CA2175250A1 - Sepsis model - Google Patents

Abstract

A double knockout, double transgenic non-human mammal is provided. The mammal is useful as a model for evaluating therapeutics to treat or prevent sepsis.

Description

WO95/14377 ~2 1 75~ 117 SEPSIS MODEL
R~ t Rt~ROUND

Field O_ The Invention This invention relates to a nu11 ~ mammal produced by r~ ; nAnt DNA terhn~~ y . More 10 spen~ftnA11y, the lnvention concerns a non-human mammal that is susceptible to sepsis and other diseases, and that is useful for screening potential th~rAretlt~
compounds .
15 Description of Related Art In V~vo Screen1na Syst~m~
Evaluating a chemical compound for its potential as a human th~rAreu~ 1- necessitates data and 20 information about the compound's efficacy in an ln vivo system. Ideally, the in vivo system used for data collection would be a human being; however, for ethical and pr~ c reasons, l~hor~Atory animals, and not human beings, are typically used as in vivo screening systems 25 for drug development.
While laboratory animals can be helpful for use as model systems to assess the efficacy of a potential therapeutic and to identify the possible side-effects of the therapeutic, such models have 30 limitations. One significant 1;m~tAt~oa concerns the differences between laboratory animals and humans in - disease resistance and susceptibility. These differences are often due to inter-species differences in the structure and function of the immune system.
35 Therefore, to evaluate a chemical compound for its therapeutic value in humans, it would be use~ul to have WO 95/14377 P~IIIL 1 ~ 117 ~15~r` 2- ~
an animal model with an immune system that resembles the human immune system.
TrAnRaPn~C anr~ ~nonkr~ut TerhnololJies Recent advances ln re, ~ nAn~ DNA technology have enabled researchers to genetically manipulate animals such as mice. The techniques of transgenic mammal generation and knockout mammal generation have been employed by researchers to produce animals that either do not express an endogenous gene (knockout animals ), or to produce animals that contain one or more exogenous or heterologous genes ~transgenic animals) .
Production of trAnRg~n 1 c mammals involves the insertion of a novel nucleic acid sequence, often called a transgene, into one or more chromosomes of the mammal.
The transgene, typically comprised o$ DNA, codes for a p~rticular polypeptide. The ~rAnsg~n~ is typically inserted via microin~ection into the pronucleus of an egg where it is incorporated into the DNA of the developing embryo. This embryo is then implanted into a "surrogate host" for the duration of gestation. The offspring of the surrogate host are evaluated for the presence of the novel DNA.
Expression of the transgene, ~ . e ., production of the protein encoded by the transgene DNA sequence, may confer a new phenotype on the mammal. Depending on the transgene (s) inserted into the mammal, the control elements (such as promoter and enhancer/silencer) used and the pattern and level of expression of the transgene (s), the mammal may become more or less susceptible to a particular disease or series of diseases. Such transgenlc mammals are valuable for screening and testing compounds that may be useful in treating or preventing the disease (s), and/or for developing methods useful in the treatment or ~l~A~nf~q~ R
of the disease.

WO95/14377 21~ o r~ t ~l7 Production of a knockout mammal rectuires insertion of a nucleic acid sequence (usually DNA) that is designed to suppress expression of the gene to be "knocked out" into an undiffer~nt;~ted cell line termed 5 an embryonic stem cell, or ES cell. After the nucleic acid sequence has been inserted lnto the ES cell, the ES
cell is injected into a developing l; An embryO
where it hopefully will be integrated into, and become part of, the e_bryo durlng development. The embryo is 10 then I l~nted into a surrogate host for the ~ r~t;t~n of qestation .
^ System r, ^ -ts The immune system of every l ;~n species 15 i8 comprised of many specialized cells that act together in a highly complex manner to protect the mammal f rom dangerous foreign substances such as a variety of pathogens, toxins, and the like.
Many cell types of the immune system exert 20 their effects and recognize foreign substances in part via a series of proteins known as the MHC ~ma~or histocompatibility complex). The mouse MHC is known as the H--2 complex ("E-2n) . The human MHC is referred to as the HLA complex ( "HLA" ) . The HLA complex is 25 comprised of more than 100 genes (Klein et al., Sc~.
Am., 269:78-83 [1993]).
A distinguishing feature of the MHC is that many of the MHC loci (a locus is defined as the chr ~s 1 location of a gene) have an unusually large 30 number of alleles, or variations, in the nucleotide and corresponding amino acid setIuences. The MHC proteins are generally divided into two classes called MHC class I ("MHCI") and MHC class II ("MHCII") . MHC class I
proteins are membrane bound proteins that are expressed 35 on the surface of almost all nucleated cells; MHC class II proteins are membrane bound proteins that are WO 95114377 r~ l c ~117 ~`17~ 4-expressed only on the surface of certain types of immune system cells, namely dendritlc cells, macrophages, and B
cells .
The MHC class II proteins play a critical role 5 in the immune system's recognition and attack of foreign substances. When most forelgn substances enter the body, they are initially recognized by antigen presenting cells ~APCs ) such as macrophages which bind to the substance, ingest it and process it. The lO processed portions of the substance may be attached to MHC class II proteins on the surface of the macrophage.
This prf~ ntAt ~ o~ of "display" of the substance bound to the MHC on the surface of the macrophage enables other cells of the immune system, such as certain T cells, to 15 recognize and respond to the fore$gn s11hst~nce. T cells become "activated" and are induced to proliferate and to release, ~ic that are harmful to the foreign substance. In addition, activation of T cells serves to activ2te other cells of the immune system to also mount 20 an attack on the substance.
T cells, one type of immune system cell, express a variety of proteins on their cell surface.
~any of these proteins are involved in recognition and binding of extracellular substances, and/or in cell 25 signaling . Two important T cell surface l e~ are CD4 and CD8. CD4 is a glycoprotein monomer of 1ec~11Ar weight about 55kD. The murine CD4 is encoded by L3T4 and is about 26 kb in size. CD8 is a disulfide-linked heterodimer. In humans, the genes encoding the two CD8 30 monomers are referred to as CD8 alpha and CD8 beta. In the mouse, the monomers comprising the CD8 heterodimer are encoded by the genes Lyt-2 and Lyt-3. Lyt-2 is about 4 . 4 kb in size and contains 5 exons .
Immature T cells express both CD4 and CD8 on 35 their cell surface. As the cells mature, they cease to express either CD4 or CD8. Thus, mature T cells are WO9S/14377 ~ 5~ r~" 1~ ll7 classified based on whether they expreæs CD4 or CD8.
Generally, mature T cells that express only CD4 are called T helper cells, and recognize and respond to antigens bound to MEIC class II molecules. ~ ner~lly, mature T cells that express only CD8 are called cytotoxic T cells, and recognize and respond to antigens bound to ME~C class I -~l eC~
MHC, CD4, and CD8 l~r~ q differ in their amino acid sequence between species. Thus, one species may recognize a pathogen or substance as foreign and mount a response to it, while another species may not.
In addition, there are intra-species differences in the re3ponse to any one toxin or pathogen (i.e., different species and even members of the same species may be extremely sensitive to a particular toxin or pathogen, while others are more tolerant of its presence in the body). This is due to the many different allelic variations of the MHC loci, ~as discussed above). Each individual member of a species expresses only one or at most two of these allelic forms at each ~C locus, and different allelic forms differ in their ability to present or bind different pathogens.
In an attempt to understand the inter-species differences in activity and recognition of molecules of the immune system, researchers have generated laboratory animals that 1) contain a human transgene encoding a protein of the immune system, and/or 2) do not express one or more endogenous genes encoding a protein ~s) of the immune system.
Nishimura et al. (J. Immunol., 145:353-360 [1990] ) describe a mouse carrying the transgenes encoding the alpha and beta chains of the human I~LA-DQw6 protein, a member of the ME~C class II protein family.
Barzaga-Gilbert et al (J. Exp. Med., 175:1707-1715 [1992]) describe a mouse rrnt:~n~n~ a transgene encoding human CD4. A few of the lines of W095/l4377 r~,-~, Ic ~17 .
5~ 6 -~ransgenic mlce that were generated expressed both the human and murine CD4 molecules on T cells, but at di f f erent levels .
Killeen et al. (EMBO J., 12:1547-1553 [1993]) describe a mouse lacking expression of endogenous CD4 but cnnt;~n;ng a transgene encoding human CD4.
Robey et al. (Cell, 64: 99-107 [1991] ) describe a transgenic mouse expressing murine CD8 under the control of the human CD2 regulatory seouences.
Teh et al. (Nature, 349:241-243 [1991]
describe a mouse rontA~n~ng the murine CD4 transgene under the control of the murine lck promoter. The transgenic mice generated reportedly expressed CD4 in all thymocyte subsets and in all peripheral T-cells.
Krimpenfort et al., U. S. Patent No. 5,175,384 issued December 29, 1992, describe a tr;:lneg~n~ C mouse wlth a substantial depletion of mature T cells or plasma cells .
PCT patent application no 92/22645 pllhl 1 ~h~
December 22, 1992, describes transgenic mice reportedly ~ of;cient such that they are able to r-1nt,sl;n a tissue/organ transplant from another species more readily as compared to wild type mice.
Rahemtulla et al. (Nature, 353:180-184 [1991] ) describe a mouse that does not express endogenous CD4.
Fung-Leung et al. (Cell, 65:443-449 [1991] ) describe a mouse that lacks expression of the endogenous CD8 molecule.
Srh;lhAm et al. (Eur. J. Immunol., 23:1299-1304 [1993] ) describe a mouse lacking expression of both endogenous CD4 and CD8.
The presence of dlsease causing organlsms and/or their toxins in human blood or tissues can result in a condition known as sepsls. The symptoms of sepsis .. .. .. ...... _ .. ...... . . _ _ . _ . ... .

