CA2116012A1

CA2116012A1 - Transgenic non-human animal model for developing and testing therapies to treat sepsis

Info

Publication number: CA2116012A1
Application number: CA 2116012
Authority: CA
Inventors: Sanna M. Goyert
Original assignee: Individual
Current assignee: North Shore University Hospital Research Corp
Priority date: 1991-08-21
Filing date: 1992-08-21
Publication date: 1993-03-04
Also published as: WO1993004168A1

Abstract

A method is provided for developing therapies to effectively treat sepsis and for studying the cellular and molecular mechanisms that result in sepsis, using an in vivo system comprised of transgenic non-human animal whose germ cells and somatic cells contain and express the human gene for the CD14 myelomonocytic differenctiation marker. The gene is introduced into an embryo of the non-human animal or of an ancestor of the non-human animal. The resulting non-human animal is characterized by a heightened sensitivity toward endotoxin-induced sepsis that is mediated by the gene product of the human CD14 gene.

Description

W093/04168 PCT/US92J07031 ~

2116Dl2 ~

TRANSGENIC NON-HUMAN ANIMAL MODEL
FOR DEVELOPING AND TESTING THERAPIES TO TREAT SEPSIS
FIELD 0~ THE INVENTION
This invention generally relates to transgenic -animals, and to the analysis of human gene products using transgenic animals that carry and express human genes.
This invention also relates to methods and therapies for effectively preventing or treating medical conditions ~uch as sepsis, and for studying the cellular and molecular mechani~ms that result in sep~is.

BACKGROUND OF THE INVENTION -~
A ~ariety of pathological medical conditions (e.g., sepsis, auto-immune diseases, tissue rejection) are caus~d or greatly exacerbated by the undesirable side effects of normal immune reaction~. These harmful effect are often mediated by cellular receptors that detect and transmit to the cell information about the ~omposition of the extracellular en~ironment, triggering the chain of events that result in the pathological state.

Sepsis is a life-threatening medical condition caused by infection or trauma. It is characteriæed initially by chills, profuse sweating, fever, weakness, and hypotension, followed by leukopenia, intravascular coagulation, shock, adult respiratory distress syndrome, multiple organ failure, and often, death. R. Ulevitch, et al., J. Trauma 30: S189-92 (1990).

211~012 Sepsis can be caused (induced) by certain substances (defined as any element, molecule, chemical compound, or any mixture thereof) liberated during infection or trauma. Pathogenic bacteria, viruses, and plants can elaborate sepsi~-inducing substances.

The lipopoly~accharides ("LPS"; al YO, "endotoxinsn) that are typically present on the outer membrane of all gram-negative bacteria are among the most studied and best understood sep~is-inducing sub~tances.
While the preci~e chemical structures of ~PS molecules obtained from different bacteria may vary in a species-specific fashion, a region called the lipid A region is common to all ~PS molecules. E. Rietschel et al., in 15 Handbook of Endotoxi~ 187-214, eds. R. Proctor and `~
E. Rietschel, Elsevier, Amsterdam (1984). This lipid A
region mediates many, if not all, of the hPS-dependent pathophy~iologic changes that characterize ~epsis.

~PS is believed to be a primary cause of death in humans afflicted with gram-negative sepsis. van Deventer et al., Lancet, 1: 605 (1988); Ziegler et al, `
J. Infect. Dis., 136: 19-28 (1987). Treatment of patients suffering from sepsis and gram-negative bacteremia with a monoclonal antibody against LPS
decreased their mortality rate. Ziegier et al., N. Eng.
J. Med., 324: 429 (1991).

