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WO1991013979A1 - Souris transgenique surexprimant l'il-4 et procede d'utilisation - Google Patents

Souris transgenique surexprimant l'il-4 et procede d'utilisation Download PDF

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Publication number
WO1991013979A1
WO1991013979A1 PCT/US1991/001279 US9101279W WO9113979A1 WO 1991013979 A1 WO1991013979 A1 WO 1991013979A1 US 9101279 W US9101279 W US 9101279W WO 9113979 A1 WO9113979 A1 WO 9113979A1
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animal
transgene
operator
transgenic
repressor
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PCT/US1991/001279
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Philip Leder
Robert Tepper
Douglas A. Levinson
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President And Fellows Of Harvard College
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Publication of WO1991013979A1 publication Critical patent/WO1991013979A1/fr

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Definitions

  • the field of the invention is transgenic animals.
  • the cytokines are highly pleiotropic effectors of immunity and inflammation which play a major role in the growth and differentiation of lymphoid and hematopoietic cells responding to a foreign antigenic challenge.
  • interleukin-4 IL-4
  • IL-4 one cytokine in particular, termed interleukin-4 or IL-4, exerts a broad range of proliferative and differentiation-inducing activities in both mature and immature T cell subsets, B cells, and hematopoietic precursors (reviewed by Paul, FASEB 1:456-461, 1987).
  • the invention features, in one aspect, a transgenic non-human vertebrate animal (preferably a mammal such as a rodent, e.g., a mouse) having cells (e.g., somatic cells and germline cells) coutaining a transgene encoding IL-4, which transgene was introduced into the animal, or an ancestor of the animal, at an embryonic stage.
  • a "transgene” is defined as a piece of DNA which is inserted by artifice (i.e., by a means other than sexual propagation) into a cell, and becomes part of the genome of the animal which develops from that cell.
  • S uch a trensgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic animal, or may represent a gene homologous to a natural gene of the transgenic animal, but which is inserted into the animal's genome at a location which differs from that of the natural ho olog.
  • a "trensgenic animal” is an animal having cells that contain a transgene, which transgene was introduced into the animal, or an ancestor of the animal, at an embryonic stage.
  • embryonic stage any point from the moment of conception (e.g., as where the sperm or egg bears the transgene) throughout all of the stages of enbryonic development of the fetus, and preferably refers to a stage within the ' first eight days following conception.
  • the transgene contains a heterologous promoter region: for example, a mammalian immunoglobulin promoter region (which may include both a mammalian immunoglobulin enhancer and a mammalian immunoglobulin promoter) , or a promoter region which includes the SV40 eanly region promoter; the promoter of a mammalian actin gene, CD-2 gene, c-fos gene, Thy-1 gene, elastase gene, or metallothionein gene; or the long- terminal repeat of the mouse mammary tumor virus (MMTV-LTR) .
  • MMTV-LTR mouse mammary tumor virus
  • the heterologous promoter region does not contain a naturally-occurring IL-4 promoter;
  • the IL-4 is a mammalian IL-4 (e.g., that of a mouse or of a human) ;
  • the transgene is expressed predominantly in lymphoid tissues of the transgenic animals [i.e., transgene mRNA is more abandant in lymphoid tissues (e.g., spleen, thy us, and lymph nodes) than in other types of tissues of the transgenic animals];
  • the transgenic animal exhibits a heightened allergic response compared to wild-type animals of the same species; and the transgenic animal is pridisposed to develop an inflammatory lesion of the eyelid.
  • transgenic animals which exhibit a heightened allergic response may be used in a method for testing an anti-allergy treatment, which method involves exposing the animal to the anti-allergy treatment and determining the effect of the treatment on the allergic response of the animal.
  • a “promotor” is a ' segment of DNA 5• to the transcription start site of a gene, to which RNA polymerase binds before initiating transcription of the gene.
  • promoter region is meant the entire part of the transgene which is 5' to the transcription start site of that transgene.
  • a “heterologous promoter region” is a promoter region that is not identical to the corresponding naturally-occurring promoter region for the given gene (e.g., a promotor region which does not include the naturally-occurring promotor) .
  • the invention also features an "operator / represser " " transgenic non-human vertebrate animal
  • a transgene having an untranslated region consisting of all parts of the transgene which are not translated into protein, including the promoter region, the 5' untranslated region [between the transcription start site and the initiation codon] , and all introns, which includes a heterologous DNA segment of 6 or more (e.g., up to 100) base pairs, - which heterologous DNA segment consists of a prokaryotic or eukaryotic operator, a eukaryotic transcription factor binding site, a palindromic sequence, or a sequence having dyad symmetry, which trensgene was introduced into the animal, or an ancestor of the animal., at an embryonic stage.
  • This heterologous DNA segment functions to reduce, or "attenuate,” the level of transcription of the transgene, a function which is useful, for example, where a mouse with unattenuated expression of the transgene would be incapable of sexual reproduction (as where such unattenuated expression is fatal to the transgenic mouse, or otherwise prevents it from reaching sexual maturity) .
  • the heterologous DNA segment is present as one to ten copies in the untranslated region (more preferably, one to three copies), and is selected from the following group: the operator of the E. coli lac operon, the E. coli tet operon, the E. coli met operon, or the E.