WO 95/l4377 F~I/IL~ l.'~ ~ 117 ~7~Q
7 -- ~ ~
induced by such organisms as E coli, l~leoslella rr~ iAf', Pseudomonas aerug~nosa, Enterooacter aero~enes, and Neisseria menin~tidis can include fever, diarrhea, a drop in blood pressure, leaky blood vessels, 5 an~/or disseminated blood clotting in a variety of organs. It has been estimated that the mortality rate from sepsis is about 35 percent (Aldridge, TI13Tech, 11:373-375 [1993]), and up to 50 percent in septic shock .
One severe form of sepsis is termed septic shock and results when an individual with sepsis experiences a large drop in blood pressure. Treatment for septic shock requires ~ Ate restoration of blood pressure which is typically ~ qh~d by 15 administering fluids and/or inotropic agents, and broad spectrum antibiotics.
There are a number of toxins that have been identified as the causative agents in ~qep3is. Among these are exotoxins (toxins secreted by the invading 20 organism) and endotoxins (toxins that are a c~ ~ of the cell wall of the invading organism, e . g., LPS, or lipopolysaccharide) .
One group of exotoxins that have been studied extensively is the Staphylococcal enterotoxins and 25 related enterotoxins. These enterotoxins are proteins that are made by various species of both Streptococci and Staphylococcus, and share a similar -hAn~ om of action in activating the body ' s immune system to mount an attack on the pathogen. Members of this group of 30 enterotoxins are known as "superantigens". The mechanism of superantigen binding to MHCII and T cells is distinct from that of other antigens. Superantigens bind simultaneously to the T cell receptor and the M~CII
molecule, thereby activating a large num~ber of T cells 35 irrespective of the antigen binding specificity of the T

WO 9S/14377 P~ 17 ~7 52~ 8 -cell. This results in the release of large amounts of cytokines ln the body, which ultimately leads to shock.
The - - -h~n ~ cm of binding for conventional antigens is to bind to a very well defined groove in a 5 specific ME~CII molecule; this antigen/Mi7CII complex is then recognized only by a highly specific subset of T
cells, i.e., those that express a receptor on their surface that is specific for the particular antigen/MHCII complex (l in 107 T cells). Typically l0 therefore, fewer T cells are activated by a normal antigen binding as compared with Sllr~r~nt; ~7en re3ponses, which trigger up to 40 percent of the total T cell population .
While broad spectrum antibiotic therapy is 15 often used to treat sepsis, it is not always effective, and can have a num.ber of detrimental side effects.
Depending on the cause of sepsis, steroids may also be indicated to modulate the activity of the immune system.
In ~ ;t;~n, blood products such as fresh frozen plasma 20 or anticoagulants such as heparin may be used to counter the effects of the immune system's complement cascade activity which leads to clotting.
There is a need in the art to develop a - l; P,n model for in vivo evaluation of certain human 25 diseases such as sepsis where the model l) ls susceptible to the disease, and 2) closely mimics the progression of the disease as it occurs in humans.
SrlMMARY OF TEIF INVENTION
In one aspect, the present invention provides a non-human mammal or its progeny lacking expression of endogenous CD4 and CD8, wherein the mamm.al has inserted a nucleotide sequence comprising the DNA encoding human 35 CD4 or a biologically active fragment thereof and a nucleotide sequence comprising the DNA encoding an .. , .. . . . _ _ _ _ _ _ _ _ _ _ _ _ _ _ .

W0 95/l4377 ;~ ~ r ~ 17 _ g _ allele of the HLA DQ locus or a biologically active fragment thereof.
In another aspect, the invention provides a process for preparing a mammal or its progeny comprising 5 suppressing expression of the nucleotide sequence encoding endogenous CD8 in the mammal; suppressing expression of the nucleotide seqt ence encoding endogenous CD4 in the mammal; inserting nucleotide sequences for 2m allele of the HLA DQ locus alpha and 10 beta chains, or biologically active fr;~ thereof into the mammal; and inserting a nucleotide sequence for human CD4 or a biologically active fr~gment thereof into the mammal .
In yet another aspect, the invention provides 15 a method of screening a compound for its anti-sepsis effect, comprising exposing a mammal lacking expression of endogenous CD4 and CD8, wherein the mammal has inserted a nucleotide sequence comprising the DNA
encoding human CD4 or ~ biologically active fragment 20 thereof and a nucleotide sequence comprising the DNA
encoding an allele of the HLA DQ locus or a biologically active fragment thereof to a compound or organism that has the capacity to induce sepsis administering to the mammal a th~r~retlt;r~l ly effective amount of the 25 compound and screening the mammal for sep3is or sepsis-like symptoms.
PRT~r DESrRTPTION OF T~ FIGURF:~
Figure 1 depicts the knockout construct used to suppress expression of Lyt--2, the mouse CD8 gene ~mCD8-/--). The dark boxes represent exons of Lyt-2 (indicated by Roman numerals) with the shaded areas denoting coding regions. The neomycin phosphotransferase gene is indicated by the hatching.
Selected restriction enzymes are indicated.

WO 9~14377 1 ~ 17

2 \1 5~ - lo -Figure 2 depicts the knockout construct used to suppress expression of the mouse CD4 gene (mCD4--/-) .
The neomycin phosphotransferase gene is indicated by the hatching. The dark boxes represent exons of the mCD4 5 gene (indicated by Roman numerals). Selected restrLctlon enzymes are indicated.
Figure 3 depicts the transgene construct used to generate human CD4 (hCD4 ) transgenic mice . The hCD2 lO 5 ' promoter region and the hCD2 3 ' regulatory region are indlcated as are the hCD4 cDNA insert and the hCD2 minigene consisting of the hCD2 exon I genomic sequence and the hCD2 cDNA codlng reglon for the L~ 1 n~ r of the gene. Selected restriction enzymes are also indlcated.
Figure 4 depicts the breeding scheme used to generate a double knockout, double transgenic mouse.
hCD4 represents a human CD4 transgenic mouse; DQw6 represents a human DQw6 (alpha and beta chalns) 20 transgenlc mouse; mCD4 represents the mouse endogenous CD4; mCD8 represents the mouse endogenous CD8 (Lyt-2 gene) . The symbol "hCD4+/-" refers to a mouse heterozygous for the hCD4 transgene; the symbols "mCD4/8-/-" and "mCD4/8+/-" refer to a mouse that is a 25 heterozygote knockout of CD4 and CD8.
Figure 5 depicts a dose-response curve measuring T cell response or proliferation (indicated as 3H thymidine uptake) when stimulated by decreasing 30 amounts of SEB in freshly killed ex v~vo cells of different mice genotypes. The concentration of SEB is indicated on the X axis. Dark circles represent the double knockout, double transgenic mouse (mCD4-/-, mCD8-/-, DQw6+, hCD4+), open clrcles represent the double 35 knockout (mCD4-/-, mCD8-/-) with the hCD4 transgene, . , . . . . ~

WO 95/14377 ~ P~ 117 dark squares represent C57BL/6 wild type, and the open rontAg^nA1 represents double knockout mice.
DET~TT ~n DE~CRTPTION OF TTT~ INVENTION

The term "knockout" refers to partial or complete reduction of the expression of zt least a portion of a polypeptide encoded by an endogenous DNA sequence in a single cell, selected cells, or all of the cells of a mammal .
The term "knockout construct" refers to a nucleotide sequence that is rl~ci~n~d to decrease or suppress expression of a polypeptide encoded by endogenous DNA sequences in a cell. The nucleotide sequence used as the knockout construct is typically comprised of (l) DNA from gome portion of the ._n~
gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and (2) a marker sequence used to detect the presence of the knockout construct in the cell. The knn-k~lt construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position 80 as to prevent or interrupt transcription of the native DNA sequence. Such insertion usually occurs by homologous re~ ~ 1 nAt 1 ~n ( i . e ., regions of the knockout construct that are homologous to endogenous DNA sequences hybridize to each other when the knockout construct is inserted lnto the cell and recombine so that the knockout construct is incorporated into the corr~r^n~lln~ position of the endogenous DNA) . The knockout construct nucleotide sequence may comprise l) a full or partial sequence of - one or more exons and/or introns of the gene to be suppressed, 2) a full or partial promoter sequence of the gene to be suppressed, or 3) combinations thereof.
Typically, the knockout construct is inserted into an embryonic stem cell (ES cell) where the cell is an wo g5/l4377 r~ 1~ 117 ~7 undifferentiated cell, usually derived from an embryo of the same species as the developing embryo into which it is subsequently injected and is integrated into the 3~:S
cell genomic DNA, usually by the process of homologous 5 recombination. This ES cell is then injected into, and integrates with, the developing embryo.
The phrases "disrupting of the gene", "gene disruption", "suppressing expressionn, and "gene suppression", refer to insertion of a nucleotide 10 sequence into one region of an endogenous gene (usually one or more exons) and/or the promoter region of a gene 80 as to decrease or prevent expression of that gene in the cell. Insertion is usually accomplished by homologous recombination. For purposes herein, "gene", 15 "nucleotide sequencen, and "nucleic acid sequence" all refer to the sequence of nucleotides or codons encoding a polypeptide or fragment of a polypeptide. By way of example, a nucleotide sequence knockout construct can be prepared by inserting a nucleotide sequence comprising 20 an antibiotic resistance gene into a portion of an isolated nucleotide sequence that is complementary to the endogenous DNA sequence ~promoter and/or coding region) to be disrupted. When this isolated nucleotide sequence c~n~n~n~ the antibiotic resistance sequence 25 construct is then inserted into a cell, the construct will typically integrate into the genomic DNA. Thus, many progeny of the cell will no longer express the gene at least in some cells, or will express it at a decreased level, as the nucleotide sequence of the gene 30 is now disrupted by the antibiotic resistance gene.
The term "marker sequence" refers to a nucleotide sequence that is ~ sed as part of a larger nucleotide sequence construct ~i.e., the "knockout construct") to disrupt the expression of the gene ~8) of interest ~such 35 as, for example, CD4 and/or CD8), and (2) used as a means to identify those cells that have lncorporated the .. ..... . .... . ....