~PS causes polymorphonuclear leukocytes, endothelial cells,~and cells of the monocyte/macrophage lineage to rapidly elaborate and release a variety of cell product~, among these a variety of immunoactive immunoregulatory (i.e., capable of initiating, modulating or mediatin~ any aspect of an organism's humoral or cellular immNne response~ or proce~ses) substances known as cytokines. One particular cytokine, alpha-cachectin or tumor necrosis factor (TNF), is apparently a primary ' `

mediator of septic shock. Beutler et al., N. Enq. J.
Med., 316: 379 (1987). Intravenous injection of ~PS into experimental animals and man produces a rapid, transient relea~e of TNF. Beutler et al ., J. Immunol., 135: 3972 (1985); Mathison et al ., J. Clin Invest. 81: 1925 (1988). Pretreatment of animals with anti-TNF antibodie reduces lethality, ~uggesting that TNF i~ a critical mediator of septic shock. Beutler et al., Science, 229:
869, (1985); Mathison et al., J. Clin. Invest. 81: 1925 (1988).

Receptors, especially membrane receptors, play a critical role during ~epsis. Several monocyte/
macrophage ~urface antigens that possess receptor and signal transduction functions have been identified; many of them are cell differentiation markers (i.e., they are characteri~tically preQent only in defined stages, especially end Qtages, of cells of a defined lineage and $unction). One such antigen, CD14, is a 55-kD
glycoprotein expres~ed by monocytes, macrop~-ages, and activated granulocytes. It is recognized by several different monoclonal antibodies. S. M. Goyert et al., :
J. Immunol 137: 3909 (1986); A. Haziot et al., J.
Imm~nol. 141: 547-552 (1988).
The characteristic cell type- and stage-specific expression of CD14 in mature cells of the myelomonocytic lineage suggests an important effector function. In addition, the observation that certain conditions such as hyperthermia lM. Kappel et al., Clin.
Exp. Immunol. 84: 175 tl991)] or tissue rejection lJ.
Bogman et al. La~çç~, 238: ii (1989)] lead to the - proliferation of CD14-positive monocytes, suggests that CD14 bearing cells are important elements in the immune response to these medical conditions.

CD14 is linked by a cleavable phosphoinositol WOg3/04168 PCT/US92/07031 tail ~A. Haziot et al ., J. Immunol. 141: 547-552 (1988)]
to the exoplasmic surface of mature monocytes, macrophages, granulocytes and dendritic reticulum cells, of renal nonglomerular endothelium, and of hepatocytes in rejected livers. A soluble form of CD14 is pre~ent in normal sera and in the urine of nephrotic patients.
Bazil et al ., Eur. J. Immunol. 16: 1583 (1986).

Human and murine CD14 have been cloned and sequenced. E. Ferrero and S. M. Goyert, Nuc. Acids Res.
16: 4173 ~1~88); S. M. Goyert et al ., Sciene 239: 497 -(1988); M. Setoguchi et al., N. Nasu, S. Yoshida, Y.
Higuchi, S. Akizuki, and S. Yamamoto, Biochem. Biophys.
aÇ_ 1008: 213-22 (1989). The sequence analysis revealed -~
15 that CD14 belongs to a family of leucine-rich membrane- -~
bound and soluble proteins have receptor and cell adhe~ive functions. M. Setoguchi et al., Bioc~ç~L
Bio~y~. Acta 1008: 213-22 (lg89); E. Ferrero, C.L.
~- Hsieh, U. Francke and S~M. Goyert, J. Immunol. 145: 133 20 ~ ~1990). Antibodies to CD14 reduced human monocyte chemiluminescence, caused the internalization of CD14 molecules, and caused transient increases in interleukin-1 synthesis, cytosolic calcium concentration and monocyte H202 production. F. Lund-Johansen et al., FEBS_Lett. 273:
55 (1990). Interleukin 4 has been shown to down-regulate the expression of CD14. R. Lauener, S. Goyert, R. Geha and D. Vercelli, Eur. J. Immunol. 20: 2375 (1990).
Together, these observations suggest that CD14 may possess an intrinsic, regulatable capacity to engage in signal transduction.