  • the heterologous DMA segment is located in the promoter region, and includes the operator of the E. coli lac operon or, alternatively, the following palindromic sequence:
  • the invention features a transgenic non-human vertebrate animal (preferably a mammal such as a rodent; e.g., a mouse) having cells (e.g., somatic cells and germline cells) containing a transgene encoding a heterologous repressor protein (e.g., the E. coli lac repressor protein), which transgene was introduced into the animal, or an ancestor of the animal, at an embryonic stage.
  • a repressor protein is a protein which is capable of binding to a specific DNA sequence within a given gene, thereby reducing (i.e., "repressing") the level of transcription of that gene.
  • a "heterologous repressor protein” is a repressor protein which meets one or both of the following criteria: (1) it is a.repressor protein other than one which is synthesized by wild-type animals of the same species as the transgenic animal, or (2) the gene encoding it is inserted into a position in the genome of the transgenic animal other than the position of the corresponding natural gene of wild-type animals of the same species. Animals transgenic only for such a heterologous repressor protein gene are referred to as
  • the cells also contain a second transgene having an operator sequence to which the heterologous repressor protein is capable of bimding, in which case the animal is herein termed "operator +/repressor+”'
  • operator + /repressor + animals are preferably made by the following method: (1) a transgenic non-human vertebrate recipient animal is provided which has cells containing a transgene encoding a repressor protein (e.g., the E.
  • transgene preferably IL-4 or human growth hormone
  • an additional transgene preferably IL-4 or human growth hormone
  • operator + /repressor + transgenic animals may be made either by sexually crossing an operator + / repressor " animal with an appropriate operator " / repressor animal, or by injecting fertilized oocytes of operator " / repressor “1” animals with DNA encoding a gene bearing the appropriate operator.
  • the binding of the repressor protein, and thus the attenuation of transcription may be reversed in operator-!- , repressor "1" double-transgenic animals by (a) mating the double-transgene animal to a wild-type animal, thereby producing offspring approximately one-half of which lack the repressor transgene, which half includes some which retain the other transgene; or (b) introducing into the double-transgenic animal an inactivator of the repressor protein: where the repressor protein is the E. coli lac repressor protein, the inactivator is isopropyl thio- ⁇ -D-galactoside ("IPTG”) .
  • IPTG isopropyl thio- ⁇ -D-galactoside
  • the inactivator of the repressor can be any entity which, when it interacts with the repressor protein in vivo, causes the repressor protein to lose its ability to repress transcription.
  • the IL-4-transgenic animals of the invention which overexpress IL-4 to varying degrees in lymphoid tissues, provides a means to study the effect of overexpression of IL-4 in a reproducible in vivo system. The tendency of a large percentage of these amimals to develop an inflammatory lesion of the eyelid having histologic features seen in allergic disorders makes the IL-4-overproducing animals of the invention a useful animal model for human allergic disorders. Such an animal model can be used to study the nature of the allergic response, and to test proposed means of preventing, controlling or curing allergies in humans.
  • transgenic animals having attenuated levels of expression of a foreign gene have general applicability to the field of transgenic animal generation, as they permit control of the level of expression of genes, full expression of which may be lethal to the subject animal, or which may otherwise prevent reproduction and maintenance of the animal's germ line.
  • the operator+ /repressor ⁇ method of the invention "damps down" expression of the associated gene to a level dependent upon the number of such operators insented into the promoter region, permitting the creation of a series of strains displaying a gradient of levels of expression.
  • the operator +/repressor+ method of the invention provides a reversible "on-off" switch that largely prevents expression of the gene associated with the inserted operator until such time as the system is derepressed by the addition of an exogenous repressor-inactivating molecule, or by removal of the repressor gene by mating the double transgenic animal with a wild-type animal, resulting in segregation of the two transgenes in the offspring such that some (approximately 1/4) of the offspring carry the operator- bearing gene but not the repressor gene.
  • the operator +/repressor+ method ensures that transgenic strains bearing even potentially lethal transgenotypes can be easily maintained without selection against the lethal transgene.
  • the use of the repressor inactivator to control the timing of expression of the foreign geme provides a means for investigating the effect of the particular gene product on, for example, behavior, learning, immunological stimulation or suppression, the etiology of various diseases such as cancer, or a particular stage of embryological development.
  • an established animal strain expressing the transgenic repressor gene would be useful as a source of oocytes into which to transfer any gene bearing an appropriate operator sequence, or to cross sexually with a second animal already transgenic for a foreign gene bearing the operator sequence, in order to generate any desired operator + /repressor + double transgenic animal.
  • Fig. 1 is a representation of the structures of DNA constructs used " in the generation of IL-4 transgenic lines.
  • Fig. 2 is the sequence of a double-stranded 18bp lac operator analog with an internal Sail restriction site.
  • Fig. 3 is a photograph of an RNase protection analytic gel in which RNA from various lymphoid tissues of TG.UD and TG.UG heterozygotes, wild-type littermate controls, and IL-e-producing plasmacytoma cells ("I3L6") was probed with an IL-2 riboprobe which distinguishes endogenous IL-4 mRNA (183-nucleotide protected fragment) from transgenic IL-4 mRNA (212- nucleotide protected fragment) .
  • I3L6 IL-e-producing plasmacytoma cells
  • Fig. 4 is a series of photographs of histologic sections of thymus from wild-type mice (A and C) , transgenic TG.UD mice (B and D) , and bone- marrow- reconstituted mice (E and F) .
