The full length Ly-6E.l//ocZ construct exhibited reproducible, high level, tissue speciAc expression in mid-gestation embryos in all 4 BL transgenic lines with minimal background, ectopic expression. In order to determine whether the removal of sequences including DNasel hypersensitive sites + 6.7, + 8.7 and + 8.9 affects the spatial pattern of transgene expression, embryos containing the truncated Ly- ôE .lIlacZ construct were stained with X-gal at day llp .c . Stained whole mount embryos at day llp .c . from all XN transgenic lines are shown in figure 21. For comparison, the first panel in figure 21 shows a non-transgenic X-gal stained day llp .c . embryo demonstrating the absence of non-specific staining and a XN23 transgenic embryo displaying extensive ectopic X-gal staining. Mesonephros specific expression was observed only in embryos of one line, XN23. In addition, XN23 adults alone exhibit kidney specific expression of /acZ, suggesting that common genetic regulatory elements may be used, or that there is a direct relationship between lacZ'*' mesonephros cells and lacZ'*’ kidney cells, although anterior mesonephros cells do not give rise to adult renal structures (A. Medvinsky, per s. comm, and Kaufman, 1992). Intense X-gal staining in the tail, similar to that observed for BL transgenic lines was observed only in XN229 transgenic embryos (figure 19), although weaker tail staining can be detected in XN37 and XN231 embryos. Upon closer inspection, whole mount X-gal stained XN229 embryos display tail staining in hindgut endoderm, mesoderm and notochord as is seen in BL embryos, whereas XN37 embryos display only notochord and mesodermal staining and XN231 embryos display only endodermal staining. No Ly-6E. 1 specific adult expression, however,
Figure 21. X-gal staining of XN truncated Ly-6E. 1//ûcZ containing embryos.
Embryos were isolated from all breeding lines of XN transgenic mice (XN23, XN37, XN224, XN225, XN229, and XN231) at day 11 p.c., fixed and stained with X-gal. For reference, the embryo of line XN23 is depicted adjacent to a non- transgenic embryo that does not exhibit X-gal staining.
X N 2 3 X N 3 7 X N 2 2 4
m
is seen in either XN229 or XN231 lines. In addition, embryos containing XN constructs seem to be more susceptible to position effects, exhibiting stronger and more widespread ectopic expression than embryos of BL Ly-6E .l//ûcZ transgenic lines.
Although not providing conclusive evidence, analysis of transgenic mice containing the truncated Ly-6E.l//ocZ construct suggests that the basic elements directing Ly-6E. 1 specific expression may still be present in the XN construct. The probability that specific aspects of the BL expression pattern could arise in a cohort of fewer than ten XN animals by chance is miniscule. Each of the broad classes of expression detected in full-length Ly-6E. l//ûcZ-containing transgenic mice is seen in XN mice, although not coincident in animals of the same line, ie, XN229 = tail only (hindgut, notochord and mesoderm); XN23 = mesonephros and kidney; XN37 = tail mesoderm, notochord and activated lymphocytes; XN231 = hindgut only. It is possible that the element deleted from the BL construct renders the transgene less susceptible to position effects from the surrounding genetic elements at the site of integration. Alternatively, it may be a powerful enhancer element that, in the BL mice masks position effects and its absence renders expression in XN mice too low to be detectable in most situations.
3, Lv-6E.l/nivc transgenic mice.
3.1 Introduction.
Cell lines are a useful tool for the study of rare cell populations, providing relatively large amounts of material enabling biochemical studies to be undertaken on homogeneous cell populations. In an attempt to generate haematopoietic precursor cell lines for such biochemical studies and to investigate the role of the cell cycle in haematopoietic homeostasis in vivo, Ly-6E. Uc-myc transgenic mice were generated.
The c-myc gene is the cellular homologue of v-myc, a viral oncogene associated with avian retroviruses that has been shown to cause leukaemias and carcinomas (Cole, 1986). Genetic abnormalities found in human Burkitt’s lymphoma and rodent plasmocytomas have been reported in which the c-myc gene is translocated into the immunoglobin locus, effectively placing c-myc under genetic control of the immunoglobin heavy chain enhancer (Cory, 1986). Under experimental conditions, transgenic mice expressing c-myc controlled by the immunoglobin heavy chain enhancer are predisposed to malignancy in pre-B and mature B lymphocytes, suggesting an important role for c-myc in the control of cell proliferation (Adams et al. , 1985; Langdon et al. , 1986). The c-myc gene encodes a transcription factor that forms heterodimers (Blackwood & Eisenman, 1991). The protein binds to DNA, possesses leucine zipper domains similar to those of the Eos, Jun and CREB transcription factors and basic-helix-loop-helix domains similar to those of MyoD, and
is an "immediate early growth response" factor, being rapidly induced upon mitogen stimulation of quiescent cells (Landschultz et al., 1988; Murre et a l , 1989).