In vi tro analyses have shown that ~D14 is the receptor for lipopolysaccharide (LPS or endotoxin) when ` LPS is bound to an acute phase serum protein called LBP
(LPS binding protein). LBP recognizes the lipid A region ; of LPS and forms high affinity 1:1 stoichiometric - complexes. Tobias et al ., J. Biol. Chem., 264:10867 '; "

.

2111~12 (1989). The binding of this complex to CD14 causes cells to release interleukins, tumor necrosis factor (~TNFn) ~ ~2~ and other substances which eventually cause the lethal ~3hut-down" of the cardiovascular-pulmonary-renal systems observed in qepsis. Beutler et al., N.Eng. J. ~ , 316:379 (1987); R. Ulevitch et al., J.
Trauma 30:189-92 (l990); F. Lund-Johansen et al., FEBS :~
~ett. 273: 55 (1990).

CD14 ha~ al~o been implicated in tissue rejection. Immunostaining with anti CD14 monoclonal antibodies is capable of differentiating rejection from other forms of interstitial nephritis, and ha~ been u~ed to diagno~e renal allograft rejection. J. ~ogman et al . -~an~, 238: ii (1989). In a recently published preliminary report, CD14 was detected on the surface of --hepatocyte~ in 6 out of 8 cases of liver allograft rejection, but not in ten cases of acute and chronic ; hepatitis due to virus infection, autoimmunity, or drugs.
R. Volpes et al., ~ancet, 337: 60 ~1991). In addition to demonstrating for the first time the epithelial --expression of CD14, this last result correlates the hepatocellular expression of this antigen with liver allograft re~ection.
The CD14 gene is located in a region of human chromosome 5 that is known to contain a cluster of genes that encode several myeloid-specific growth factors or growth factor receptors, as well as other growth factor and receptor genes. S.`M. Goyert et al., Science 239:
497 (1988). The mapping of the CD14 gene to this region of chromosome 5, its expression preferentially by mature myeloid cells, and its deletion in the malignant cells of patient~ having myeloid leukemias and del~5q) suggest that the CD14 antigen may play a role in the pathogenesis of myeloid di~orders.

WO93/041~ PCT/US92/07031 2116~12 The lack of a suitable animal model that closely approximates the human condition has been a major ;~
obstacle to the development of an effective treatment for human ~epsis. The only means for testing the potential efficacy of putative therapeutic agents has been extrapolation from the results of studies in animal models, in ~itro testing of cells derived from human and animal ti~sues, and epidemiological studies with human patients.
,, The animal models in which ~epsis has been studied have necesQarily involved the animal homologue of proteins such as CD14 rather than the human proteins. As a result, therapies developed and tested in animal models in which sepsis is initiated by the interaction between the animal homologue and LPS may-not be applicable to the human s$tuation.

Very little is known about the correlation between in ~itro testing and in vivo efficacy. It may vary conQiderably, since by their very nature the in `~
vitro analyses are greatly simplified experimental constructs that cannot duplicate the complex cellular interactions that occur within an organism.
The studies with human patients neceYsarily involve a non-~elected population of afflicted indi~iduals. The interpretation of these studies is generally complicated by the fact that many of the patients suffer from a variety of additional medical problems. Not only is the composition of this population beyond the control of the experimenter, but the ~`
experimenter is ethically limited as to the range of studies that can be performed and the treatments that can - 35 be developed.