W0 95/14377 r~.,~. ~'t ~ 117 iz~

knockout construct into the genome. The marker sequence may be any sequence that serves these purposes, although typically it will be a sequence encoding a protein that confers a detectable trait on the cell, such as an 5 antibiotic resistance gene or an assayable enzyme not typically found in the cell. Where the marker sequence encodes a protein, the marker se~uence will also typically contain either a homologous or heterologous promoter that regulates its expression.
The term "transgene" refers to an isolated nucleotide sequence that may or may not be operably linked to a promoter, and that is inserted into one or more cells of a mammal or 1~ An embryo . The transgene may be comprised of a nucleotide sequence that 15 is either homologous or heterologous to a particular nucleotide sequence in the mammal ' s ~nr~ n~llR genetic ^-t~r~Al~ or that is a hybrid sequence (i.e. one or more portions of the transgene are homologous, and one or more portions are heterologous to the mammal ' s genetic 20 material~. The transgene nucleotide sequence may encode a polypeptide or a variant of a polypeptide, found endogenously in the mammal, it may encode a polypeptide not naturally occurring in the mammal ~i.e. an exogenous polypeptlde), or it may encode a hybrid of endogenous 25 and exogenous polypeptides. Where the transgene is operably linked to a promoter, the promoter may be homologous or heterologous to the ma;nmal and/or to the transgene. Alternatively, the promoter may be a hybrid of endogenous and exogenous promoter elements 30 (~.n~Anr~rf:, silencers, suppressors, and the like) .
The term "CD4" refers to a cell surface glycoprotein expressed primarily on T helper cells of l ~ An species, and is believed to be involved in cell signaling primarily via recognition and binding as 35 a co-receptor to MHCII molecules that have bound antigen and are recognized by the T cell receptor. As used

3~7 E~~ 17 ~17~5 - 14 -hereln, human CD4 may have the wild-type nucleotlde sequence, or it may be an lnsertlonal, ~ et~cn~1, and/or substltutional variant thereof. slmllarly, mouse CD4 may have the wlld type nucleotlde sequence, or lt may be an lnsertlonal, deletlonal, and/or substitutional variant thereof. As used herein, insertional, deletional and/or substitutional variant lncludes, lnter alia, allellc variants.
The term "CD8~ refers to a cell surface glycoprotein that ls typically comprised ln l iAn specles of two heterologous monomer polypeptides. CD8 is expressed primarlly on the surface of cytotoxlc T
cells, and 18 belleved to be involved ln cell 81~Jn~1 ~n~T
primarlly vla recognition and binding as a co-receptor to MHCI molecules that have bound antigen and are r~o~n~ 7ed by the T cell receptor. As used herein, the human CD8 monomers may have the wild type alpha and beta chain nucleotide sequences, or may be insertional, ~I.ol et ~ r~n~ 1, and/or substitutional variants thereof, including, ~nter al ia, allelic variants . r~ouse CD8 monomers may have the wild type nucleotide sequences of the Lyt-2 and Lyt-3 genes, or may be insertional, deletional, and or substitutional variants thereof, including, lnter alia, allelic variants.
The term "allele of the }ILA DQ locus" refers to any and all variants of the human ma~or histo ~h~ 1 ~ty complex class II (MHCII) DQ locus, and includes all polypeptides, or monomers of that locus ~i.e., alpha and beta chains), which are combined to generate the final protein known as the product of the DQ locus. By the way of example, the DQw6 allele is comprised of alpha and beta monomers, and the monomers together comprise one allelic product of the DQ locus.
The term "operably linked" refers to the arrangement of various nucleotide sequences relative to each other such that the elements are functionally _ ... _ .. _ . , . .. . .. .. . .. . _ _ _ _ _ _ _ _ _ _ _ _ . _ _ .

W0 951143M 2,~ ~3 1 t G t l7 connected and are able to interact with each other.
Such elements may include, without limitation, one or more promoters, enhancers, polyadenylation sequences, and transgeneæ. The nucleotide sequence elements, when properly oriented, or operably linked, act together to l Ate the activity of one another, and ultimately may affect the level of expression of the transgene ~8) . By modulate is meant increasing, decreasing, or r~ntA;n;nq the level of activity of a particular element. The position of each element relative to other elements may be expressed in terms of the 5 ' terminus and the 3 ' terminus of each element, and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements.
The term "biologically active fragment" refers to a nucleotide sequence that is less than the full-length genomic or cDNA nucleotide sequence of a gene or transgene, but contains a sufficient portion of the full length nucleotide sequence such that the gene (or transgene) product of the biologically active fragment possesses at least a portion of the biological activity possessed by the gene product of the full length se~lu~nc~ .
The terms "rodent" and ~rodents" refer to all members of the phylogenetic order Rodent~a including any and all progeny of all future generations derived therefrom .
The term "murine" refers to any and all members of the family Muridae, I nr~ ng rats and mice .
The term "progeny" refers to any and all future g~n~rA~ nC derived and descending from a particular mammal, I.e., a mammal -~ntAin~nq a knockout construct inserted into its genomic DNA. Thus, progeny of any successive generation are ; nc~ llfl~d herein such that the Wo 95/14377 P~ 117 progeny, the Fl, F2, F3, generations and so on indefinitely are ~ n~ d $n this definition .
The term "mammal" refers to all members of the class r - l ;A except humans and includes progeny of the mammal.
The terms "immun~ l At~" and ~ Ati~n"
refer to changes in the level of activity of any ,olle,~Ls of the immune system as compared to the average activity of that component for a particular specie~. Thus, as used herein, 1 ~ 1A~ n refers to an increase or a decrease in activity.
T ' l AtiOn may be detected by assaying the level or function of B cells, any or all types of T cells, antigen presenting cells, and any other cells believed to be involved in immune function. Additionally or alternatively, ~ 1 At t ~m may be detected by evaluatlng 1) the level of expresslon of partlcular genes belleved to have a role in the immune system, 2 ) the level of particular, _ ~c such as cytoklnes (lnt.o~ ; nc and the llke) or other molecules that have a role in the immune system, and/or 3) the level of partlcular enzymes, protelns, and the llke that are lnvolved ln lmmune gy8tem fllnct ~ ~n ~ n~ .
The term "sepsls" refers to the presence of dlsease causlng organlsms and/or their toxins ln the blood and/or tlssues of a mammal.
The term "antl-sepsls effect" refers to a sltuation wherein the symptoms of sepsis or the sepsis conditlon are reduced or ellmlnated.
The term "therapeutic regimen" refers to a treatment deslgned to achieve a particular effect ~.e., reduction or eliminatlon of a detrlmental condltion or di3ease. The treatment may include admlnlstratlon of one or more compounds elther slmultaneously or at dlfferent tlmes, for the same or different amounts of time. Alternatlvely, or addltionally, the treatment may _ _ . . .

WO95/14377 2~ }~ "7 include exposure to other therapies such as radiation, a particular diet, physical therapy, and the like.
Rnnokout Teohn~ logy 1. Selection of Rnookout Gene (s) This invention contemplates a mammal in which at least two genes of the immune system have been disrupted or knocked out. Tnr~ d within the scope of this invention however, is a mammal with more than two genes knocked out.
In the present invention, the gene(s) to be knocked out or disrupted are 5f~1 ~ctF-rl from CD4 and CD8 . At least some sequence information on the genes to be disrupted must be available for preparation of both the knockout construct and the 3creening probes. The sequence information may be f rom a species other than the species to be genetically ~~n;r~llAted by insertion of the knockout construct, provided that it is reasonably believed that the nucleotide s~ nc~ from each species will be substantially homologous. Usually, the nucleotide sequence comprising the knockout construct will be comprised of one or more exon and/or intron regions from the genomic DNA sequence, and/or a promoter region. However, the DNA to be used may alternatively be a cDNA sequence provided that the cDNA
is suffiiciently large. Generally, the DNA used in the knockout construct will be at least about l kilobase (kb) in length and preferably 3-4 kb in length, thereby providing sllff~ nt complementary sequence for homologous recombination or hybridi~ation to genomic DNA
when the knockout construct is introduced into ES cell (discussed below) .
Typically, for ease of preparation of a double knockout mammal, one gene will be knocked out from each WO 9S/14377 P~ [ ll7 of two mammals of the same species. The mammals will then be bred with each other, and the of f spring interbred to ultimately generate a single mammal with both selected genes knocked out. Alternatively, a 5 single mammal may be initially generated with more than one gene knocked out (e.~. by in~ecting more than one knockout construct into the ES cell, as discussed below) .
Nhere more than two genes are to be knockout out, 10 the above procedures may be followed, 1. e., several mammals may be prepared, each containing one knockout construct, and the mammals can be crossed and backcrossed appropriately, or one mammal ~'nnt~n~n~ all of the knockout constructs may be generated.
By way of example, mice not expressing either copy of gene A ~i.e., gene A knockout; ~ y~uus) are bred with mice not expressing either copy of gene B (l.e., gene B knockout; hl yyuuS) resulting in offspring "Fl"
that are heterozygous for both mutations (i.e., have one 20 of two copies of both mutant genes [A+/--, B+/-] ) . The offspring (Fl) can then be mated, and if the genes segregate separately (in M~n~ n fashion) then one-sixteenth of the offspring (F2) will be h- Zy~uuS for both ~t1~nC and thus will be double knockouts (i.e., 25 A-/-, B-/-). Mice with these genotypes may be screened by suitable assays to identify the absence of expression of each gene product.
The nucleotide sequence (s) comprising the knockout construct (s) can be obtained using methods well known in 30 the art such as those described by Sambrook et al.
(Molecular Clonln~: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989] ) .
Such methods include, for example, screening a genomic library with a cDNA probe that has a suitable level of 35 homology to the genomic sequence such that the genomic clone can be ~ n~ f i ed. Alternatively, if a cDNA

WO 95114377 2 1 ~ ~ "~. ~.t ~ 117 sequence is to be used as part of the knockout construct, the cDNA may be obtained by screening a cDNA
library with oligonucleotide probes or antibodies (where the library is cloned into an expression vector) . If a 5 promoter sequence is to be used in the knockout construct, synthetic DNA probes can be designed for screening a genomic library ~-nntA~n~n~ the promoter sequence, or ~ suitably homologous promoter may be used as a screening probe.
Another method for obtaining the nucleotide sequence comprising the knockout construct is to manufacture this sequence synthetically, using methods such as those described in Engels et al. ~Agnew Chem.
Int. Ed. Eng.) 28:716-734 [1989]. These methods 15 include, fnter alla, the phosphotriester, phosphoramidite, and E~-rhnsphnn~te methods of nucleic acid synthesis.
The nucleotide sequence comprising the knockout construct must be generated in sufficient quantity for 20 later genetic r-n~rlllation~ Amplification of the nucleotide sequence to achieve the desired quantity may be cnnrll~cted by 1) placing the sequence into a suitable vector and transforming bacterial or other cells that can rapidly amplify the vector, 2) by PCR amplification, 25 or 3) by synthesis using methods set forth by Engels et al, supra.
2. Preparation of l~nockout Constructs The nucleotide sequence comprising the knockout construct is typically digested with one or more restriction enzymes selected to cut at a location(s) such that a new DNA sequence encoding a marker gene can be inserted in the proper position within this nucleotide sequence. The proper position for marker gene insertion is that which will serve to prevent or WO95/14377 1~-,~5 1.~ 117 ~Z5~ ~

decrease expression of the native gene. This positLon will depend on various factors such as the position of the restriction sites within the sequence to be cut, and whether an exon sequence or a pr3moter sequence, or both 5 is (are) to be interrupted (~.e., the precise location of insertion necessary to inhibit promoter function or to inhibit synthesis of the native exon1. Preferably, the enzyme selected for cutting the DNA will generate a longer arm and a shorter arm, where the shorter arm is lO at least about 300 base pairs (bp) in length. In some cases, it may be desirable to actually remove a portion or even all of one or more exons of the gene to be suppressed so as to keep the length of the knockout construct comparable to the original genomic sequence 15 when the marker gene is inserted in the knockout construct. In these cases, the genomic DNA is cut with appropriate restriction ~n~ nllcl~Ace-c such that a fragment of the proper size can be removed. The L~ ~ n1 n~ pieces are ligated with the marker sequence, 20 as discussed below.
The marker gene can be any nucleotide sequence that is detectable and/or assayable, however typically it is an antibiotic resistance gene or other gene whose expression or presence in the genome can easily be 25 detected. The marker gene is usually operably linked to its own promoter or to another strong promoter from any source that will be active or can easily be activated in the cell into which it is inserted; however, the marker gene need not have its own promoter attached as it may 30 be transcribed under the control of the promoter of the gene to be suppressed. In addltion, the marker gene will normally have a polyadenylation (polyA) sequence attached to its 3 ' end; this sequence serves to t~rm~ nAte transcription of the gene . Preferred marker 35 genes are any antibiotic resistance genes such as neo WO 95/14377 1'~ ~3 1.'~ - ~17 2~ ~

