Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1998-10-16
2004-11-30
Fredman, Jeffrey (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S008000, C800S010000, C800S013000, C800S018000, C800S021000, C800S025000, C435S069100, C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06825394
ABSTRACT:
The present invention relates to cell lines and transgenic animals. More particularly it relates to a method for producing a rat cell line, a method for producing a transgenic rat, a transgenic rat, a rat cell line, cells and tissue obtained therefrom and uses therefore.
Currently there are few permanently growing or immortalised cell lines of epithelial or neuronal origin which are fully characteristic of the differentiated phenotype or produce differentiated products at the levels seen in viva. For example, there are immortalised cell lines of human and rodent origin from the breast but these fail to express many of the characteristics of fully differentiated cells in vivo (Cancer Metastasis Rev. 6. 55-83, 1987 and Histol, Histopathol. 8 385-404, 1993). Those of the Central Nervous System (CNS) have limited proliferation capabilities whilst those that do proliferate are almost exclusively derived from neuroblastomas of peripheral nerves and are not representative of the CNS.
The present in vitro methods for examining differentiated epithelial and neuronal cell behaviour involves repeated establishment of primary cell cultures from rodent fetal tissues. Unfortunately these cultures rapidly senesce, contain mixed populations of cells and invariably are overgrown by contaminating, more rapidly growing cell types such as fibroblasts (J. Cell Biol. 73. 561-577, 1977 and In Vitro, 25, 23-36, 1989). They are therefore unsuitable for most molecular analyses where total cell extracts are required such as, for example, assays for messenger RNA, specific proteins, ligand-receptor interactions, and are wasteful in time and animals used for their production. The ability to produce immortalised populations of such cells would therefore be an enormous advantage for basic and applied biological studies. Since the rat is the laboratory animal used in most pharmacological studies, it is the animal of choice for any experimentally-derived cell systems for the pharmaceutical and toxicological industries.
Unfortunately the very process of immortalisation or transformation mitigates against the expression of the differentiated phenotype, since biological systems are almost invariably programmed only to differentiate after proliferation has ceased. Thus primary cultured cells, if they can be induced to proliferate, may be immortalised by chemical agents or more specifically by transforming or immortalising genes carried by oncogenic viruses (J. Virol 15., 613-618, 1985) eg. breast cells. (Dev. Biol. 136 167-180, 1989), but the resultant cell lines fail to express the levels of differentiated products or structures seen in vivo (Histol, Histopathol. 8, 385-404, 1993, J. Cell Biol. 91., 827-836, 1981, Eur.J. Biochem. 133, 707-715, 1983, J. Natl. Cancer Inst. 76, 246-256, 1986). If the primary cultures do not possess much ability to proliferate (eg. neuronal cells) then introduction of immortalising genes, by whatever route, fails to immortalise the cells, since several rounds of replication of the DNA are required to integrate successfully any transfected DNA into that of the host (J. Virol 15, 613-618, 1975).
There are essentially two approaches to the generation of cell lines.
1) One can isolate primary tissue cells from the tissue of interest and culture these in vitro. One can then attempt to immortalise these cells in vitro using various techniques before they senesce, using microinjection or other transfection techniques.
2) One can attempt to generate a transgenic animal from which to derive cell lines.
Primary Cell Transfection:
The most common way of immortalising primary tissue cells is to transform these with an immortalising gene construct. Such genes are commonly found in a variety of viruses. There are various problems with this technique. One needs a large number of primary cells as transformation is inefficient. Transformation will result in integration of the transforming construct into arbitrary sites in the genomic DNA. The genomic environment of the integration determines its levels of expression and experience shows that this tends to be highly variable so transformations of the same primary tissue often give rise to cell lines with widely differing properties. The immortalisation of cells also tends to suppress terminal differentiation which is disadvantageous if the cell line is supposed to mimic the behaviour of its tissue of origin.
One can use a conditional immortalising gene construct. An example is a temperature sensitive mutant, tsA58 (P. Tegtmeyer, J. Virol. 15, 613-618, 1975) which is found in the early region of the Simian Virus 40, and which encodes a thermolabile protein, the Large Tumour (LT) antigen which is capable of immortalising cells only at its permissive temperature (C. A. Petit, M. Gardes & J. Feunteun, Virology 127, 74-82, 1983, P. S. Cat & P. A. Sharp, Mol. Cell. Biol. 9, 1672-1681, 1989). This allows transfected primary tissue cells to be grown indefinitely in culture at the permissive temperature but if cells are required for experimentation then they can be cultured at the restrictive temperature and one would hope that they express genes in the manner of their primary tissue of origin. This does not overcome the problem of multiple and heterologous integration sites of one's transformation constructs.
Cell Lines from Transgenic Animals:
Construction of a transgenic animal results in an organism that has an engineered construct present in all cells in the same genomic integration site. Thus cell lines derived from a transgenic animal will be consistent in as much as the engineered construct will be in the same genomic integration site in all cells and hence will suffer the same position effect variation. This is a small improvement over primary cell immortalisation.
(i) Tumour Derived Cell Lines:
The first and simplest approach has been to transfect cultured murine embryonic stem cells with an immortalising gene construct under the control of a cell type specific promoter. Transformed ES cells can then be injected into a blastocyst from a host mother and the host embryo reimplanted into the mother. One hopes to get a chimeric mouse whose tissues are composed of cells derived from types of embryonic stem cell present in the embryo. Usually the mice from which the ES cells for transformation are derived are chosen to have a different coat colour from the host mouse into whose embryos the transformed cells are to be integrated. Chimeric mice will then have a variegated coat colour. Such mice are then crossed with an appropriate strain in the hope that the germline will also be chimeric and that offspring mice will carry the transgene. It is then hoped that the transgenic mice will develop tumours in the tissues in which the promoter is activated. These tumours can then be cultured as cell lines.
It has been shown that mice transfected with a construct of the SV40 LT antigen under the control of the metallothionein promoter developed tumours of the choroid plexus, and that cell lines can be isolated from these transformed tissues (R. L. Brinster et al, Cell 37, 367-379, 1984). Similarly, it has been shown that in mice transfected with an LT-antigen construct under the control of the 5′ regulatory sequences of the insulin gene, tumours developed in the beta-islets of the pancreas (D. Hanahan, Nature 315, 115-122, 1985). This is a clear example of immortalising gene being targeted to the tissue of interest.
Despite these and other successes with this approach it is not ideal. Tumour formation is associated with multiple genetic abnormalities and chromosomal rearrangements (B. Vogelstein et al, N. Engl. J. Med. 319, 525-532, 1988). More often than not the resultant cells no longer express the relevant terminally differentiated genes, or at least not appropriately.
(ii) Conditional Immortalisation of Cell Tissues
A second approach is to transform one's ES cells with a conditional immortalising gene coupled to a broad specificity promoter so that ostensibly the construct is expressed in all tissues in the mouse. If a temperature sensitive immo
Barraclough Barry Roger
Davies Barry Robert
Kilty Iain Charles
Rudland Philip Spencer
Schmidt Guenter
Baker & Botts L.L.P.
Fredman Jeffrey
Kaushal Sumesh
LandOfFree
Conditionally immortalized cell lines derived from... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Conditionally immortalized cell lines derived from..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Conditionally immortalized cell lines derived from... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3356569