The techniques for generating transgenic -~
:

W093/~168 PCT/US92/07031 2111~U12 animals (i.e., animals that stably express genes which have been introduced into their germ line or into an ancestor~s germ line) have been perfected over the last decade and are becoming more and more easily accessible.
(Expression iB here defined a~ the detectable presence or production of a particular gene product in certain ti~sues of the transgenic animal and its progeny.) Since 19~1, a wide variety of tran~genic mice containing genes such aq human globin genes, rabbit globin genes, chicken transferrin genes, immunoglobulin genes, rat growth hormone genes, thymidine kinase genes, and human growth hormone genes have been described in the ~cientific literature. U.S. patent 4,736,~66 (issued on April 12, 1988 to inventors ~eder and Stewart and assigned to Harvard University) diaclosed a non-human transgenic mammal, preferably a mouse, containing an acti~ated oncogene ~equence which, when incorporated into the genome of this mammal, increased its susceptibility to de~elop neoplasm such as malignant tumors. These transgenic mammals have been found to be useful to test materials ~uspected of being carcinogenic by exposure to carcinogenic substances and determination of neoplastic growth as an indicator of carcinogenicity.

For the preceding reasons, it is an object of this invention to produce transgenic animal models for underætanding human ailments such as sepsis, for developing methods for the early detection and effective -treatment (including vaccines and drugs) of human ailment~ such as sepsis, for the protection of individuals not yet sick, and for the development of therapies for tho~e already afflicted.

lt is also an object of the preQent invention to produce transgenic an;mals that stably incorporate and express human genes that mediate sepsis, in par~icular transgenic animals that express the gene product of thè
- ' :

2116~12 ;:

human CD14 gene.

It i8 a further object of the invention to provide methods and means for studying the mechani~ms of human ailments such as sepsis, as a model for diseases caused by host immune respon~e to exogenous and endogenous triggers of the immune system. Such methods and means expressly include methods for the in vivo testing of substances that cause, mediate, ameliorate or counteract sep~is, comprising administering said substances to a eukaryotic animal which expresses a human gene that encodes a molecule that mediate~ either sepsis or the organism's response to sepsis. ("Cause" is here defined as initiating molecular events that result in the symptoms of ~epsis, or that are implicated in the organism' 8 response to sepsis; "mediate~ means effecting any molecular events that form part of the causal cain of events that result in the symptoms of sepsis, or that are implicated in the organism's response to sepsis;
~ameliorate~ means a reduction in the severity of any of the symptoms of sepsis, and "counteract" means a generalized reduction and eventual nullification of the symptoms of sepsis.) Finally, it is an object of this invention to examine the role CD14 plays during tissue rejection, and `
to determine the molecular mechanism for this role.
':

SUMM~RY OF THE INVENTION
A new animal model has been developed which allows the direct testing of novel therapies for sepsis.
In the preferred embodiment, transgenic animals express - proteins that mediate sepsis, in particular the CD14 myelomonocytic differentiation marker.
~ An example of the invention are transgenic mice ;~ which express high levels of the gene product of the : ~ "

W0~3/04168 PCT/US92/07031 ' ~ll fi O 12 ~

human CD14 gene on their monocytes, macrophages and granulocytes. As a result of the human CD14 expression, ~' these mice are more susceptible than normal mice to sepsis induced by LPS. The increased sensiti~ity of these mice to sepsis induced by LPS is the result of direct interactions with the gene product of the human CD14 gene.

These transgenic mice should allow the in vivo development and testing of effective therapies to human CD14 mediated sepsis. This is the first animal model to allow such studies.

BRIEF DESCRIPTION OF DRAWINGS
15 A. Figure 1. MoM~V-hCD14 gene construct.
B. Figure 2. Southern blot analysis of genomic DNA ,~
obtained from the tails of tran~genic and control mice. -' C. Figure 3. Pluorescence-activated cell sorter (FACS) analysis of cells obtained from transgenicand control mice.