(the neomycin resistance gene) and beta-gal (beta-galactosidase) .
After the nucleotide sequence comprising the knockout construct has been digested with the 5 appropriate restriction enzymes, the marker gene sequence is ligated into it using methods well known to the skilled artisan, 3uch as tho3e de3cribed in Sambrook et al., supra. The ends of the DNA fragments to be ligated together must be ,~t;hl~; this is achieved by 10 either cutting the fragments with enzymes that generate ; hl e ends, or by blunting the ends prior to ligation. Blunting is conducted using any of several methods well known in the art, such as for example by the use of Klenow fragment (DNA polymerase I) to fill in 15 sticky ends.
~ he ligated knockout construct may be inserted directly into embryonic stem cells (discu3sed below), or it may fir3t be placed into a suitable vector for amplification prior to in3ertion. Preferred vectors are 20 tho3e that are rapidly amplified in bacterial cells such as the pBluescript II SK vector (Str:~-tag~n~, San Diego, CA) or pGEM7 (Promega Corp., Madison, WI) .
3 . TrAncfection of r ry~n~ c Stem Cell -This invention contemplates production of knockout mammals from any species of non-human mammal tn~ lt~ n~
without limitation, rodents such as rat3, ham3ters, and mice. Preferred rodents include members of the Muridae 30 family, including rats and mice.
Generally, the embryonic stem cells (ES cells) used to produce the knockout mammal will be of the same species as the knockout mammal to be generated. Thus for example, mouse embryonic stem cell3 will usually be 35 used for g~n~rA~ n of knockout mice.

WO 95/14377 ~ ~S 1 ~ ~ :17 ~i15~ - 22 -Embryonic stem cells are generated and r-~ntA~n~d using methods well known to the skilled artisan such as those described by DQet~rh---n et al. (,T. EmQryol. Exp.
~orpAol. 87:27-45 [1985]) . Any line of ES cells can be used, however, the line chosen is typically selected for the ability of the cells to integrate into and become part of the germ line of a developing embryo so as to create germ line trAnRm~ Ysion of the knockout construct .
Thus, any ES cell line that is believed to have this capability is suitable for use herein. One mouse strain that is typically used for production of ES cells, is the 129J strain. A preferred ES cell line is murine cell line D3 (~ r;rAn Type Culture rQllert~rn~ 12301 Parklawn Drive, Rockville, ~D 20852, catalog no. CRL
1934). The cells are cultured and prepared for knorkrt~t construct lnsertion using methods well known to the skilled artisan such as those set forth by Robertson (in: Teratocarclnomas and Enibryonlc Stem Cells: A
Practical Approach, E.J. Robertson, ed. IRL Press, Washington, D.C. [1987] ) by Bradley et al. (Current Topics ln Devel. Biol., 20:357-371 [1986]) and by Hogan et al. (MAn~ Atin~7 the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1986] ) .
Insertion of the knockout construct into the ES
cells can be accomplished using a variety of methods well known in the art ~nrl~ n~ for example, electroporation, microinjection, and calcium phosphate treatment (see Lovell-Badge, in Robertson, ed., supra).
A preferred method of insertlon is electroporation.
Each knockout construct to be inserted into the cell must first be in the linear form. Therefore, if the knockout construct has been inserted into a vector, linearization is accomplished by digesting the DNA with a suitable restriction ~nr~rnl~rl~-AYe selected to cut only WO g5ll4377 P~" I ~ ~ 'l7 2~

within the vector sequence and not within the knockout construct sequence.
For in3ertion, the knockout construct is added to the ES cells under appropriate conditions for the 5 insertion method chosen, as is known to the skilled artisan. Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.
If the ES cells are to be electroporated, the ES
10 cells and knockout construct DNA are exposed to an electric pulse using an electroporation machine and following the r-nllfArturer~s gll~ ;n.os for use. After electroporation, the ES co-lls are typically allowed to recover under suitable incubation conditions. The cells 15 are then screened for the presence of the knockout construct .
Screening can be accomplished using a variety of methods. Where the marker gene is an antibiotic resistance gene, the ES cells may be cultured in the 20 presence of an otherwise lethal c~nc~ntrAtlnn of antibiotic. rrhose ES cells that survive have presumably integrated the knockout construct. If the marker gene is other than an antibiotic resistance gene, a Southern blot of the ES cell genomic DNA can be probed with a 25 sequence of DNA ~l~s1gne~ to hybridize only to the marker sequence. Alternatively, PCR can be used. Finally, if the marker gene is a gene that encodes an enzyme whose activity can be detected (e.g., beta-galactosidase), the enzyme substrate can be added to the cells under 30 suitable conditions, and the enzymatic activity can be analyzed. One skilled in the art will be familiar with other useful markers and the means for detecting their presence in a given cell. All such markers are contemplated as being ~ nrl ~ l within the scope of the 35 teaching of this invention.

WO95/14377 ~ r~ t~ll7 The knockout construct may integrate into several locations in the ES cell genome, and may integrate into a different location in each ES cell's genome due to the occurrence of random insertion events. The desired 5 location of insertion is in a complementary position to the DNA sequence to be knocked out. Typically, less than about 1-5 percent of the ES cells that take up the knockout construct will actually integrate the knockout construct in the desired location. To identify those ES
10 cells with proper integration of the knockout construct, total DUA can be l'YtrACted from the ES cells using standard methods such as those described by Sambrook et al., supra. The DNA can then be probed on a Southern blot with a probe or probes designed to hybridize in a 15 specific pattern to genomic DNA digested with (a) particular restriction enzyme (s) . Alternatively, or additionally, the genomic DNA can be amplified by PCR
with probes specifically rl~qn~-d to amplify DNA
fragments of a particular size and se~Iuence (i.e., only 20 those cells ~ nt~n~ng the knockout construct in the proper position will generate DNA fr~5 q of the proper size).

4. In1ection/I l~ntation of E ' ryos After suitable ES cells containing the knockout construct ln the proper locatlon have been ~ nt ~ f ~
the cells can be inserted into an embryo. Insertion may be ~c~ h -d in a variety of ways known to the 30 skilled artisan, however a preferred method is by microinjection. For microin~ection, about 10-30 cells are collected into a micropipet and in~ected into embryos that are at the proper stage of development to permit lntegratlon of the foreign ES cell containing the 35 knockout construct into the developing embryo.
, . ..... . .. . .... . .. . ... .. _ .. _ . ,,, . , .. , . , . ,, ,,,,, _ _ W0 95114377 ~ 7~ r~ 17 The suitable stage of development for the embryo used for insertion of ES cells is very species dependent, however for mice it is about 3.5 days. ~he embryos are obtained by perfusing the uterus of pregnant

5 females. Suitable methods for accomplishing this are known to the skilled artisan, and are set forth by Eradley ~in Robertson, ed., supra).
While any embryo of the right age/stage of development is suitable for use, preferred embryos are 10 male. In mice, the preferred embryos also have genes coding for a coat color that is dif~erent from the coat color encoded by the ES cell genes. In this way, the offspring can be screened easily for the presence of the knockout construct by looking for mosaic coat color 15 (~n~i~cnt~ng that the ES cell was incorporated into the developing embryo). Thus, for example, if the ES cell line carries the genes for white fur, the embryo selected will carry genes for black or brown fur.
After the ES cell has been introduced into the 20 embryo, the embryo may be implanted into the uterus of a pseudopregnant foster mother for gestation. While any foster mother may be used, the foster mother is typically selected for her ability to breed and reproduce well, and for her ability to care for the 25 young. Such foster mothers are typically prepared by mating with vA~ectl ~ 7ed males of the same species . ~he stage of the pseudopregnant foster mother is important for successful implantation, and it is species dependent. For mice, this stage is about 2-3 days 30 pseudopregnant.
5. Screen~n~ for Presence of ~nr~ckout G~ne Offspring that are born to the foster mother may be 35 screened initially for mosaic coat color where the coat color selection strategy (as described above) has been WO95/14377 ~ 117 L\l 5~

employed. In addition, or as an alternative, DNA from tail tissue of the offspring may be screened for the presence of the knockout construct using Southern blots and/or PCR as described above. Offspring that appear to 5 be mosaics may then be crossed to each other, if they are believed to carry the knockout construct in their germ line, in order to generate h~ IZyy~ S knockout animals. If it is unclear whether the offspring will have germ line tr~n~m~RR;On~ they can be crossed with a l0 parental or other strain, and the offspring can be screened for heterozygosity. The heterozygotes are identified by Southern blots and/or PCR amplification of the DNA, as set forth above.
The heterozygotes can then be crossed with each 15 other to generate homozygous knockout offspring.
~omozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice. Probes to screen the 20 Southern blots can be designed as set forth above.
Other means of identifying and ~hArA~tf-r~7~nlr the knockout offspring are avallable. For example, Northern blots can be used to probe the mRNA for the presence or absence of transcripts encodlng elther the gene knocked 25 out, the marker gene, or both. In additlon, Western blots can be used to assess the level of expresslon of the gene knocked out in various tissues of these offspring by probing the Western blot with an antibody against the protein encoded by the gene knocked out, or 30 an antibody against the marker gene product, where this gene is expressed. Finally, in s~tu analysis (such as fixlng the cells and labeling wlth antlbody) and/or FACS
~fluorescence actlvated cell sortlng) analysls of varlous cells from the offsprlng can be conducted uslng 35 sultable antlbodles to look for the presence or absence of the knockout construct gene product.
. . _ ..... .. . _ _ _ . _ _ . _ _ _ WO95/14377 r~ 0~ 17 2~ 7~2~B