  • Fig. 5 is a flow cytometric analysis of the surface antigens of thymocytes from wild type (A) ; TG.UD heterozygote (B, C, D) ; and TG.UG mice ' (E) , performed by double-color staining with antibodies against the indicated surface antigens.
  • Fig. 6 is a bar graph illustrating the proliferative response of spleen cells from four IL-4 transgenic lines and wild-type controls at three spleen cell concentrations.
  • Fig. 7 is a pair of bar graphs illustrating the mean concentrations of serum IgE (A) and serum IgGl and IgG2a (B) for IL-4 tra sgenic lines and wild- type controls.
  • Fig. 8 is a series of photographs illustrating the inflammatory eye lesion observed in many IL-4 transgenic mice, both acroscopically (A, right side; a wild-type control mouse is shown for comparison in A, left side) and at lOOOx magnification of a stained tissue section (B and C) .
  • Fig. 9 is an illustration of the genes utilized in Example 3. IL-4-Overexpressing Transgenic Mice
  • IL-4-overexpressing transgenic mice Three classes of IL-4-overexpressing transgenic mice were generated in accordance with the invention.
  • One class termed “operator /repressor " ,” bears a murine IL-4 gene in which the natural promoter region of the IL-4 gene has been replaced with a promoter region heterologous to the IL-4 gene (i.e., having a sequence not identical to the natural promoter region of the IL-4 gene) .
  • This construct causes the recipient transgenic mice to overexpress IL-4 to such an extent that every founder animal of this class died within two weeks of birth.
  • the second class of transgenic animals is herein termed the “operator "1" /repressor " " class.
  • transgenic mice of this class which are described in Example 2 bear the same IL-4 gene with the same heterologous promoter region as the transgenic mice described in Example l, but in addition have either one or three copies of an E. coli lac operator sequence inserted into the promoter region, which reduces the level of expression of the IL-4 gene in the recipient mouse.
  • the third class of transgenic animals, the "operator /repressor " class is illustrated by the double-transgenic mice of Example 3, which bear the same lac operator-containing IL-4 transgene described in Example 3, in addition to a lac repressor transgene.
  • Repressor protein expressed by the lac repressor transgene in the double-transgenic animal binds to the operator sequence(s) present in the IL-4 promoter region, thus repressing transcription of the IL-4 transgene in vivo.
  • the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the .deposited plasmid, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer.
  • Applicants' assignee acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
  • transgenic DNA was extracted from tail sections according to a protocol modified from Davis et al. (Meth. Enzymol. 65:404-411, 1980), using a single phenol-chloroform extraction prior to ethanol precipitation.
  • the presence of transgenic DNA was detected by Southern blot analysis (Southern, J. Mol. Biol. 98:503-517, 1975) of EcoRl-digested DNA using a 40 0 bp Stvl probe (containing the first IL-4 exon) radiolabeled with ⁇ -[ 32 P]dCTP using the random hexamer priming method (Feinberg and Vogelstein, Anal. Biochem. 132:6-13, 1983).
  • mice Nine of 66 mice (14%) so analyzed carried the transgene. Despite their normal gross appearance at birth, all the founder animals displayed severe runting and died within the first two weeks of life. The postnatal lethal phenotype was poorly characterized due to the rapid demise of the animals; however, gross and histologic analysis of a single founder (TG.TA) demonstrated severe hypoplasia of the thymus with absence of a definable cortex and medulla and marked lymphocyte depletion in the spleen.
  • TG.TA gross and histologic analysis of a single founder demonstrated severe hypoplasia of the thymus with absence of a definable cortex and medulla and marked lymphocyte depletion in the spleen.
  • constructs were designed which were analogous to Ig.IL4 except that they had, in order to attenuate expression from the promoter, single or multiple copies of a lac operator (lac O) sequence from E. coli inserted into the immunoglobulin promoter.
  • lac O lac operator
  • the constructs were prepared as follows: a double-stranded oligonucleotide which encodes an 18 bp lac operator analog (Fig. 2) was synthesized (Sadler et al., Proc. Natl. Acad. Sci. USA 80:6785-6789, 1983). An internal Sail restriction site was engineered at the 3' and for the purpose of cloning additional operator sequences.
  • the oligonucleotides were inserted into the 3' BanI site within the human IgH promoter of the Ig.IL4 fragment and cloned into the plasmid vector pBluescript (pBT; Stratagene, La Jolla, CA) .
  • the introduced operator sequence resides between the TATA box region of the promoter and the usual transcriptional start point (see Fig. 1) .
  • the triple operator construction, pIg.03.IL4 was created by cloning a tandem repeat of the' operator sequence into the Sail site of pIg.O.IL4, which lies immediately 3' of the already-existing lac 0 sequence in the promoter (see Fig. 1) .
  • Both plasmids contain a Sall-Xmal fragment of pBT polylinker sequence immediately 5* of the first IL-4 exon.
  • the Ig.O.IL4 and Ig.03.IL4 recombinants used for microinjection were isolated from their respective plasmids by digestion with NotI and Asp7l8.
  • IL-4 expression in J558L plasmacytoma cells transiently transfected with either of these two constructs was approximately 10- fold lower than IL-4 expression in Ig.IL4 - transfected plasmacytoma cells, as measured by both a bioassay for IL-4 which measures its ability to stimulate proliferation of the HT2 indicator T cell line (Lichtman et al., Proc. Natl.