DETAILED DESCRIPTION OF THE INVENTION

A. Isolat~on and charac~erizatiou of human CD14 cDNa.

A human CDNA library was constructed in pCD, the Okayama-Berg eukaryotic expression vector lH. Okayama and P. Berg, Mol. Cell Biol. 3: 280 (1983)] using messenger RNA ~MRN~) isolated from human,M4-AML
(myelomonocytic) cells. S. M. Goyert et al ., Science 239: 497 (1988). 1.0 to 2.65 kb cDNA inserts were size-~elected in low-melting agarose gels according to T.
Yokota et al., ,Proc. Natl Acad. Sci. 81: 1070 (1984)~ S.
M. Goyert et al., Science 233: 497 (1988). Escherichia coli (RRl) were then trans,formed with the cDNA and plated on agar. A total of 1056 colonies were randomly W093/04168 P~T~US92/07031 selected, transferred individually to small liquid cultures, and grown overnight at 37C. The 1056 liquid cultures were consolidated into 44 pools of 24 liquid cultures each. Each pool was grown in 500 ml of Luria broth containing 100 ug of ampicillin per milliliter, and plasmid DNA was isolated from each pool and used to transfect COS 7 cells as described by S. M. Goyert et al., Science 239: 497 (1988).

The COS 7 cells transfected with the 44 plasmid pools were analyzed for cell surface expression of CD14 by indirect immunofluorescence using a monoclonal antibody (Mo539) to CD14, [Dimitiu-Bona et al ., J .
Immunol. 130: 145 (1983)] and a fluoresceinated sheep -~
antibody to mouse immunoglobulin. Five of the clones derived from these pools were positive for human CD14 expression. S.M. Goyert et al., Science 239: 497 (1988).
Each of the 24 plasmids from one of the positive pools was iQolated on a cesium chloride gradient, transfected individually into COS 7 cells, and screened for CD14 expression as described above. One cDNA
clone, labelled pCD-CD14, was found to expre~s CD14. S.
M. Goyert et al ., Science 239: 497 (1988).
To confirm that the pCD-CD14 clone encoded -authentic CD14 molecules, immunoprecipitates prepared ~-from pCD-CD14-transfected COS 7 cells and from M4-AML -cells expressing endogenous CD14 were compared by SDS-polyacrylamide gel electrophoresis. The molecules precipitated from both sources were nearly identical in size. S. M. Goyert et al., J. Immunol. 137: 3909 (19~6).
In addition, the PCD-CD14 probe was found to hybridize to a single mRNA species that showed an expression profile identical to CD14: it was present in monocytes, granulocytes and M4-AML cells, but not in less mature myeloid cells represented by the leukemic cell lines k62 W093/04168 PCT~US92/07031 211~12 (undifferentiated), Us37 ~monoblast-like), HL60 (promyelocyte-like), or M2-AM~ (myeloblastic with maturation) cells or lymphocytes. S. M. Goyert et al., Science 239: 497 (1988)~ The predicted protein sequence of the pCD-CD14 clone corresponded to the partial protein sequence of CD14 determined by micro3equence analysis~

Analysis of recombinant CD14 produced by inserting pCD-CD14 into the lambda NMT vector and stably tran~fecting U251-Mg cells indicates that it is anchored to the membrane by a glycosyl phosphatidylinositol (GPI) linkage. Three forms of the CD14 protein can be isolated from these transfected cells (U251-CD14) including form I, the cell surface form, form II, the form released by an enzyme which cleaves GPI-anchored proteins (the enzyme i8 called pho~phatidylinositol phospholipase C, or PI-P~C), and fonm III, a form which is ~maller in molecular weight (48 KD Kda) than forms I (53 kDa) and II (53 kDa) and which is spontaneously released into U251-CD14 -culture supernatants. Haziot, A. et al. J. Immunol. 141:
547-552 (1988).

B. Identification and ana~yQi~ of the human CD14 gene.
The human CD14 gene was isolated from a size-selected (6 kb average) Eco RI genomic library constructed in the lambda vector gtWes. S. M. Goyert et al., Science 239: 497 (1988). DNA sequence analysis demonstrated that the human CD14 gene contains a single intron of 88 base pairs immediately after the ATG
translational start site. E. Ferrero and S. M. Goyert, Nuc. Acids Res. 16: 4173 (1988). The initiation codon is flanked by a sequence which shows homology to the - consensus sequence C(C)AoCCATCC for a translation initiation site [as defined by M. Kozak, Nucl. Acids Res.
15: 8125-8148 (1987)] and is separated from the rest of the coding region by the 88 bp intron.