TranqSrene Techn-~lo~y 1. Selecti~n of TrAnqc~ene ~B) Typically, the transgene ~s) u3eful in the present invention will be a nucleotide sequence encoding a polypeptide lnvolved in the immune response, hematopoiesis, inflammation, cell growth and proliferation, cell lineage differentiation, and/or the 10 stress response. Preferred transgenes are those that comprise polypeptldes of the human immune system, such as CD4, and any allelic forms of the 31LA DQ locus. The most preferred transgenes are human CD4 and the ~LA DQ
locus allele DQw6 (both the alpha and beta chains).
Tn~ d within the scope of this invention is the insertion of two or more transgenes into a mammal.
The transgenes useful in this invention may be prepared and inserted individually, or may be g~norAt -d together as one construct for insertion. The transgenes 20 may be homologous or heterologous to both the promoter selected to drive expression of each transgene and/or to the mammal. Further, the transgene may be a full length cDNA or genomic DNA sequence, or any fragment, subunit or mutant thereof that has at least some biological 25 activity i.e., exhibits an effect at any level ~biochemical, cellular and/or morphological) that is not readily observed in a wild type, non-transgenic mammal of the same species. Optionally, the transgene may be a hybrid nucleotide sequence, i . e ., one constructed f rom 30 homologous and/or heterologous cDNA and/or genomic DNA
~r~; n~q. The transgene may also optionally be a mutant of one or more naturally occurring cDNA and/or genomic se~uences, or an allelic variant thereof.
Each transgene may be isolated and obtained in 35 suitable quantity using one or more methods that are well known in the art. These methods and others useful WO 9S/14377 P~ 17 a - 28 -for isolatlng a transgene are set forth, for example, ln Sambrook et al. (l~olecular Clonlng: A Laboratory ~qanual, Cold Sprlng Harbor Laboratory Press, Cold Sprlng Harbor, NY [1989] ) and in Berger and ~Cimmel ~Methods ln 5 Enzymology: Guide to Mr~l~c~ r Clon~ng Techn~ques, vol.
152, Academic Press, Inc., San Diego, CA [1987]).
Where the nucleotlde sequence of each transgene ls known, the transgene may be 5yn~h~cl7~a, ln whole or in part, using chemical synthesls methods such 10 as those ~osnr~hPd ln Engels et al. (Angew. ChemInt.
Ed. Engl., 28:716-734 [1989]). These methods lnclude, lnter alla, the phosphotrlester, phosrhnrAm~ te and H-rh~SPhf~nAte methods of nuclelc acld synthesls.
Alternatlvely, the ~ rAn.q~ n~ may be obtalned by screenlng an appropriate cDNA or genomlc llbrary uslng one or more nuclelc acld probes (ol ~on~l~ lentldes~ cDNA
or genomlc DNA f ra j A wlth an acceptable level of homology to the transgene to be cloned, and the like) that wlll hybridlze selectlvely wlth the ~rAnS~n~ DNA.
Another sultable method for obtalnlng a trAnAg~n~ ls the polymerase chaln reactlon (PCR) .
However, successful use of thls method requlres that enough lnformatlon about the nucleotlde sequence of the ~rAnAg~n~ be avallable so as to design suitable ollgonucleotlde primers useful for amplification of the appropriate nucleotlde sequence.
Where the method of cholce requlres the use of ollgonucleotlde primers or probes (e.g. PCR, cDNA or genomic library screening), the ollgonucleotlde sequences selected as probes or prlmers should be of adequate length and suf f lclently unamblguous so as to mln1m~7~ the amount of non-speclfic binding that wlll occur durlng llbrary screening or PCR. The actual sequence of the probes or primers is usually based on conserved or hlghly homologous sequences or regions from WO9~/14377 2 1 7~ ~ 117 the same or a simllar gene from another organism.
Optionally, the probes or primers can be degenerate.
In cases where only the amino acid sequence of the transgene is known, a probable and fllnct~nAl 5 nucleic acid sequence may be inferred for the transgene using known and preferred codons for each amino acid residue. This sequence can then be chemically synthe3 ized .
I'his invention contemplates the use of lO transgene mutant sequences. A mutant transgene i3 a transgene rr~ntA~n~ns one or more nucleotide substitutions, deletions, and/or insertions as compared to the wild type sequence. The nucleotide substitution, deletion, and/or insertion can give rise to a gene 15 product (~.e., protein) that is different in its amino acid sequence from the wild type amino 2cid sequence.
Prl rAr?lt1r~n of such mutants is well known in the art, an~ is described for example in Wells et al. (Gene, 34 :315 [1985] ), and in Sambrook et al, supra.
2. Selection of Recrulatory El ntS
The transgenes of the present invention are typically operably linked to promoters, where a promoter 25 is selected to regulate expression of each transgene ln a particular manner.
Each transgene may be regulated by the same or by a different promoter. The selected promoters may be homologous (i.e., from the same species as the mammal to 30 be transfected with the transgene) or heterologous (~.e., from a source other than the species of the mammal to be transfected with the transgene). As such, the source of each promoter may be from any 1ln1r~11ll1Ar, prokaryotic or eukaryotic organism, or any vertebrate or 35 invertebrate organism. The more preferred promoters of this invention are human and mouse promoters that WO95114377 r~ LS1~ ~17 ~ ~ 5~5~ 30 _ regulate expression of genes of the lmmune system such as the human CD2 promoter, the human CD4 promoter, the HLA DQw6 alpha and beta promoters, the mouse CD4 promoter, the mouse p561ck promoter, the mouse I-E alpha 5 promoter, and the mouse H-2k promoter. The most pref erred promoters are the human CD2 promoter and the HLA DQw6 alpha and beta promoters.
The promoters of this invention may be used alone or in combination with homologous and/or 10 heterologous enhancers and/or silencers in order to permit a tighter regulation of expression.
The nucleotide sequences of the promoters of this invention may be obtained by any of several methods well known in the art. Typically, promoters useful 15 herein will have been previously identified by mapping and/or by restriction ~n~lrn~lrl~Aqe diqestion and can thus be isolated from the genomic DNA of the proper tissue 60urce uslng the appropriate restriction ~n~lrnl~rl~AR~q. In gome cases, the promoter may have 20 been seqn~nr~fl. For those promoters whose nucleotide sequence is known, the promoter may be synthF-q~ -~cl using the methods ~ qrr~h~d above for transgene synthesis.
Where all or only portions of the promoter sequence are known, the promoter may be obtained using 25 PC~ and/or by screening a genomic library with suitable oligonucleotide and/or promoter sequence rL~ q from the same or Another species.
Where the promoter sequence is not known, a fragment of DNA cr~ntA~n~ng the promoter may be isolated 30 from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be A~.~ l; qh~r~ by restriction F.Tl~lr,n~lrl ~A qe digestion using one or more carefully selected enzymes to isolate the proper DNA fragment.
35 After digestion, the desired fragment is isolated by agarose ~el purification, Qiagen~l9 column (Qiagen Corp., W0 95/14377 ~ f9 r`,l/lLS l't - ~17 - 31 - ' ' ' ` ~
Chatsworth, CA~ or other methods known to the skilled arti3an. Selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary ækill in the art.
3. Selection of Oth~r Vector C on~nts In addition to the transgene and the promoter, the vectors useful for preparing the transgenes of this lO invention typically contain one or more other elements useful for (l) optimal expression of transgene in the mammal into which the transgene is inserted, and ~2) amplification of the vector in bacterial or mammalian host cells. Each of these elements will be positioned 15 appropriately in the vector with respect to each other element so as to r~y~m~7e their respective activities.
Such positioning is well known to the ordinary skilled artisan. The following elements may be optionally included in the vector as appropriate.
i . Sign~ 1 Se~uence ~
For those ~ 8 of the invention where the polypeptide encoded by the transgene is to be 25 secreted, a small polypeptide termed signal sequence is frequently present to direct the polypeptide encoded by the transgene out of the cell where it is synthesized.
Typically, the signal sequence is positioned in the coding region of the transgene towards or at the 5 ' end 30 of the coding region. Many signal sequences have been identified, and any of them that are fllnrt~rm~l and thus ~- nt ~ h3 e with the transgenic tissue may be used in con~unction with the transgene. Therefore, the nucleotide sequence encoding the signal sequence may be 35 homologous or heterologous to the transgene, and may be homologous or heterologous to the transgenic mammal.

W095114377 r~1,~ 1c ~17 5~ ~

Additionally, the nucleotide sequence encoding the signal sequence may be rhPm~ ~A 1 1 y synthesized using methods set forth above. However, for purposes herein, preferred signal sequences are those that occur 5 naturally with the transgene ~ ~ . e ., are homologous to the transgene).
ii . r ~ r~ne Anr-h~rin~ Dc ~ n ~1, In some cases, lt may be desirable to have a transgene expressed on the surface of a particular intrAcP11111Ar membrane or on the plasma rAnP.
Naturally occurring rAnP proteins contain, as part of the polypeptide, a stretch of amino acids that serve 15 to anchor the protein to the rAnP. However, for proteins that are not naturally found on the membrane, such a stretch of amino acids may be added to confer this feature. Frequently, the anchor domain will be an internal portion of the polypeptide sequence and thus 20 the nucleotide sequence encoding it will be engineered into an lnternal region of the transgene nucleotlde sequence. However, ln other cases, the nucleotide sequence encoding the anchor domain may be attached to the 5 ' or 3 ' end of the transgene nucleotide sequence .
25 Here, the nucleotlde sequence encodlng the anchor domain may f lrst be placed into the vector in the appropriate positlon as a separate component from the nucleotide sequence encoding the transgene. As for the slgnal sequence, the anchor domaln may be from any source and 30 thus may be homologous or hPtProlo~o11q wlth respect to both the trAnRg^nP and the transgenlc mammal.
Alternatlvely, the anchor domain may be rhPln~Ally synthesized uslng methods set forth above.

