Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1998-09-28
2001-06-05
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S069100, C435S069700, C435S320100, C435S468000, C800S287000, C800S288000, C800S298000
Reexamination Certificate
active
06242667
ABSTRACT:
BACKGROUND OF THE INVENTION
Functional analysis of cellular proteins is greatly facilitated through changes in the expression level of the corresponding gene for subsequent analysis of the accompanying phenotype. For this approach, an inducible expression system controlled by an external stimulus is desirable. Ideally such a system would not only mediate an “on/off” status for gene expression but would also permit limited expression of a gene at a defined level.
Attempts to control gene activity have been made using various inducible eukaryotic promoters, such as those responsive to heavy metal ions (Mayo et al. (1982)
Cell
29:99-108; Brinster et al. (1982)
Nature
296:39-42; Searle et al. (1985)
Mol. Cell. Biol
. 5:1480-1489), heat shock (Nouer et al. (1991) in
Heat Shock Response
, e.d. Nouer, L., CRC, Boca Raton, Fla., pp167-220) or hormones (Lee et al. (1981)
Nature
294:228-232; Hynes et al. (1981)
Proc. Natl. Acad. Sci. USA
78:2038-2042; Klock et al. (1987)
Nature
329:734-736; Israel & Kaufman (1989)
Nucl. Acids Res
. 17:2589-2604). However, these systems have generally suffered from one or both of the following problems: (1) the inducer (e,g, heavy metal ions, heat shock or steroid hormones) evokes pleiotropic effects, which can complicate analyses, and (2) many promoter systems exhibit high levels of basal activity in the non-induced state, which prevents shut-off the regulated gene and results in modest induction factors.
An approach to circumventing these limitations is to introduce regulatory elements from evolutionarily distant species such as
E. coli
into higher eukaryotic cells with the anticipation that effectors which modulate such regulatory circuits will be inert to eukaryotic cellular physiology and, consequently, will not elicit pleiotropic effects in eukaryotic cells. For example, the Lac repressor (lacR)/operator/inducer system of
E. coli
functions in eukaryotic cells and has been used to regulate gene expression by three different approaches: (1) prevention of transcription initiation by properly placed lac operators at promoter sites (Hu & Davidson (1987)
Cell
48:555-566; Brown et al. (1987)
Cell
49:603-612; Figge et al. (1988)
Cell
52:713-722; Fuerst et al. (1989)
Proc. Natl. Acad Sci. USA
86:2549-2553: Deuschle et al. (1989)
Proc. Natl. Acad. Sci. USA
86:5400-5405); (2) blockage of transcribing RNA polymerase II during elongation by a LacR/operator complex (Deuschle et al. (1990)
Science
248:480-483); and (3) activation of a promoter responsive to a fusion between LacR and the activation domain of herpes simples virus (HSV) virion protein 16 (VP16) (Labow et al. (1990)
Mol. Cell. Biol
. 10:3343-3356; Baim et al. (1991)
Proc. Natl. Acad. Sci. USA
88:5072-5076).
In one version of the Lac system, expression of lac operator-linked sequences is constitutively activated by a LacR-VP16 fusion protein and is turned off in the presence of isopropyl-&bgr;-D-thiogalactopyranoside (IPTG) (Labow et al. (1990), cited supra). In another version of the system, a lacR-VP16 variant is used which binds to lac operators in the presence of IPTG, which can be enhanced by increasing the temperature of the cells (Baim et al. (1991), cited supra). The utility of these lac systems in eukaryotic cells is limited, in part, because IPTG acts slowly and inefficiently in eukaryotic cells and must be used at concentrations which approach cytotoxic levels. Alternatively, use of a temperature shift to induce gene expression is likely to elicit pleiotropic effects in the cells. Thus, there is a need for a more efficient inducible regulatory system which exhibits rapid and high level induction of gene expression and in which the inducer is tolerated by eukaryotic cells without cytotoxicity or pleiotropic effects.
Components of the tetracycline (Tc) resistance system of
E. coli
have also been found to function in eukaryotic cells and have been used to regulate gene expression. For example, the Tet repressor (TetR), which binds to tet operator sequences in the absence of tetracycline and represses gene transcription, has been expressed in plant cells at sufficiently high concentrations to repress transcription from a promoter containing tet operator sequences (Gatz, C. et al. (1992)
Plant J
. 2:397-404). However, very high intracellular concentrations of TetR are necessary to keep gene expression down-regulated in cells, which may not be achievable in many situations, thus leading to “leakiness” in the system.
In other studies, TetR has been fused to the activation domain of VP16 to create a tetracycline-controlled transcriptional activator (tTA) (Gossen, M. and Bujard, H. (1992)
Proc. Natl. Acad Sci. USA
89:5547-5551). The tTA fusion protein is regulated by tetracycline in the same manner as TetR, i.e., tTA binds to tet operator sequences in the absence of tetracycline but not in the presence of tetracycline. Thus, in this system, in the continuous presence of Tc, gene expression is kept off, and to induce transcription, Tc is removed.
SUMMARY OF THE INVENTION
This invention pertains to a regulatory system which utilizes components of the Tet repressor/operator/inducer system of prokaryotes to regulate gene expression in eukaryotic cells. In particular, this invention provides transgenic animals having a transgene comprising a polynucleotide sequence encoding a fusion protein which activates transcription, the fusion protein comprising a first polypeptide which binds to a tet operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells.
Preferably, the first polypeptide of the fusion protein is a mutated Tet repressor, e.g., a mutated Tet repressor has at least one amino acid substitution compared to a wild-type Tet repressor. In a preferred embodiment, the mutated Tet repressor is a mutated Tn10-derived Tet repressor having an amino acid substitution at at least one amino acid position selected from the group consisting of position 71, position 95, position 101 and position 102. Most preferably, the mutated Tn10-derived Tet repressor comprises an amino acid sequence shown in positions 1 to 207 of SEQ ID NO: 2.
6. The second polypeptide of the fusion protein comprises a transcriptional activation domain, such as a transcription activation domain of herpes simplex virion protein 16.
A transgenic animal of the invention can further have a second transgene comprising a gene of interest operably linked to at least one tet operator sequence. Additionally, the transgenic animal can have a third transgene, e.g., comprising a polynucleotide sequence encoding a fusion protein which inhibits transcription, the fusion protein comprising a first polypeptide which binds to a tet operator sequence, operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells. The inhibitor fusion protein preferably binds to the tet operator sequence in the absence, but not the presence, of tetracycline.
The transgenic animal of the invention can be, for example, a mouse. In other embodiment, the animal is a cow, a goat, a sheep or a pig.
The transgene(s) of the invention can be integrated randomly or at a predetermined location within the genome of the animal.
The invention further provides a method for modulating transcription of a tet operator-linked transgene in an animal of the invention, involving administering tetracycline or a tetracycline analogue to the animal.
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Bujard Hermann
Gossen Manfred
BASF - Aktiengesellschaft
Benzion Gary
DeConti, Jr. Giulio A.
Hanley Elizabeth A.
Lahive & Cockfield LLP
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