The pCD-CD14 cDNA clone was found to consist of 1367 nucleotides with a polyadenylate tail at the 3~ end.
S~ M. Goyert et al., Science 239: 497 (1988). An initiation codon was identified at position 105, followed by an open reading frame (coding region) consisting of 1125 nucleotides flanked by 104 nucleotides of 5' untranslated sequence and 126 nucleotides of 3' untranslated sequence. Comparison with the partial protein sequence determined by microsequence analysis 10 confirms the identity of this clone as encoding CD14 and -~
indicates the pre~ence of a signal peptide of 19 amino a~ids (-19 to -1).

Southern blot analysis of DNA digested with se~eral different restriction enzymes and probed with CD14 cDNA gave single bands, suggesting that CD14 i8 encoded by a single gene. S. M. Goyert et al., Science 239: 497 (1988). -. .
The h~nan CD14 gene was detenmined to be located on chromo ome 5 by a variety of techniques.
S. M. Goyert et al., Science 239: 497 (1988). Southern blot analysis was performed using Eco R1 digests of DNA
isolated from human, mouse, and human-mouse hybrid cells.
Restriction endonuclease digested genomic DNA was separated on 0.7~ agarose gels, transferred to nitrocellulose, and hybridized with 32p_ labeled nick-translated CD14 cDNA. J. M. Chirgwin et al., Biochemistry 18: 5294 (1979)~ Filters were then washed in 0.3% standard saline citrate with 0.1% SDS at 65C.
Of 21 hybrid clones, 6 were positive for the 5.5-kb Eco RI gene fragment. These six hybrids were found by - ka~yotype analysis and testing for human isoenzymes, cell surface antigens, and DNA markers lW. J. Rettig et al., Proc. Natl. Acad. Sci. U.S.A. 81: 6437 (1984); W. J.
Rettig et al., J. Exp. Med. 162: 1603 (1985), N. C.
Dracopoli et al., Proc. Nat'l Acad. Sci. U.S.A. 83: 1822 `:

2 1 1 ~ O 1 2 Pcr/us92/o7o31 ~1986)] to contain human chromosome 5, and to have no other human chromosome in co~ranon~ None of the 15 hybrids that were negative for human CD14 contained a complete copy of human chromosome 5 In si tu chromosomal hybridization ~M. M.
LeBeau, C. A. Westbrook, MØ Diaz, J.D. Rowle, Nature (London) 312: 70 (1984)] of the 3H-labelled cDNA probe to normal human metaphase cells resulted in specific 10 làbeling only of chromo~ome 5. S. M. Goyert et al., Science 239: 497 (1988). Human metaphase cells were prepared ~rom phytohemagglutinin-stimulated peripheral _ blood lymphocytes. Radiolabeled CD14 probes were prepared by nick translation of the entire plasmid with 15 all four 3H-labeled nucleoside triphosphates to a specific activity of 1.0 X 10~ dpm/ug. Metaphase cells were hybridized at 4.0 and 8.0 ng of probe per milliliter of hybridization mixture. Autoradiograms were ex~o~ed for 11 days. All hybridizations were repeated three times 20 and gave similar results: the labeled sites were -clustered at 5q22-q32; the largest cluster of grains was located at 5q23-q31. S. M. Goyert et al., Sciçnce 239:
497 ~1988).