WO95/14377 r~l,.. ,,1.'~ 11'7 2 1 7~

iii. Ori,r,in of Replication El This component is typically a part of prokaryotic expression vectors purchased commercially, 5 and aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synth.-st 7ed based on a known sequence, and ligated into the vector.0 iv. Tranccril~tion Trrm~nAtion El~
This element, also known as the polyadenylation or polyA sequence, is typically located 15 3 ' to the transgene nucleotide sequence in the vector, and serves to terminate transcription of the transgene.
While the nucleotide sequence encoding this element is easily cloned from a llbrary or even purchased commercially as part of a vector, it can also be readily 20 syn~he5i7ed using methods for nucleotide sequence synthesis such as those described above.
v. Tntron El~ t In many cases, transcription of the transgene is increased by the presence of one or more introns on the vector. The intron may be naturally occurring within the transgene nucleotide sequence, ~ rer~l ly where the transgene is a full length or a fragment of a genomic DNA sequence. Where the intron is not naturally occurring within the nucleotide sequence (as for most cDNAs), the intron (8) may be obtained from another source. The intron may be homologous or heterologous to the transgene and/or to the transgenic mammal. The position of the intron with respect to the promoter and the transgene is important, as the intron must be WO 95/14377 P~ 17 ~7~2~ 34_ transcribed to be effective. As such, where the transgene is a cDNA sequence, the preferred position for the lntron is 3 ' to the transcrlption start sLte, and 5 ' to the polyA transcription termination sequence.
Preferably for cDNA transgenes, the intron will be located on one side or the other (l.e., 5' or 3') of the transgene nucleotide sequence such that it does not interrupt the transgene nucleotide sequence. Any intron from any source, ~ nr~ n~ any viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this invention, provided that it is compatible with the host cell (s) into which it is inserted. Also included herein are synthetic introns. Optionally, more than one intron may be used ln the vector.
vi. Selectahle ~l~rkor~ t Selectable marker genes encode proteins necessary for the survival and growth of transfected cells grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanomycin for prokaryotic host cells, and neomycin, hyyLI y~in~ or methotrexate for 11;~n cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for cultures of Bacilli .
All of the elements set forth above, as well as others useful in this invention, are well known to the skilled artisan and are described, for example, in Sambrook et al. (Molecular Cloning:A Labor~tory Manual, Cold Spring Elarbor Laboratory Press, Cold Spring ~larbor, NY [1989] ) and Berger et al., eds. (Guide to Molecular , .. . ... . _ ... . _ ... _ . _ . _ . , . , . _ _ _ _ _ . _ _ .

WO95/14377 21 7~gQI) r t.'t~117 .

Clon~n~ TecAnlques, Academlc Press, Inc., San Diego, CA
[1987] ) .
4. C- nctruction o~ Vectors The vectors most useful for pr--rArAt;-n of transgenlc mammals of thi6 invention are those that are ~ t;hle with prokaryotic cell ho3ts. However, eukaryotic cell hosts, and vectors compatible with these 10 cells, are within the scope of the invention.
In certain cases, some of the various vector elements may be already present in commercially available vectors such as pUCl8, pUC19, pBR322, the pGEM
vectors ~Promega Corp, Madison, nI), the pBluescript~l9 15 vectors such as pBIISK+/-- ~Stratagene Corp., La Jolla, CA), and the like, all of which are suitable for prokaryotic cell hosts. In this case it is n-c~A~Ary to only insert the transgene ~8) into the vector.
However, where one or more of the elements to 20 be used are not already present in the vector, they may be individually obtained and ligated into the vector.
Methods used for obtaining each of the elements and ligating them are well known to the skilled artisan and are comparable to the methods set forth above for 25 obtaining a transgene ~i . e ., synthesis of the DNA, library screening, and the like).
vector3 used for amplification of the transgene ~8) nucleotide sequences and/or for transfection of the l; An embryog are constructed 30 using methods well known in the art. Such methods include, for example, the standard techniques of restriction Pn~ n~ Ace digestion, ligation, agarose and acrylamide gel purification of DNA and/or RNA, column chromatography purification of DNA and/or RNA, 35 phenol/chloroform extraction of DNA, DNA sequencing, WO 95114377 r~ 117 polymera3e chain reaction amplification, and the like, as set forth in Sambrook et al., supra.
The final vector used to practice this invention is typically constructed ~rom a starting vector such as a commercially available vector. ~his vector may or may not contain some of the elements to be included in the completed vector. If none of the desired elements are present in the starting vector, each element may be individually ligated into the vector by cutting the vector with the appropriate restriction such that the ends of the element to be ligated in and the ends of the vector are compatible for ligation. In some cases, it may be necessary to "blunt"
the ends to be ligated together in order to obtain a satisfactory ligation. Blunting is accomplished by first filling in "sticky ends" using Klenow DNA
polymerase or T4 DNA polymerase in the presence of all four nucleotides. 'rhis procedure is well known in the art and is described for example in Sambrook et al., supra.
Alternatively, two or more of the elements to be inserted into the vector may f irst be ligated together (if they are to be positioned ad~acent to each other) and then ligated into the vector.
One other method ~or constructing the vector is to conduct all ligations of the variouY elements simultaneously in one reaction mixture. Here, many nonsense or nonf-ln~t~-nAl vectors will be g~n~r~t~d due to improper ligation or insertion of the elements, however the f--n~t1r~nAl vector may be i~lont~f~Pd and selected by restriction ~nfi~m~lrl~A~e digestion.
After the vector has been constructed, it may be transfected into a prokaryotic host cell for amplification. Cells typically used for amplification are E coll DH5-alpha (Gibco/BR~, Grand Island, NY) and WO95~14377 ~¦ 7~ r .,~s~-117 other E. coli strAins w$th characteristics slmilar to DH5--alpha .
Where l; An host cells are used, cell lines such as Chinese hamster ovary (CEIO cells; Urlab et al., Proc. Natl. Acad. Scl USA, 77:4216 [1980])) and human embryonic kidney cell line 293 (Graham et al., ,T.
Gen. Virol., 36:59 [1977]), as well as other lines, are suitable .
Transfection of the vector into the selected host cell line for amplification is accomplished using such methods as calcium phosphate, electroporAt;-,n, microin~ection, lipofection or DEAE-dextran. The method selected will in part be a function of the type of host cell to be transfected. These methods and other suitable methods are well known to the skilled artisan, and are set forth in Sambrook et al., supra.
After culturing the cells long enough f or the vector to be sufficiently amplified (usually overnlght for E. col ~ cells), the vector (often termed plasmid at this stage) is ~Rr~l~ted from the cells and purified.
Typically, the cells are lysed and the plasmid is ~-Ytr~ctP~I from other cell contents. Methods suitable for plasmid pur; f i r~t i ~n include ~nter alia, the Alk~l;nP lysis mini-prep method (Sambrook et al., supra).
5. Pre~>~ration of Pl~e:m;d For Inqerti~7n Typically, the plasmid rrnt~;n;n~ the transgene is linearized using a selected restriction ~n~irnnrl ~ e prior to insertion into the embryo . In some cases, it may be preferable to isolate the transgene, promoter, and regulatory elements as a linear f ragment f rom the other portions of the vector, thereby injecting only a linear nucleotide sequence rrn~s~;n~n~
the transgene, promoter, intron (if one is to be used), WO 95/14377 r~ 7 enhancer, polyA sequence, and optionally a signal 3equence or r~n.~ anchoring domain into the embryo.
This may be accomplished by cutting the plasmid so as to remove the nucleic acid sequence region ~ nt~n~n~ these 5 elements, and purifying this region using agarose gel - electrophoresis or other suitable purification methods.

6 . Pro~llction of Tran~g~n1 c ~ 1~
The specific line(s) of any l;~n species used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryos, and good reproductive fitness. In addition, the haplotype is a si~n~ f~ nt 15 factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or C57BL/6 x DBA/2 Fl, or FVB lines are often used (obtained commercially from Charles River Labs, Boston, MA, The Jackson Laboratory, Bar Harbor, ME, or Taconic Labs. ) . Preferred strains 20 are those with H-2b~ H-2d or H-2q haplotypes such as C57BL/6 or DBA/l . The line (s) used to practice this invention may themselves be transgenics, and/or may be knockouts (l.e., mammals which have one or more genes partially or completely suppressed). Preferably the 25 same line will be used for preparation of both the initial knockout mammals and the transgenic =als.
This will make subsequent breeding and backcrossing more ,off~c~nt .
The age of the mammals that are used to obtain 30 embryos and to serve as surrogate hosts is a function of the species used, but is readily determined by one of ordinary skill in the art. For example, when mice are used, pre-puberal females are preferred, as they yield more ernbryos and respond better to hormone in~ections.

WO9~/14377 ~I J~5~ r~ 17 Similarly, the male mammal to be used as a stud will normally be selected by age of sexual maturity, among other criteria.
Administration of hormones or other chemical compounds may be necessary to prepare the female for egg production, mating, and/or reimplantation of embryos.
The type of hormones/cofactors and the quantity used, as well as the timing of administration of the hormones will vary for eAch species of mammal. Such considerations will be readily apparent to one of ordinary skill in the art Typlcally, a primed female ~l.e., one that is producing eggs that can be fertili2ed) is mated with a stud male, and the resulting fertilized embryos are then removed for introduction of the t~An~g~on~ (s) .
Alternatively, eggs and sperm may be obtained f rom suitable females and males and used for ln v~tro fert11~7~t~cn to produce an embryo suitable for introduction of the transgene.
Normally, fertilized embryos are incubated in suitable media until the pronuclei appear. At about this time, the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, the male pronucleus is preferred.
Introduction of the transgene nucleotide sequ~nc.o into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
Following introduction of the transgene nucleotide sequence into the embryo, the embryo may be incubated ln vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention. One common method is to incubate the embryos ln vitro for WO 95/143M r~l~.., 40_ about 1-7 days, depending on the species, and then reimplant them into the surrogate host.
ReimplAntat~o~ is accomplished using standard methods. Usually, the surrogate host is anesthetized, 5 and the embryos are lnserted lnto the oviduct. The number of embryos implanted into a partlcular host will vary by species, but will usually be comparable to the number of offspring the species nAt~rAl ly produces .
Transgenic offspring of the surrogate host may 10 be screened for the presence and/or expression of the tran3gene by any suitable method. Screening is often al ~ l; Ahl~cl by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using 1~ an antibody against the protein encoded by the trJ~ncg~n~
may be employed as an alternative or additional method for screening for the presence o~ the tr~n~g~-n-o product.
Typically, DNA is prepared from tail tissue (about 1 cm is removed from the tip of the tail) and analyzed by 20 Southern analysis or PCR for the transgene.
Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may 25 be used for this analysis.
Alternative or ~ t ~ ~nA l methods for evaluating the presence of the transgene include, without limitation, suita3~le bi~-h~m~ c~ 1 assays such as enzyme and/or immunological assays, histological stains 30 for particular markers or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the tr2nsgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various 35 types of blood cells and other blood constituents.
.. _ _ _ _ _ _ _ _ . . _ _ _ _ _ _ _ . . . . . .