  • the antisense RNA probe for detection of IL-4 mRNA was generated from the plasmid pGLM-1, which contains a Hindlll- Xmal pBT polylinker sequence immediately 5' of a first exon IL-4 fragment from pIg.IL4.
  • RNAase protection was carried out as described by Melton et al. (Nucl. Acids. Res. 12:7053-7056, 1984).
  • TG.TS promoter-attenuated promoter + /repressor
  • RNA expression data for the thymus and spleen from wild-type animals and TG.UD and TG.UG heterozygates is shown in Fig. 3.
  • No endogenous IL-4 mRNA transcripts could be detected in lymphoid tissues from either wild-type or transgenic animals.
  • the level of transgene expression i.e., IL-4 mRNA
  • IL-4 mRNA correlated inversely with the number of lac operator sequences inserted into the immunoglobulin promoter.
  • the level of IL-4 transgene mRNA in the thymus was greatest in the T G .TA founder (unattenuated construct) and was somewhat lower in the TG.TS line (single operator attenuation) ; expression was further reduced in the TG.TX and TG.UD (triple operator attenuation) lines, the two of which displayed comparable degrees of IL-4 transgene mRNA in the thymus.
  • the TG.UG line also generated from the triple operator-attenuated construct, displayed an even f urther (at least 10-fold) reduction in the level of expression of the IL-4 transgene in the thymus.
  • RNA from TG.UD and TG.UG splenocyte populations enriched for B or T cells panning on dishes coated with goat anti-mouse immunoglobulin (Southern Biotechnology Associates [SBA], Birmingham, AL) was performed (Wysocki and Sato, Proc.
  • Adherent cells were predominantly B cells, the population being >80% B220 + and ⁇ 10% CD3 + by flow cyto etric analysis (see below) .
  • Non-adherent cells were further enriched for T cells by incubation with a mixture of rat anti- mouse CD4 (GK 1.5; Dialynas et al., J. Immunol. 131:2445-2451, 1983) and rat anti-mouse CD8 (53.6.72; Ledbetter and Herzenberg, Immunol. Rev. 47:11-18, 1979) supernatants followed by plating on dishes coated with goat anti-rat immunoglobulin (SBA) .
  • GK 1.5 Dialynas et al., J. Immunol. 131:2445-2451, 1983
  • rat anti-mouse CD8 53.6.72; Ledbetter and Herzenberg, Immunol. Rev. 47:11-18, 1979
  • T cell- enriched population from TG.UG contained about 10-20% residual B cells.
  • the level of expression in TG.UG splenocytes enriched for B cells was found to be significantly higher than in a T cell-enriched splenocyte population.
  • IL-4 transgene mRNA from splenic B cells from the TG.UD line, but expression was clearly o b served from a population enriched for T cells (Fig. 3, lane marked "Spleen UD-T" .
  • the differences in transgene expression in the B and T lymphocytes of TG.UD and TG.UG mice proved to be most informative with respect to understanding the phenotypes observed in these lines.
  • Thymic hypoplasia was also observed, with the total number of thymocytes recovered from individual animals at 4-6 weeks of age being the lowest for the TG.TS line ( ⁇ 10% that of littermate controls) and somewhat greater for the TG.TX and TG.UD lines (34% and 31% that of littermate controls, respectively) (Table 1) .
  • TG.UD offspring were bred to be homozygous for the transgene, they displayed a more severe thymic hyperplasia, comparable to that of TG.TS heterozygotes, suggesting a gene dosage effect.
  • the thymuses of TG.UG heterozygotes were histologically normal and contained normal numbers of cells.
  • this line differs from the affected lines in having a greatly reduced level of transgene mRNA. expression in the thymus and peripheral T cells, despite a high level of expression in the B cell compartment. Thus, the thymic abnormality correlates well with the level of overexpression of IL-4 within the thymus.
  • thymic abnormality induced by IL-4 transgene expression flow cytometric analysis of thymic cell populations (Fig. 5) was performed, as follows: Single cell suspensions from thymuses were stained with the appropriate antibodies and analyzed by standard flow cyto etry on a Cytofluorograf IIS (Ortho Diagnostic S ystems Inc., Westwood, MA). A phycoerythrin-labeled (PE) antibody to mouse CD4 (GK 1.5; Be ⁇ ton-Dickinson, Mountain View, CA) and an FITC-labeled antibody to mouse CD8 (53.6.72; Becton-Dickinson) were used for single- and double- staining procedures.
  • PE phycoerythrin-labeled
  • a hamster anti-mouse CD3 500.A2; Havran et al. , Nature 330:170-173, 1987
  • PE-avidin Vector, Burlingame, CA
  • FITC-labeled anti-CD8 FITC-labeled anti-CD8.
  • Table 1 and Fig. 5 a marked reduction in the fraction of thymocytes coexpressing the CD4 and CD8 surface markers was seen in the TG.TS, TG.TX and TG.UD lines.
