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