7 ,~ 17 Progeny of the transgenic mammals may be obtained by mating the transgenic mammal with a suitable partner, or by in vltro fertll-7~t1r~n of eggs and/or sperm obtained from the transgenic mammal. Where mating 5 with a partner is to be performed, the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both. Alternatively, the partner may be a parental line. Where in v~tro fertilization is used, 10 the fertilized embryo may be implanted into a surrogate host or incubated 1~ v~tro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
PreDaration of Kno~k~lut/Tr~n~;7 n~c ~- lq Mammals rr~ntAlnln~ more than one knockout construct and/or more than one transgene are prepared in any of 20 several ways. The preferred manner of preparation is to generate a series of mammals, each crnt~1n1n~ one of the desired knockout constructs or transgenes. Such mammals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a 25 single mammal rrntAln~n~ all desired knockout constructs and/or transgenes, where the mammal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout ~s) constructs and/or transgene (s) . By way of example, Figure 4 depicts a 30 breeding scheme for g~nrr~t ~ n~ a mouse that is a CD4, CD8 double knrrkr,ut (mCD4-/-, mCD8-/-) and contains two transgenes (human DQw6 alpha and beta chains [DQw6], and human CD4 [hCD4] ) .
Typically, crossing and backcrossing is 35 accomplished by mating siblings or a parental strain with an offspring, depending on the goal of each WO 95114377 P~ 17 ~115~ 42- ~
particular step in the breeding process. In certain cases, it may be necessary to generate a large number of off3pring in order to generate a single offspring that contains each of the knockout constructs and/or transgenes in the proper ch~1 -; 1 location. For example, the murine genes encoding CD4 and CD8 are located relatively close together on the same chromosome. Thus, to generate a mouse that has both CD4 and CD8 knocked out, one has essentially two practical choices. First, one can attempt to make a double knockout by in~ecting a single ES cell with both the CD4 and CD8 knockout constructs, and hope that each construct will integrate into the same chromosome in the same ES cell, and that this ES cell will in turn properly integrate into the embryo into which it is subsequently inserted. The probability of all of these events occurring as necessary to achieve the final product is very small.
Alternatively, one can generate two knockout mammals, one containing the CD4 knockout construct and one c~ ntA~ n~n~ the CD8 knockout construct . These mammals can then be initially bred together and successively interbred and screened until an offspring is obtained that contains both knockout constructs on the same chromosome ~in mice, this result cannot be obtained unless a cross over event has occurred at ~ust the right location, l . e., between the CD4 and CD8 genes since the genes encoding CD4 and CD8 are on the same chromosome ) .
In either situation, one hundred or more offspring from several crosses may need to be screened to identify a single mammal containing the knockout constructs and/or transgenes in the proper chromosomal location.

WO 9S/14377 r~ 117 ~ ~ / 75~5~ .

Uses of ~r7~n~genic/Kn~ckout 1 1~
The mammal and its progeny of this invention will have a variety of uses depending on the transgenes expressed and the knockout constructs they contain. The mammal may be used to screen for drugs or a therapeutic regimen useful for prophylactic or therapeutic treatment of diseases such as sepsis or other immunological disorders. Screening for a useful drug would involve first inducinq the disease in the mammal (i.e., exposing the mammal to a pathogen or toxin causing sepsis) and then administering the ~Anrl~ ~lAt~o drug over a range of doses to the mammal, and assaying at various time points for the effect(s) of the drug on the disease or disorder being evaluated. ~1tPrnAt~vely, or At~ t~OnA1 1y~ the drug could be administered prior to or simultaneously with exposure to induction of the disease.
In addition to screening a drug for use in treating a disease or condition, the mammal of the present invention could be useful in designing a therapeutic regimen aimed at preventing or curing the disease or condition. For example, the mammal might be treated with a combination of a particular diet, exercise routine, rAf~At~-n treatment, and/or one or more compounds or substances either prior to, or simultaneously, or after, the onset of the disease or condition. Such an overall therapy or reglmen might be more effective at ?t; n~ the disease or condition than treatment with a compound alone.
Assays to evaluate the efficacy of the compound and/or thl~rflretlt; ~ regimen would include, for example, looking for increased or decreased ~ and B cell levels, increased or decreased immunoglobulin production, increased or decreased levels of chemical messengers such as cytokines (e.g., tumor necrosis factor and the like), and/or increased or decreased levels of WO95114377 P~111L 1CL117 44_ expression of particular genes involved in the immune response. In addition, such criteria as blood pressure, body temperature, body weight, pulse, behavior, ~rp~rAn~ of coat (ruffled fur) and the like could be 5 evaluated.
For example, patients with sepsis often experience decreased blood pressure, fever, weight 1088 and/or blood clotting. It would be desirable to administer to the patient a therapeutic agent capable of preventing or lO decreasing such effects. A mammal of the present invention could be used to screen a variety of o~1nrl~ either alone or in ~ n~tirn~ to ~ tF-whether such reduction in disease symptoms could be decreased or alleviated.
In addition, mammals of the present invention can be useful for evaluating the development of the immune system, and for studying the effects of particular gene ~t~nnR .
The transgenic knockout mammals of this 20 invention may also be used to generate one or more cell lines. Such cell lines have many uses, as for example, to evaluate the effect (s) of the transgene knr~rkn~lt on a particular tissue or organ, and to screen compounds that may affect the level of activity of the transgene in the 25 tissue. Such compounds may be useful as therapeutics to modulate the activity of the transgene.
Production of such cell lines may be accomplished using a variety of methods, known to the skilled artisan. The actual culturing conditions will 30 depend on the tissue and type of cells to be cultured.
Various media rrnt~ n~ n~ different rnnr ntr~tions of macro and micro nutrients, growth factors, serum, and the like, can be tested on the cells without undue exper~ ~t tnn to determine the optimal conditions for 35 growth and proliferation of the cells. Similarly, other culturing rrn~ltt~rnq such as cell density, media W095114377 21 ~2~Q P~ ( 117 temperature, and carbon dioxide c~nr ntr~ti~n~ ln the ~ nr1lhAtrr can also readily be evaluated.
repair, and identifying compounds that affect this process .
Other uses of the invention will be readily apparent to one skilled in the art.
The invention will be more readily understood by reference to the following examples. These examples should not be construed in any way as limiting the scope of the invention.
EX'i~ lPI.
Example 1~ 8 ~nnr~kr~ut Mm-~e Mouse strains C57BL/6, BALB/c, and DBA were used, and were purchased from the Jackson Laboratories ~Bar Harbor, ME ) .
An approximately 2.2 kb (kilobase) HindIII-Bam~I mouse genomic DNA fragment c~mt;~n;n~ exons 1-3 of the Lyt-2 gene was ~ qo1 ~tF~cl and used as the starting material for the CD8 knockout construct. The fragment was digested with EcoRI which cuts at an internal site of exon 1. The h~ct~r;~l neomycin resistance gene (neo) was inserted into this EcoRI site. The neo DNA
construct was obtained from the plasmid pMCIneoPolA
(Thomas et al., Cell, 51:503 [1987]) by digestion of this plasmid with restriction ~nfl~nllrlP~es and ~lrtr;lrt;rn and purification of the neo DNA. A schematic diagram of this construct is depicted in Figure 1.
After insertion of the construct into a vector and amplification in E coll cells, the DNA construct plasmid was purified using standard alkaline lysis and CsCl gradient purification.
After purification of the plasmid, the Lyt-2/neo construct was linearized by restriction WO 95/14377 r~l ~s t ~ ll7 2i~5~

~nr~nn~lnl ~-AAe dlgestion, and then electroporated into D3 murine embryonic stem cells (described by Doetschman et al., J.Emoryol. Exp. Morph., 87:27-45 [1985]) using the procedure of electroporation. For electroporation, about 5 nmol of the linearized construct in was added to about 5 X 106 ES cells in a volume of about 800~Ll of culture medium. The cells were pulsed at about 0 . 34 kilovolts and 250~F, and each vial of cells was then placed on two 10 cm cell culture plates contA~n~n~
embryonic fibroblast feeder cells. The culture plates were pre-coated with 1 percent gelatin, and ~ nntA~n~d 10 ml DMEM medium (Gibco/BRL, Grand Island, NY), with 15 percent ~etal calf serum (Gibco/BRL, Grand Island, NY, or the equivalent from Hyclone Labs, Logan, UT), and leukemia inhibitory factor (Fung-Leung et al., Cell, 65:443--449 [1991]) . After two days, neo s~l~rt~nn was started by adding the antibiotic G418 at about 250-300 llg/ml to the cultures, and the media was changed approximately every two days. Cells that survived in the presence of G418 most likely contained the knockout construct with the neo gene in the proper ori~ntAt t c~n for expression. These cells were then screened to verify that they had incorporated the knockout construct into their DNA. Screening was accomplished using PCR.
The D3 cells containing the Lyt-2/neo knockout construct have been deposited with the American Type Culture Collection as ~cr~q.q~ nn number CRL-11116 .
Cell lines rnntA~n~n~ the Lyt-2/neo construct were prepared for microin jection into murine embryos by trypsin treatment following the methods described by Robertson (~reratocarc~nomas and Emoryon~c Stem Cells: A
Practlcal Approach, IRL Press, Washington, D.C. 1987).
The embryos in~ected were 3 . 5 day old embryos obtained by perfusing the uterus of female mice that had been mated with male mice.

WO95/14377 2~ gD r~l,~ ,. 117 After in~ection of the embryonic stem cells into the embryos, the embryos were lmplanted into pseudopregnant females for the duration of gestation.
The offspring were mated to either each other or to a mouse with suitable coat color so as to be able to detect mice carrying the knockout construct.
The offspring of these mice were evaluated for the presence of the knockout construct by probing a .Cn~lth~rn blot of DNA obtained from tail tissue with probes designed to detect the neo gene.
Example 2: rr)4 Rnn- knut ~n~lAe An approximately 2.8 kb fragment of the mouse genomic CD4 cDNA serluence spanning a portion of intron 4, and the entire length of exons 4, 5 and 6 was isolated. An approximately 1.2 kb bAct~rlAl neomycin phosphotransferase gene construct r nntA~n~n~ a poly-A
t~rmlnAtinn signal and the thymidine kinase promoter (from herpes simplex virus) was obtained from the plasmid pMCIneoPolA (Thomas et al., Cell, 51:503--512 [1987] ) by digestion with XhoI and SalI. The neo-poly A
insert was isolated and was then ligated into the CD4 construct previously digested with KpnI, which cuts in an internal site of exon 5 in the antisense orientation relative to the CD4 transcriptional orientation. The resulting knockout construct is depicted in Figure 2.
The construct was amplified in E coli cells and the plasmid rnntAlnln~ the construct was then purified.
The knockout construct was linearized by restriction ~n~lnnllrl~AAe digestion, and the DNA was then purified. About 5 nmol of this DNA was then .ol~rtrr,rnrated into D3 embryonic stem cells and the electroporated cells selected and screened as described in Example 1.