  • CD4 CD8 double positive cells, some of which are precursors to the mature single positive (CD4 or CD8 ) medullary thymocytes (Nikolic-Zugic and Bevan, Proc. Natl. Acad. Sci. USA 85:8633-8637, 1988; Guidos et al., Proc. Natl. Acad. Sci. USA 86:7542- 7546, 1989) , normally comprise 80-90% of the thymocyte population and are predominantly small lymphocytes within the cortex (Fowlkes and Pardoll,
  • CD4 CD8 thymocytes the majority of which in the normal thymus represent an early precursor population (Fowlkes et al.,
  • Neutralizing monoclonal anti-IL-4 antibody (11B11; Ohara and Paul, Nature 315:333-336, 1985) was prepared from ascites fluid, as previously described (Tepper et al., Cell 57:503-512, 1989), yielding a final protein concentration of approximately 1 6 mg/ml.
  • the effect of combined in utero (delivered via the maternal circulation) and neonatal administration of large doses of the anti-IL-4 monoclonal antibody was determined by the following methodology: Antibody (0.5 cc) was administered, by intraperitoneal (i.p.) injection to a female TG.UD heterozygote on the day following mating to a wild-type FVB/N male and at weekly intervals until delivery.
  • IL-4 in combination with phorbol myristate acetate (PMA) has been shown to induce the in vitro proliferation of adult CD8 + and (to a lesser extent) CD4+ thymocyte populations, CD4 + CD8 + cells being unaffected (Zlotnick et al.,
  • thymus also responds to IL-4 in vitro by proliferation and differentiation to mature CD8 + cells; again, however, PMA is required.
  • CD4 ⁇ CD8 " thymocytes can pro d uce IL-4 in vitro when stimulated with the calcium ionophore A23187 plus PMA (Zlotnick et al., 1987; Ransom et al., J. Immunol. 139:4102-4108, 1987).
  • IL-4 mRNA By in situ hybridization analysis, IL-4 mRNA can be demonstrated in 5% of unstimulated day 13 and almost 50% of unstimulated day 15 fetal thymocytes, but not at other times during fetal development; interleukin 2 ("IL-2") expression follows the same temporal distribution (Carding et al.,
  • IL-4 may act selectively to expand a mature CD8 + thymocyte population by promoting maturation of precursors or, alternatively, by inducing proliferation of differentiated CD8 + cells.
  • the marked reduction in CD4 + CD8 + thymocytes, the severity o f which reduction varies directly with the level of IL- 4 expression may normally play a role in intrathymic cell death, a physiologic process which occurs during the C D 4 + CD8 + stage of thymic differentiation (Rothenberg and Lugo, Dev. Biol. 112:1-17, 1985).
  • FITC fluorescein-labeled
  • SBA FITC-labeled goat anti-mouse kappa
  • a PE-labeled antibody to mouse CD4 and an FITC-labeled antibody to mouse CD8 were used for single- and double-staining procedures.
  • the severity of the T cell deficiency correlated with the degree of thymic hypoplasia, with TG.TS heterozygotes and TG.UD ho ozygotes being most deficient and TG.UG heterozygotes displaying no T cell reduction, despite a high level of IL-4 transgene expression in the spleens of these animals.
  • splenocytes from four IL-4 transgenic lines and from wild-type controls were cultured at 0.25-4xl0 5 cells per well in 0.2 ml RPMI 1640 medium supplemented as described above, using Con A at a final concentration of 2.5 ⁇ g/ml. Cultures were performed in flat-bottom microculture plates at 37°C in 5% humidified CO for 72 hr. For the last 6-8 hr, each well was pulsed with l ⁇ C i of [3 H] _ thymidine (Amersham) .
  • IL-4 activity was not detectable in culture supernatants from these populations by bioassays using two IL-4 responsive T cell lines, HT2 and C T4 S , which are sensitive to IL-4 concentrations of lOU/ml or greater.
  • the level of transgenic IL-4 mRNA was significantly lower than the level of endogenous IL-4 mRNA observed with Con A- induced wild-type spleen cells or transgenic IL-4 mRNA from uninduced TG.UG spleen or TG.UG splenic B cells (Fig.
  • spleen WT CON A, UG TOT, and UG B lanes signals also not detectable by bioassay.
  • the failure to detect secretion of biologically active IL-4 in vitro may be due to the insensitivity of the bioassay or a result of the absorption of IL-4 to the surface of cultured splenocytes.
  • the CD8 + fraction of splenic T cells appeared to be more severely reduced than the CD4 fraction in the T cell-deficient IL-4 transgenic mice (Table 2) .
  • IL-4 plays a role in the directional maturation of precursor thymocytes to mature CD8 cells, as suggested by the IL-4 transgenic strains of the invention, additional signals must be required to promote their egress to the periphery. It is also possible that IL-4 itself may interfere with the normal process of egress from the thymus, the signals for which are poorly understood.
  • TG.TS a single transgenic line, TG.TS, displayed a marked reduction in the B lymphocyte population of the spleen, as demonstrated by the lack of cell surface staining for B220 and kappa light chain (Table 2) and by an observed failure of spleen cells to proliferate in response to lipopolysaccharide (LPS) , a polyclonal B cell itogen. Marked lymphoid depletion in the spleen was noted histologically in this line, and as a result of the combined T and B cell immunodeficiency, early death (by six weeks of age) due to infection was observed.
  • LPS lipopolysaccharide
  • B cell reduction is observed only in the line with the most severe T cell deficiency. This raises the possibility that normal B cell development might be interrupted by the absence of critical cellular interactions or helper cytokines provided by T cell populations.