The human CD14 protein sequence contains five potential sites for N-linked glycosylation and contains a 10 fold repeat of a leucine rich motiff (~XXLXLX).
Comparison of the CD14 nucleotide and predicted protein sequences to all sequences in the Bionet data bank initially revealed no significant homologies. More recently, the murine equivalent of CD14 has been sequenced. M. Setoguchhi, N. Nasu, S. Yoshida, - Y. Higuchi, S. Akizuki, and S. Yamamoto, Biochem.
Bio~hys. Acta 1008: 213-22 (1989); E. Ferrero, C.L.
Hsieh, U. Francke and S.M. Goyert, J.I~nunol. 145: 133 (1990). There is a 66~ amino acid sequence identity between the murine and human CD14s. The murine gene is located on mouse chromosome 18, which like the human gene -also contains at least five genes encoding receptors.

C. Tran~genic Animal8.
A plasmid express on ~ector was constructed in which the human CD14 gene was placed under the control of the long terminal repeat (LTR) of the Moloney Murine Leukemia Virus ("MoLTR"). A HindIII-SmaI restriction fragment that contained the MoLTR was obtained from the plasmid pZIP-NeoSV(X)l. Cepko et al., Cell 37: 1053 (1984); Lang et al., Cel~ 51: 675 (1987). This fragment was then ligated into the HindIII-SmaI sites of the plasmid pUC18 (Bethesda Research Laboratories). The ECoRl genomic fragment of CD14 was blunt-ended and in~erted into the SmaI site of the pUC-MoLTR plasmid.
The MoLTR-hCD14 inQert used for injecting into zygotes `~
~see Fig. 1) was excised from the pUC18-MoLTR-hCD14 plasmid (deposited with the American Type Culture Collection on August 21, 1991 under the terms of the ~udapest Convention, and designated as ATCC ) u3ing HindIII-KpnI and purified on low melting agarose. It was dialyzed and diluted to 2000 copies per pico-liter.

The MoLTR-CD14-containing linearized fragment was injected into fertilized mouse embryos and then implanted into pseudopregnant mice according to routine procedures. J. Gordon and F. Ruddle, Methods i n Enzymology 101: 411 ~1983); B. Hogan, F. Constantini and E. Lacy, Mani~ul~ing the Mouse EmbryQ: A Laborato~y Manual Cold Spring Harbor Laboratory (1986). Embryos were obtained from F2 hybrids of a B6CBA F1 X B6CBA F1 cross. All microinjections were performed using a Leitz microinjection system including a Leitz microscope equipped with Leitz micromanipulators. Approximately 0.1-0.5 microliters of the DNA solution (200,000 to 1,000,000 copies of the fra$ment) were microinjected into each embryo. Following micro-injection, the embryos were ' ' WO93/W168 2 11 G ~ 1 2 PCT/US92/07031 cultured for about 4-5 hours before implantation.

Fifteen (15) embryos were implanted into the oviduct of a pseudopregnant C57BL6/CBA Fl female 5 (produced by mating with a sterile male~ using transfer pipettes. The pups that were born were tested for the presence of the tranQgene. Genomic DNA was extracted from their tails, digested with EcoRl, and Southern blot analyqis was performed as described in S. M. Goyert et al., Science 239: 497 (1988). The Southern blots were probed with human CD14 cDNA labeled with 32p by random primer labelling (Fig. 2). Out of a total of 4 pups, one (labelled TG1) was transgenic (i.e., the Southern blot detected the presence of the human CD14 gene in its 15 tiQQues). ~-Peripheral blood cells, peritoneal macrophages, and spleen cells obtained from the transgenic mouse and from control mice of the same strain were stained with fluoresceinated antibodies, and examined in a fluore~cence activated cell sorter (FACS). Human CD14 immunoreactivity was detected only in the cells obtained from the transgenic mouse (See Fig. 3). Subsequent analyseQ have confirmed that the gene product of the human CD14 gene is expreQsed on the surface of monocytes and granulocytes in the spleen, lung, brain, kidney, bone marrow and peripheral blood.