Studies on the function of c-myc demonstrated that it has a role in both cell proliferation and programmed cell death, apoptosis (Evan et al., 1992). Whilst induction of c-myc expression is sufficient to drive quiescent cells into the cell cycle (Eilers et al., 1991), specific inhibition of c-myc expression by the addition of antisense oligonucleotides demonstrated that it is essential for cell proliferation (Heikkila et a l., 1987; Loke et a l., 1988). As c-myc is both necessary and sufficient for cell proliferation, it can be thought of as a single step to cellular transformation. The induction of an apoptotic pathway by c-myc expression may provide an inbuilt safeguard against the potentially oncogenic effects of a single mutation deregulating its expression. Thus, it is thought that cells expressing c-myc are primed for programmed cell death, the avoidance of which relies upon a further, positive survival signal (Evan et al., 1992). This is consistent with the theory that apoptosis is the default pathway for all cells to follow unless averted by survival factors (Raff, pers. comm.).
The oncogenic properties of c-myc have been utilised for tissue directed oncogenesis experiments using both retroviral and transgenic approaches (Langdon et al., 1986; Stewart et al., 1984; Leder et al., 1986; Alexander et al., 1987). In some of these studies, c-myc expressing cells have been maintained in vitro and yielded stable cell lines (Adams et a l., 1985; Spanopoulou et a l., 1989; Harris et a l., 1988). Targeted expression of c-myc using the regulatory elements of the Thy-1 gene resulted in the production of both T lymphoid and thymic epithelial cell lines (Spanopoulou et al., 1989). The unexpected transformation by c-myc of thymic
epithelial cells is an example of targeted oncogenesis providing a convenient source of rare cell types for detailed biochemical analysis. Similarly, Ly-6E. transgenic mice may allow the characterisation and manipulation of rare Ly-6E .l^ cells, such as haematopoietic progenitors, in vitro. The high degree of penetrance of c-myc in targeted oncogenesis experiments is demonstrated by the fact that 9 out of 10 independent Thy-l/m yc transgenic mice displayed thymic tumours (Spanopoulou e ta l., 1989).
As suggested by the results of the Thy-Vmyc study, c-myc induced transformation is not restricted to cells of the lymphoid compartment. Transgenic mice expressing c-myc under control of the erythroid specific GATA-1 regulatory elements exhibit a severe, early onset erythroleukemia (Skoda et a l., 1995) whilst c- myc expression can induce mammary tumours when targeted to the mammary gland in transgenic mice (Schoenberger et al., 1988). However, not all c-myc expressing transgenic mice develop tumours and it seems that certain tissues and organs are more susceptible to c-myc induced transformation than others (Adams & Cory, 1991; Roland & Morello, 1993). The precise cell type in which the transgene is expressed and the level of expression are crucial factors to the outcome of targeted oncogenesis experiments.
To utilise the cell transformation potency of c-myc for the analysis of haematopoietic stem and progenitor cells, transgenic mice were generated in which c-myc expression was under control of the regulatory elements of the 14 Kb Ly-6E. 1 gene. The tissue specific expression pattern of the Ly-6E. 1 transgene should yield cell lines transformed by c-myc which will be representative of immature haematopoietic stem and progenitor cells. Detailed biochemical analyses and
comparisons between such cell lines may suggest precursor/progeny relationships within the heirarchy of differentiating haematopoietic stem cells.
In addition to the generation of cell lines, the analysis of Ly-6E. transgenic mice may provide an insight into haematopoietic stem cell maintenance in vivo. The mechanisms involved in the lifelong maintenance of the haematopoietic system remain unclear (Orlic & Bodine, 1994; Spangrude et al. , 1991; Ogawa, 1993; Lord & Dexter, 1995). One theory is that a cohort of haematopoietic stem cells exist as a pool of quiescent cells which seed the bone marrow during development and are sequentially, clonally activated throughout adult life (Kay, 1965; Heilman et a/., 1978; Brecher et al. , 1986). If there exists a quiescent pool of haematopoietic stem cells residing in the adult bone marrow that are awaiting recruitment, the expression of c-myc may drive these cells into cell division and to differentiate. However, if the haematopoietic stem cell pool were not sustained by progenitors capable of continually seeding this compartment, the reservoir of haematopoietic stem cells in Ly-6E .l/m yc transgenic mice may be depleted. In addition, a greater number of commited progenitors should be detectable as the haematopoietic stem cell is forced into cell proliferation and differentiation.