Prospects for directing temporal and spatial gene expression in transgenic animals |
Summary Molecular mechanisms of development and disease can be studied in transgenic animals. Controlling the spatial and temporal expression patterns of transgenes, however, is a prerequisite for the elucidation of gene function in the whole organism. From the several binary systems which have been established to permit conditional activation of transgenes, the tetracycline responsive expression system has been the most successful one in animals. Transgenic mice carrying a tetR/VP16 hybrid gene (tTA) under different promoters have been used to temporally activate several-thousand fold the expression of genes under the control of a promoter containing tetop sequences. The importance of tetracycline regulatable systems goes beyond their ability to direct temporal gene expression in the context of the whole animal. In combination with Cre/lox recombination tools it will facilitate the deletion of genes from the genome in specific cells and at specific timepoints. This approach is required to test the function of genes whose presence is critical for embryonic development.
Why do we need inducible gene expression systems? Gain of function and loss of function
experiments in animals have been used to elucidate the role of mammalian gene products in
organ development and oncogenesis. Although it is possible to direct the expression of
transgenes to defined tissues by employing specific genetic regulatory elements,
conditional control of gene expression has been a challenge. For example, the promoter of
the whey acidic protein (WAP) gene can target gene expression to mammary alveolar cells
(1), but its temporal activation during puberty and pregnancy cannot be controlled by
experimental conditions in vivo (2). The inability to directly control the temporal
expression of transgenes has important consequences for transgenic studies designed to
elucidate the role of proteins involved in development. The phenotype observed will
frequently reflect the stage of development at which the transgene encoding the regulatory
protein was first activated. For example, if expression of the transgene encoded protein
causes lethality at a defined stage of development, it will not be possible to study its
effect on later developmental stages. This is particularly important in cases in which a
protein can play different roles dependent on the stage of development and/or the cell
type. For example, depending on the time of expression during pregnancy, TGF-ß can
interfere with either ductal or alveolar development of the mammary gland (3-5).
Another application for inducible gene expression lies in the field of gene deletions. A
substantial number of genes are required for mouse development, and their deltion from the
genome in ES cells can result in early embryonic lethality. Through the combination of an
inducible system with the Cre/lox technology it will be possible to delete genes at
specific timepoints during development.
Tetracycline responsive system The development of regulatory circuits based on the
tetracycline resistance operon tet from E.coli transposon Tn10 opened new approaches to
controlling gene expression in eukaryotic cells (6-8). Transactivator proteins (tTA and
rtTA) composed of either the wild-type or a mutant tet repressor, respectively, and the
activating domain of viral protein VP16 of herpes simplex virus activate transcription
from a minimal promoter fused to seven tet operator sequences from Tn10 (tetop) (see model
below). The HCMVIE1 enhancer/promoter and the MMTV-LTR were used to direct tTA expression
in transgenic mice, tetop controlled target gene expression was activated in different
cell types (8, 9). Transcriptional activation in this constellation can be repressed by
administration of tetracycline to the animal and is activated upon withdrawl of the
antibiotic.
The classical tetracycline responsive system requires the continues presence of
tetracyline to keep the target gene silent, a scenario which may be difficult to maintain
in certain experimental settings. The identification and isolation of a mutant
tetracycline repressor with altered DNA binding properties by Hillen and colleagues
provided the tools to built an expression system that is silent in the absence of
tetracycline and induced in its presence (6). The tet repressor in this reverse system
only binds to the tetop in the presence of tetracycline. This system has only been tested
in tissue culture cells, but experiments in transgenic mice are well underway.
Since the expression of tTA transgenes appears to be low in general, Shockett and
colleagues (10) modified the tet responsive system to permit an
Inducible gene deletion and targeting Genes can be deleted from the genome of ES cells
using homologous recombination and mice can be derived from such cells. The deletion of
many genes from the genome of ES cells, however, is incompatible with the formation of a
mouse, which demonstrates the importance of these molecules for developmental processes.
Methodologies are being developed that permit the deltetion of genes from the mouse genome
at a specified time during ontogeny. Kuhn and colleagues (11) generated a transgene
encoding the Cre enzyme which was under the control of the mouse Mx1 gene. This promoter
is fairly inactive in healthy mice and can be induced by interferon a and b. These mice
were crossed with mice carrying a DNA polymerase b gene flanked by loxP sites. Although
the activation of the Cre enzyme was partially dependent on the presence of exogenous
interferons, deletion of the polymerase b gene was seen in only a small percentage of
cells. This observation highlights a current problem in transgenesis that not every cell
within a given tissue expresses the transgene.
Currently several groups combine the tet responsive system with the CRE/lox recombination
system. This combination has advantages over the Mx1 system described by Kuhn and
colleagues, in that no or only little basal activity of Cre should be expected.
Hurdles General problems linked to transgenesis together with those specific to the
tetracycline system need to be overcome in order to successfully control gene expression.