13XAMPL~13 ~ The founder transgenic mouse described above was bred to produce a line of transgenic mice. Transgenic mice were tested for their in vivo sensitivity to LPS.
- Transgenic mice and normal control mice were injected intraperitoneally with varying doses LPS:

~ice ug LPS per % Viable gram body we~gh~ -~

WO93/04168 PCT/US92/07031 ~

Normal 30 16.6 100.O '.
Transgenic lO 0 All the transgenic mice died at a dose of LPS (10 ug LPS
per gram body weight) which was not lethal to any of the normal mice of the same strain.
' These studies are the first to show in vivo that the gene product of the human CD14 gene is a major contributor to the induction of sepsis and death. The observed increase in sen~itivity to LPS is the result of events directly mediated by the human CD14 gene product.
These experimental results confinm that the CD14 transgenic mice can be used as a model for human CD14 mediated sepsis.
:.
Modifications and variations of the transgenic animal models for screening of therapeutic compounds (for example, the in vitro use of cells derived from the transgenic animals, or transgenic animals that have not incorporated the gene into their germ line) will be obvious to those skilled in the art from the foregoing detailed description of the invention. Such modifications and variations are intended to come within the scope of the appended claims.

Claims

17 We claim:

1. A transgenic animal in which the human C14 gene is expressed.

2. A transgenic non-human animal model for testing substances that cause, mediate, ameliorate or counteract sepsis, comprising a eukaryotic animal which expresses a human gene that encodes a molecule that mediates sepsis.

3. The transgenic animal of claim 2, where the animal is a transgenic mammal.

4. The transgenic mamma} of claim 3, where the mammal is a transgenic rodent.

5. The transgenic rodent of claim 4, where the rodent is a transgenic mouse.

6. The transgenic animal model of claim 2 wherein said substance that causes or mediates sepsis is LPS, LBP, or sepsis-mediating cytokines such as interleukins and tumor necrosis factor (TNF).

7. The transgenic animal model of claim 2, wherein the sepsis-mediating molecule is a protein.

8. The transgenic animal model of claim 7, wherein the protein is selected from the group consisting of cellular differentiation markers, cell surface receptors for immunoregulatory substances that mediate sepsis present on the surface of immunoresponsive cells and regulatory proteins which control gene expression.

9. The transgenic animal model of claim 8 wherein the protein is encoded by the human gene for the myelomonocytic differentiation antigen CD14.

10. The transgenic animal model of claim 9 wherein the encoded CD14 is expressed on the surface of the transgenic animal cells.

11. A transgenic animal model for studying the events of tissue rejection that are mediated by the human CD14 gene and its gene product, comprising a transgenic animal in which the human CD14 gene is expressed.

12. A method for testing a substance that causes, mediates, ameliorates or counteracts sepsis comprising administering said substance to a eukaryotic animal which expresses a human gene that encodes a molecule that mediates sepsis.

13. A method according to claim 12 wherein said substance that causes or mediates sepsis is LPS, LBP, or sepsis-mediating cytokines such as interleukins and tumor necrosis factor (TNF).

14. A method according to claim 12 wherein said sepsis-mediating molecule is a protein.

15. A method according to claim 14 wherein said protein is selected from the group consisting of cellular differentiation markers, cell surface receptors for immunoregulatory substances that mediate sepsis present on the surface of immunoresponsive cells and regulatory proteins which control gene expression.

16. A method according to claim 15 wherein said protein is encoded by the human gene for the myelomonocytic differentiation antigen CD14.

17. A method according to claim 16 wherein said encoded CD14 is expressed on the surface of the transgenic animal cells.

18. A method for studying CD14 mediated tissue rejection comprising utilizing a transgenic animal in which the human CD14 gene is expressed.

19. A method for assaying for compounds capable of causing or inhibiting sepsis comprising providing transgenic animal cells which express a human CD14 gene.

20. A method according to claim 19 further comprising culturing the cells in the presence of a potential sepsis-causing or sepsis-inhibiting compound.