  • TG.UG line In the immunocompetent TG.UG line, the high level of constitutive IL-4 expression in the spleen resulted in marked splenomegaly (3-4 fold increase by weight compared with littermate controls) with expansion of both B and T lymphocyte populations as well as non-lymphoid cells. A lesser degree of splenic hyperplasia was also observed in the TG.UD and TG.TX lines. The development of comparable degrees of splenic hyperplasia (2-4 fold increase by weight and cell number) can be induced in wild-type animals within
  • ELISA determinations on serum samples from mice in each of the transgenic lines were performed, as follows: serial dilutions of serum from 1 :1 0 0 to 1:500 were added to microculture wells coated with 10 ⁇ g/ml of purified rat anti-mouse monoclonal antibody, EM 95 (Baniyash and Eshhar,
  • Serum levels were calculated from a standard curve using a purified mouse monoclonal IgE (ATCC # TIB142; Rudolph et al., Eur. J. Immunol. 11:527-529, 1981). Serum IgGl and IgG2a levels were measured as described (Boom et al, J. Exp. Med. 167: 1350-1363, 1988), using serum dilutions of 1:500 to 1:5000, with mouse monoclonal antibodies as standards. As shown in Fig.
  • IL-4 has been shown to enhance the cell surface expression and secretion specifically of IgGl and IgE in vitro by LPS-stimulated B cells (Vitetta et al., J. Exp. Med. 162:1726-1730, 1985;
  • the abnormality was bilateral and present in 12/12 TG.TS heterozygous and 5/5 TG.UD homozygous animals examined, while in TG.UD and TG.TX heterozygotes the lesion was present in 35% of over 50 transgenic offspring studied, and was typically unilateral.
  • FIG. 8B histologic analysis of the eyelid lesion was carried out as follows: tissues were fixed in phosphate- buffered formalin, blocked in paraffin, sectioned at 4 ⁇ , and stained with hematoxylin and eosin (Fig. 8B) , or toluidine blue (Fig. 8C) .
  • Microscopic analysis of the lesion tissues revealed a dense inflammatory infiltrate involving the subepithelial stroma, and composed of mononuclear cells and a striking number of eosinophils (Fig. 8B) .
  • Toluidine blue staining also demonstrated the presence of an excessive number of tissue mast cells in a lesion from a TG.UD mouse (Fig. 8C) .
  • IgE plays an important role in the initiation of allergic reactions by interacting with the high-affinity Fee receptor (FceRI) on mast cells and basophils (Metzger et al., Prog. Immunol. 5:493-501, 1983; Conrad et al., J. Immunol. 130:327-333, 1983). Upon cross-linking of IgE-bound Fc receptors, these effector cells release chemotactic and inflammatory mediators.
  • FceRI high-affinity Fee receptor
  • IL-4 has also been shown to enhance the expression of the low- affinity receptor for IgE (FceRII, CD23) on human B lymphocytes and monocytes (Kikutani et al., Cell 47:657-665, 1986; Defrance et al., J. Exp. Med. 165:1459-1467, 1987; Vercelli et al. , J. Exp. Med. 167:1406-1416, 1988) and induces a specific isoform (FceRIIb) on these cells (Yokota et al., Cell 55:611-618, 1988).
  • FceRIIb specific isoform
  • the ocular inflammatory lesion was not observed in the TG.UG line, despite a marked elevation in serum IgE - levels.
  • the TG.UG line is distinct in that it displayed no thymic abnormality or peripheral T cell deficiency, with expression of the transgene being predominantly within the B lymphocyte lineage. This suggests that deregulated expression of IL-4 within cells of the T lymphocyte lineage may be necessary to induce the allergic-like phenotype.
  • the frequency of IL- 4-producing T cell clones derived from the conjunctiva of patients with vernal conjunctivitis, an ocular allergic disorder is greatly increased in comparison with controls (S.
  • IL-4 is normally produced by a subset of CD4 + T lymphocytes
  • its deregulated expression within the T lymphocyte lineage in several of the transgenic mouse lines of the invention may have particular relevance to the understanding of its actions in both physiologic and pathologic host responses and in the ontogeny of T cells.
  • the notion that the specific types of humoral and cellular effectors elicited in response to an antigenic challenge may depend on the preferential production of specific ly phokines (Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989) is testable by the construction of transgenic strains analogous to the IL-4-overexpressing strains of the invention.
  • pIgIL4 pIgEPOIL-4 (bearing the Ig.O.IL-4 gene, which includes one copy of the lac 0 sequence)
  • p l gEPIA bearing the mouse immunoglobulin heavy chain enhancer, the human immunoglobulin heavy chain promoter, and a gene encoding the E.
  • coli lac repressor protein linked to the SV40 polyA site coli lac repressor protein linked to the SV40 polyA site
  • pIgEPIAneo identical to plgEPIA except that 3• to the SV40 polyA-encoding sequence and in opposite orientation is a gene encoding neomycin resistance, expression of which is driven from the SV40 early promoter
  • pIgEP0IL-4 plus plgEPIA pIgEPOIL-4 plus pIgEPIAneo
  • pIgIL4 plus pIgEPIAneo Transient transfection was accomplished by electroporation (Potter, Proc. Natl. Acad. Sci.
  • the lac operator /repressor method of controlling expression was also tested in a different in vitro system, using murine erythroleukemia (MEL) cells tranfected with (a) a lac repressor gene driven by a friend leukemia virus (FLV) promoter, and (b) a GH gene driven by an FLV promoter into which had been inserted one, two, or three lac 0 sequences.