General hurdles of transgenesis include position dependent variations in the level and
homogeneity of gene expression, even within a line. Heterogenous expression of transgenes
within defined cell populations has been observed frequently. Extensive mosaic transgene
expression occurs in mammary tissue of mice carrying hybrid genes under the control of the
MMTV-LTR (12, 13) the WAP (14) and the b-lactoglobulin gene promoter (15). The degree of
heterogeneity is not only reflected in the promoter elements used, which are also subject
to a heterogenous expression within the endogenous loci (16) but also upon the site of
integration. Mosaic gene expression may not be a problem in experiments studying the
consequence of oncogene expression or that of a secreted protein. However, it poses
serious problems in cases, such as the temporal excission of genes from the genome using
the Cre/lox system, in which each cell within a given lineage or organ has to express the
transgene.
Traditionally, the binary systems are based on two transgenes which are independently
integrated into the genome. This results in the excaserbation of any problems associated
with transgenesis. Most importantly, the activation of the reporter gene by the
transactivator will only be seen in cells in which both genomic integration sites are
actually recognized by the transcription machinery. This conclusion is consistent with our
findings that tTA induced expression of a tetop-lacZ reporter gene is extremely mosaic in
certain tissues, including muscle (8) and mammary gland (9). However, in other tissues,
such as seminal vesicle and salivary gland, almost homogenous expression has been observed
(9). This suggests that an important determinant of the degree of mosaicism is the
expression pattern of the transcriptional regulatory region used to activate the tTA gene.
At the same time, markedly heterogenous b-galactosidase staining was found in mammary
tissue of double transgenic lactating mice. This indicates that the degree of heterogenity
from an individual promoter can vary from between tissues. Homogenous transgene expression
may be achieved by choosing other promoter elements to control tTA expression. In contrast
to the MMTV-LTR, the promoter/upstream region of the mouse WAP gene generally conveys a
homogenous expression in lactating mammary tissue (16). The introduction of sequences
which can mediate more reliable transgene activity, such as MAR elements, could provide
more homogeneous expression of tTA (2). Another route of achieving reproducible expression
pattern may be the introduction of the tTA into an endogenous gene via homologous
recombination in ES cells.
In general, tetracycline delivery is performed through the implantation of slow release
pellets and does not appear to be a major problem. However, complete abrogation of gene
expression in some tissues, for example skin, may require higher levels of tetracycline
administration or the use of different tetracycline analogues. While tetracycline is
broadly distributed in tissues, the pharmacokinetics of tetracycline delivery for the
tet-responsive system in individual tissues may vary. This could be relevant for studies
related to embryonic development where rapid gene induction and repression is be required.
Model for the Positive and Negative tetracycline Responsive Gene Expression System
Image design by Priscilla A. Furth and Lothar Hennighausen
References
1. Pittius, C. W., Hennighausen, L., Lee, E., Westphal, H., Nichols, E., Vitale, J. & Gordon, K. (1988) Proc. Natl. Acad. Sci. U. S. A. 5874-5878.
2. Burdon, T., Sankaran, L., Wall, R. J., Spencer, M. & Hennighausen, L. (1991) J. Biol. Chem. 266, 6909-6914.
3. Jhappan, C., Geiser, A. G., Kordon, E. C., Bagheri, D., Hennighausen, L., Roberts, A. B., Smith, G. H. & Merlino, G. (1993) EMBO J. 12, 1835-1845.
4. Kordon, E., McKnight, R. A., Jhappan, C., Merlino, G., Hennighausen, L. & Smith, G. H. (1995) Dev. Biol. 167, 47-61.
5. Pierce, D. F.,Jr., Johnson, M. D., Matsui, Y., Robinson, S. D., Gold, L. I., Purchio, A. F., Daniel, C. W., Hogan, B. L. M. & Moses, H. L. (1993) Genes Dev. 7, 2308-2317.
6. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W. & Bujard, H. (1995) Science 268, 1766-1769.
7. Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547-5551.
8. Furth, P. A., St.Onge, L., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H. & Hennighausen, L. (1994) Proc. Acad. Natl. Sci. U. S. A. 91, 9302-9306.
9. Hennighausen, L., Wall, R., Tillmann, U., Li, M. & Furth, P. A. (1995) J. Cell. Biochem. 59, 463-472. (detailed data)
10. Shockett, P., Diflippantonio, M., Hellman, N. & Schatz, D. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6522-6526. (detailed data)
11. Kuhn, R., Schwenk, F., Auget, M. & Rajewski, K. (1995) Science 269, 1427-1429.
12. Hennighausen, L., McKnight, R. A., Burdon, T., Baik, M., Wall, R. J. & Smith, G. H. (1994) Cell Growth Diff. 5, 607-613.
13. Muller, W. J., Sinn, E., Pattengale, P. K., Wallace, R. & Leder, P. (1988) Cell 54, 105-115.
14. Li, B., Greenberg, N., Stephens, L. C., Meyn, R., Medina, D. & Rosen, J. M. (1994) Cell Growth and Differentiation 5, 711-721.
16. Robinson, G. W., McKnight, R. A., Smith, G. H. & Hennighausen, L. (1995) Development 121, 2079-2090.
1. transgenic MMTV-tTA mice
2. 
Contributed by
Lothar Hennighausen and Priscilla Furth
tel. (301) 496-2716
FAX (301) 496-0839
(e-mail: mammary@.nih.gov)
Submitted April, 1996 |
