion gene and the bacterial tRNA suppressor gene (sUpF) has been described earlier. Helper cell lines were infected to establish stably transfected amphotropic virusproducing cell lines (Stocking et al., 1993).

Fig. 1: Establishment of growth-factor independent TF -I cell mutants.

Cloning of retroviral integration sites by inverse PCR

Since all our mutants contain more than one retroviral integration we intended to clone large numbers of integration sites in order to eliminate integration events not affecting genes involved in factor independency. To achieve this, we initially attempted to select for supF containing A phages of genomic phage libraries. Due to the finding that the supF gene had suffered mutations ( data not shown) probably during establishement of the packaging cell line, this technique could not be used. The first technique applied was therefore cloning by inverse PCR as is depicted in Figure 2. It involves cutting of genomic DNA by a restriction enzyme, ligation favouring intramolecular reaction (dilute solution), heating to 94°C for 30 min (nicking), and amplification of the circular genomic DNA using two primers which recognise retroviral sequences.

Fig. 2: Inverse PCR strategy for cloning retroviral integration sites.

Genomic fragments in the range of 0.15 kb to 1.4 kb were obtained. Southern-blot and sequence analysis confirmed that these fragments respresent retroviral flanking sequences (data not shown). However, Southern-blot analysis revealed as well, that several of the cloned fragments contained repeat elements. Most of these repeat elements were identified by sequence analysis as to be Alu elements. The estimated copy number of 700 000 Alu elements per haploid genome predicts a density of one Alu repeat every 4 kb of genomic DNA (reviewed by Makalowski et al., 1994). At the cytogenetic level, Alu repeats are concentrated in R bands, the most transcriptionally active areas of the genome. Alu elements are found in introns of most of known actively transcribed genes. It appears that integration of the retroviral vectors occurred with some preference close to repeat sequences, mainly Alu elements.

Cloning of retroviral flanking sequences by kanamycin gene complementation.

Mainly due to inheritent problems encountered by the IPCR cloning technique such as the presence of repeat elements within short fragments, which in part did not allow any further analysis, we established a second cloning protocol. We made use of the neomycin resistance gene, which collfers kanamycill resistallce to bacteria. Since no bacterial ribosomal bindillg site preceeded the neomycin gene, selecting for the complete proviral sequence including 3'- and 5'- flanking sequences could not be achieved. However, a cloning strategy for 3' flanking which involves cutting the genomic DNA by Nco I, ligation into a plasmid vector containing the 5' end of the kanamycin gene and a bacterial promoter depicted in Fig. 2 could be established. To obtain the highest possible transformation efficiency, the following parameters were optimized: bacterial strain, ligation conditions, transformation conditions, DNA concentration, and field strength (data to be shown elsewhere).

Fig. 3. Construction of the vector used for the cloning of 3' -flanking DN As by kanamycin gene complementation.

Restriction of genomic DNA from TF-I mutants by Nco I and subsequent Southemblot analysis using the Ncol/BamHI 3' fragment of the retroviral vector (see fig. 2) revealed that all identified fragments were larger than 2.5 kb, the size predicted by the vector part of the cloned fragment ( data IlOt show-n). It is therefore suggested, that deletions or recombinations of the 3' part of the retroviral vector did occur with a rather low frequency if at all. Moreover, all fragments cloned by the kanamycin gene complementation approach did match in size with the corresponding genomic fragment seen by Southern-blot analyis (data not shown). Southern-blot analysis revealed as well, that approximately 50% of the cloned fragments do contain Alu repeats. been published to date. Its chromosomal localisation, however, has been assigned to 21q22 (Potier et a]., 1993) in the vincinity of the gene for familial amyotrophic lateral sclerosis (Eubanks et a]., 1993; Gregor et a]., 1993).

Fig. 4: Retroviral insertion into the Glutamate receptor GluR5.

It is generally agreed that receptors for the major excitatory neurotransmitter glutamate may play not only key roles in synaptic machanisms for distribution of information, and in neurodegeneration ( reviewed by Barnard, 1992; Gasic and Heinemann, ]991 ; Sommer and Seeburg, 1992; Wisden and Seeburg, 1993) but also in mouse embryo developement as well, since the transcripts of GluR5 and GluR6 were detected by Northern analysis in mouse embryos of 11 days gestation (Gregor et al., 1993). This finding and the observation that GluR5 protein forms ion channels in Xenopus oocytes that are responsive to L-glutamate (Bet tier, et al. 1990) allows speculating that the GluR5 mayas well playa prominent role in the development of the hematopoietic system, in particular be involved in Ca2+ homeostasis. Based on these considerations we test at present whether a) the GluR5-R does playa role in differentiation and or proliferation of hematopoietic cells and b) whether common integration events can be identified.

Acknowledgements

This work was supported by a grant of the Deutsche Krebshilfe and AMGEN. The Heinrich-Pette-Institut is financially supported by Freie und Hansestadt Hamburg and Bundes ministerium für Gesundheit.

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