  • MEL murine erythroleukemia
  • FLV friend leukemia virus
  • a TH (repressor ) transgenic mouse was sexually crossed with a TX (operator ) transgenic mouse, which constitutively expressed IL-4 from an Ig promoter into which one copy of the lac 0 sequence has been inserted.
  • Offspring of this mating were found to distribute among four genotypic classes (operator + / repressor , operator /repressor " , operator " / repressor , operator ⁇ /repressor ⁇ ) , as expected if the two transgenes originally inserted on different chromosomes.
  • transgenic IL-4 mRNA produced in the brains of mice from two of these classes were compared by RNase protection analysis and found to be at comparably low levels near the lower limits of detectability.
  • the IL-4-overexpressing transgenic animals of the invention are useful as animal models for certain immunological abnormalities, to test proposed treatments for human diseases characterized by such abnormalities.
  • those animals of the invention which exhibit a heightened allergic response, including the tendency to develop an allergy-like inflammatory lesion of the eyelid, can be used to test proposed treatments for human allergies.
  • the two expression-attenuating methods of the invention can be applied to the generation of many different types of transgenic animals expressing any given transgene in virtually any tissue, where expression in the absence of such expression- attenuation is higher than is desired, or where experimental control over the level of expression at any given time is desired. These methods are particularly useful where unattenuated expression of the transgene is lethal to the transgenic animal, or otherwise prevents the animal from being able to reproduce and thus maintain the transgenic ger line.
  • the operator +/repressor+ system of the invention expression from which can be regulated at will by the use of a repressor inactivator (such as IPTG) , offers fine-tuned temporal control of expression of the transgene.
  • IPTG repressor inactivator
  • the methods of generating an operator /repressor " or operator + /repressor + transgenic animal set forth in Examples 2 and 3 , respectively, can be applied not only to a gene encoding murine IL-4, but also to any other transgene.
  • Examples of likely candidate genes include those encoding other interleukins, other hormones, regulatory factors, neurotransmitters, enzymes, structural proteins, viral proteins, and oncogenes.
  • the operator /repressor system utilized can be that of the E.
  • Minor changes in the operator or repressor sequences which do not interfere with repressor control of transcription are within the invention.
  • the means of reversing repression will differ according to the operator/repressor system used, with IPTG or other stable analogs of lactose being useful with the lac operator/repressor system and other entities being useful with other systems.
  • the identities of repressor inhibitors for each known operator/repressor system are well known in the art, or can be developed and tested by known means.
  • the identity of the "operator" in the operator / repressor " system may differ from the particular DNA segment utilized in Example 3, the lac operator.
  • Some of the potential substitutes for the lac operator would include the operator of the E. coli tet operon, the E. coli met operon, and the E. coli gal operon; the phage lambda operator; the phage 434 operator; the phage 21 operator; the phage 22 operator; the yeast STE6 operator; the dyad symmetry element of the human c-fos promoter; the AP- l transcription factor binding site; and the estrogen receptor binding site.
  • the "operator" may be, rather than a true operator or protein-binding site derived from a natural gene, a DNA sequence which is heterologous to (i.e., does not occur naturally within) the promoter region, and which contains a palindromic or dyad-symmetry sequence, types of DNA sequences frequently found in these natural operators and protein binding sites. It is thought that the operator /repressor " system reduces the level of transcription by making the binding of RNA polymerase to the promoter less efficient, whether by providing a site to which endogenous DNA-binding factors will serendipitously bind and thereby interfere with binding by RNA polymerase, or by introducing DNA secondary structure that interferes with transcription.
  • heterologous DNA segments having palindromic or dyad-symmetry character may be used in the operator /repressor " method of the invention.
  • the sequence utilized, whether a natural operator or otherwise, should contain at least 6 and up to 100 base pairs, with 8- 50 base pairs being a useful range and 10-30 being preferred.
  • Each heterologous DNA segment can be tested for ability to attenuate expression by synthesizing it, incorporating it into a promoter or other untranslated part of the gene of interest, and generating transgenic animals bearing that "operator + " gene.
  • the sequence can be present as a single copy in the transgene, or can be two or more copies which are linked or separate, or even which differ from one another in length and/or sequence.
  • the number of copies of this heterologous DNA segment present in a given untranslated region determines the degree of attenuation of transcription of that gene, so that in some cases a series of transgenic animal lines, such segment, would be useful. Although in theory any number of such heterologous DNA segments may be used in a given transgene, in practice ten copies is the upper limit for optimal attenuation.
  • the heterologous DNA segments can be inserted into any portion or portions of the promoter region, from the 5' end of the promoter (e.g., within 1,000 bp to the 5' side of the so-calied "TATA box") to the transcription start site, and can be adjacent to one another or spaced apart within the promoter.
  • heterologous DNA sements could be inserted anywhere in the 5' untranslated region (between the transcription start site and the translation initiation codon) ; in an intron; or even, by careful manipulation of redundant codons in a given gene to produce, for example, a exon sequence that is palindromic (or has dyad symmetry) and still encodes the same protein.
  • the latter manipulation could be accomplished by a computer search of the transgene exon sequences for appropriate adjacent codons that, when substituted with other codons encoding the same residues as the original codons, form, for example, a palindromic sequence.
  • the invention includes not only the particular strains of IL-4-transgenic mice disclosed herein, but also other transgenic mouse strains or any other transgenic non-human vertebrate animals, which are transgenic for murine IL-4 or heterologs of murine IL-4 (including the IL-4 of any mammalian species, such as human) .
  • the promoter region attached to a particular IL-4-transgene can be any promoter sequence other than the one which naturally occurs immediately 5' to the transcription start site for that IL-4 gene (i.e., any heterologous promoter region) .
  • useful promoter regions include mammalian immunoglobulin promoters (whether attached to mammalian enhancer regions or not) ; promoters for the actin family of genes; the mammalian CD-2, Thy-l, elastase, c-fos, or etallothionein • promoter; the long-terminal repeat of MMTV; or the SV40 early-region promoter. Selection of a particular promoter region will depend upon the type of tissue in which expression is to be targeted, with immunoglobulin promoters, such as the immunoglobulin heavy chain promoter, useful for targeting lymphoid tissues such as thymus and spleen. What is claimed is:

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Abstract

Animal vertébré transgénique comportant des cellules contenant un transgène codant l'IL-4, ledit transgène ayant été introduit dans l'animal ou dans un ancêtre de l'animal, à un stade embryonnaire; et procédés de régulation de niveau d'expression d'un transgène chez un animal transgénique.
PCT/US1991/001279 1990-03-05 1991-02-28 Souris transgenique surexprimant l'il-4 et procede d'utilisation WO1991013979A1 (fr)

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US5714146A (en) * 1992-08-26 1998-02-03 Board Of Regents Of The University Of Washington IL-4 bone therapy
EP0624100A1 (fr) * 1992-11-03 1994-11-17 Synaptic Pharmaceutical Corporation Adn codant un recepteur de serotonine humain (5-ht 4b?) et ses utilisations
EP0624100A4 (en) * 1992-11-03 1995-11-29 Synaptic Pharma Corp Dna encoding a human serotonin receptor (5-ht 4b?) and uses thereof.
US6432655B1 (en) 1992-11-03 2002-08-13 Synaptic Pharmaceutical Corporation Method of obtaining compositions
US6376243B1 (en) 1992-11-03 2002-04-23 Synaptic Pharmaceutical Corporation DNA encoding a human serotonin receptor (5-HT4b) and uses thereof
US6300087B1 (en) 1992-11-03 2001-10-09 Synaptic Pharmaceutical Corporation DNA encoding a human serotonin receptor (5-HT4B) and uses thereof
US6783756B2 (en) 1993-06-14 2004-08-31 Abbott Gmbh & Co., Kg Methods for regulating gene expression
US6252136B1 (en) 1993-06-14 2001-06-26 Basf Aktiengesellschaft Transgenic organisms having tetracycline-regulated transcriptional regulatory systems
US5866755A (en) * 1993-06-14 1999-02-02 Basf Aktiengellschaft Animals transgenic for a tetracycline-regulated transcriptional inhibitor
US6914124B2 (en) 1993-06-14 2005-07-05 Tet Systems Holding Gmbh & Co. Kg Tetracycline-regulated transcriptional activator fusion proteins
US6242667B1 (en) * 1993-06-14 2001-06-05 Basf Aktiengesellschaft Transgenic organisms having tetracycline-regulated transcriptional regulatory systems
WO1995003402A1 (fr) * 1993-07-22 1995-02-02 Merck & Co., Inc. EXPRESSION DE L'INTERLEUKINE-β HUMAINE DANS UN ANIMAL TRANSGENIQUE
EP0724628A4 (fr) * 1993-07-22 1997-07-02 Merck & Co Inc Modele animal transgenique pour le traitement de troubles cognitifs
EP0724628A1 (fr) * 1993-07-22 1996-08-07 Merck & Co. Inc. Modele animal transgenique pour le traitement de troubles cognitifs
US5824837A (en) * 1993-07-22 1998-10-20 Merck & Co., Inc. Expression of human interleukin-1β in a transgenic animal
EP0787179A1 (fr) * 1994-10-20 1997-08-06 Merck & Co., Inc. ANIMAUX TRANSGENIQUES PRESENTANT UNE DEFICIENCE EN INTERLEUKINE-1$g(b)
EP0787179A4 (fr) * 1994-10-20 1999-05-19 Merck & Co Inc ANIMAUX TRANSGENIQUES PRESENTANT UNE DEFICIENCE EN INTERLEUKINE-1-g(b)
US8530188B2 (en) 2006-02-03 2013-09-10 Fujifilm Diosynth Biotechnologies (UK) Limited Expression system
JP2016136959A (ja) * 2006-02-03 2016-08-04 フジフィルム・ダイオシンス・バイオテクノロジーズ ・ユーケイ・リミテッド 発現系
US9677103B2 (en) 2006-02-03 2017-06-13 Fujifilm Diosynth Biotechnologies Uk Limited Expression system
US11098335B2 (en) 2006-02-03 2021-08-24 Fujifilm Diosynth Biotechnologies Uk Limited Expression system
US11485957B2 (en) 2017-10-10 2022-11-01 Nantbio, Inc. Modified EC7 cells having low toxicity to viral production payloads
JP2022180439A (ja) * 2017-10-10 2022-12-06 ナントバイオ，インコーポレイテッド ウイルス生成ペイロードに対して低い毒性を有する改変ｅｃ７細胞

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