WO95114377 r~ ~ 117 ~\~ 5~5~ - 48 -The D3 cell line ~ ntAIn;nq the CD4 knockout construct has been deposited with The American Type Culture Collection as Accession Number C~L-11114.
After identification of ES cell3 c ~ntA~n~nq 5 the CD4/neo construct, the cells were microin~ected into 3.5 day old murine embryos; the embryos were obtained from females that had been mated with males. The embryos were then implanted into pseudopregnant foster mothers for the duration of gestation.
The offspring of these mice were evaluated for the presence of the knockout construct by probing a Southern blot of DNA obtained from tail tissue.
Example 3: cn4/rn8 Dollhle Rnock~ut ~ ce The CD4 and CD8 knockout mice described above were used to generate a mCD4-/-/mCD8-/- double knockout mouse. mCD4+/-mCD8+/+ mice were bred with mCD4+/+mCD8-/- mice to obtain mCD4+/-mCD8+/- heterozygous F1 20 offspring. The heterozygote siblings were crossed, and the offspring were screened as follows. About 20 ~Ll of blood was obtained from the tail vein of each mouse and was collected into heparinized capillary tubes. The blood was washed once in Immunof luoescence Staining 25 Buffer ("IBS", consisting of calcium and r-7n~si free phosphate buffered saline [Sambrook et al., supra], O.1%
sodium azide, and 5% fetal calf serum). Each blood sample was then ~ ncllhated with the following monoclonal antibodies (PE is phycoerythrin, and FITC is fluorescein 30 isothiocyanate):
1) PE-con~ugated anti-human CD4 (Leu3a; Becton-Dickinson Co.) 35 2) FITC--con~ugated anti--HLA DQ (LeulO; Becton Dickinson Co.) Wo 95/14377 r~ 117 ~ ~75~5~) 3) PE-con~ugated anti-mouse L3T4 (Becton-Dlckinson Co. ) 4) FITC con~ugated anti-mouse Lyt-2 (Becton-Dickinson Co.) All ; ncllh~t ~ nnc were at 4C for 20 minutes .
Red blood cells were then lysed using FACS Lysis Buffer (Becton-Dickinson Co. ) for 2 minutes, and then washed in IBS twice.
The cells were then analyzed by f low cytometric analysis using Lysis II software (Becton--Dickinson Co. ) . Wild-type and heterozygote CD4/CD8 knockout mutants were distingui3hed from each other on the basis of intensity of staining; hl -12.yyuuS CD4/CD8 knockout mutants were ia~nt ~ f ied by the complete absence of either CD4+ or CD8+ cells (based on the lack of immunofluroescence staining with the antibodies).
The freauency of offspring h~ /Zyyl~uS for the mutation on both loci was 2 percent (4 out of 200).
This is lower than the freauency expected for llnl~nk~d genes (one-s~ ~rte~onth) because both genes are located on chromosome 6 (Parnes, Adv. Immunol. 44:265 [1989]) .
Example 4: Hl~r-n ~`~4 Traneaen~ c Mn~lae The human CD4 (hCD4 ) transgenic mice were prepared as follows:
An approximately 2 . 8kb human CD4 cDNA seauence (Maddon et al., Cell, 42:93-104 [1985]) was inserted into an artificially created EcoRI site of a human CD2 promoter-regulator cassette (Greaves et al., Cell, 56:979-986 rl989]). This cassette contains about 4.8 kb of hCD2 5 ' seauence and about 5 . 5 kb of hCD2 3 ' 35 seq-~nce. Insertion of the CD4 cDNA into the CD2 cassette resulted in removal of the CD2 first exon, WO9~/14377 P~ It~117 ~ 52~ --leaving the CD2 5 ' promoter sequence and the CD2 3 ' regulatory intron sequence3 intact. The final construct as depicted ln Figure 3 i3 COpy number ~ n~ and is specifically expressed in lymphocytes, regardle3s of the 5 integration site in the genome of the transgenic mouse.
This construct was in~ected into CBA/Ca x C57BL/10 embryos using the procedures described above.
The offspring were screened for the presence of the tr~nS~enl~ construct, and those carrying the construct 10 were backcrossed to C57BL/6 mice for five g~n~ratinnc.
The offspring of the F5 generation were then bred into the CD4/CD8 double knockout mice to generate mice that are CD4/CD8 double knockouts cnnt~nlng the human CD4 trAn~n~ .
Example 5: F ~r~Tc}I DOw6 Tran~a~-n~c r~imlqe A C57B46 mouse carrying the transgenes human HLA-DQw6 alpha and HLA-DQw6 beta was prepared as 20 follows.
The gene encoding the HLA-DQw6 alpha chain was isolated from a genomic library of DNA prepared from a human B ly ,hnhl Iqtoid cell line (EB-TOK) previously transformed with EBV (Epstein-Barr virus), using lambda 25 charon 4A as a cloning vector. This cell line is h~ yy~us for the HLA--DR2--DQw6--Dw12 haplotype.
The gene was obtained as an approximately 13 kb EcoR1 fragment cnnt~tn~ns about 0.8kb of 5' sequence, the full length coding region, and about 6kb of 3 ' 30 llntr;.n~l~ted sequence.
The gene encoding the DQw6 beta chain was isolated as described by Tsukamoto et 21.
(ImnZunogenet~cs, 25:343-346 [1987] ) . An approximately 18kb EcoRI fragment of the DQw6 beta chain nucleotide 35 sequence was ligated to a EcoR~-PstI cDNA sequence WO 95/14377 ~ ~ 7~ cl,~5 ~ t ~ ~l7 .

cnnt~1n~n~ exon 6 of DQw6 beta and some 3 untranslated se~auence in order to generate a complete DQw6 beta gene.
Both the DQw6 alpha and beta genes were llnearlzed by restriction ~n~lnnllrl~Rce digestion with 5 Eco~I.
A mlxture of the DQw6 alpha and beta genes was then microin~ected into embryos of C57B4 6 mice . The embryos were implanted into 8 foster mother for the duratlon of gestation.
The pups were screened for the presence of the transgenes as follows. Tail tissue was obtained from each mouse and DNA from the tissue was prepared using the SDS-proteinase K method. The DNA was digested with BamElI and then subjected to electrophoresis on a 0.9%
15 agarose gel. Southern blot analysis was then performed to identify those pups rnntA~n~n~ the transgene.
MEICII tr~nQg--n~c mice were then backcrossed to C57BL/6 mlce for ten generatlons. The offspring of the F10 g n~rRti~n were then crossed with each other to 20 obtain h- - yy~uS DQw6+/+ transgenic mice. The h. zyy lLes were then bred with the CD4/CD8 double lcnnrkmlt mice to obtain mice that are CD4/CD8 double knockouts and contain the DQw6 alpha and beta transgenes .
Example 6: cn4-~-/cn8-~- hnow6 ht~n4 ~nuqe The CD4/CD8 double knockout mlce containlng the human CD4 transgene were bred with the CD4/CD8 30 double knockout mlce rnntR~n~n~ the DQw6 transgenes to obtain offspring that are double knockout double transgenic mice. The mice were typed for the presence of the knockout constructs and transgenes uslng the immunofluorescence staining procedures set forth above.
35 The complete breeding scheme used to generate this ~ouse is depicted in Figure 5.

WO 95114377 r~l,.. I t ~ ll7 7 5 ~5~ 52 -Example 7: Srrel~n~ ncr for Sepsic Suqceptihilitv The effect3 of SEB (Staphylococcus enterotoxin B, Sigma Chemical Co., St. Louis, MO), LPS
5 ~lipopolysaccharide, Sigma Chemical Co. ), and anti CD3 monoclonal antibody (Pharmingen, San Diego, CA) were evaluated in wild type, trAn~n~c, knockout, and double knockout, double trzmsgenic mice, all of which were between 6 and 12 weeks old. Spec1f~Ally, the following 10 mice were evaluated:
1) human CD4+, DQw6+, mouse CD4-/-, mouse CD8-/-(hCD4+, DQw6+,mCD4-/-,mCD8-/-) 15 2) human CD4+, mouse CD4-/-, mouse CD8-/-(hCD4+,mCD4--/-,mCD8--/--) 3) DQw6+, mouse CD4-/-, mouse CD8-/-(DQw6+,mCD4-/-,mCD8-/-) 4) mouse CD4-/-, mouse CD8-/-(mCD4-/-,mCD8-/-) 5) DQw6 6) wild type (C57BL/6) 7) wild type (BALB/c) Wild-type rodents are generally resistant to the effects of enterotoxins. Therefore, administration of about 20mg (in 100 ~11 PBS) of the sensitizing agent D-galactosamine (D-gal) was given to each mouse intraperitoneally about 5-15 minutes prior to in~ection of the test substance. Each test substance was administered intr~peritoneally in about lO0 ~l of PBS.

WO 9~/14377 P~ 17 217~5~

The amount of each test substance that was administered i8 indicated in Table 1.
The number of mice that died within 72 hours after exposure to SE:B or LPS is 8hown in Table 1.

Table 1:
Itou~l D- ~l D- ~l D-gal D- ~l ~2gmg) (2gmg) (20mg) (2gmg) ~SEB ~LPS ~ ~nt i - CD 3 ~2 IJ,a) ~0.1 ~I,a~ ~2.5 v,a~
hCD4+ 6/8 3/8 DQw6+
mCD4 -/-15 I~n8-/-hCD4 +
mCD4-/- 0/8 7/9 5/9 I ~n8-/-DQw6 +
mCD4-/-- 3/8 1/6 3/6 ~n8-/-25 mCD4-/- 0/7 4/8 2/8 -nB-/-DQw6+ 0/6 2/4 6/7 C57B~/6 0/8 5/8 6/7 35 BALB/c 0/14 2/5 6/8 Surprisingly, the double knockout, double 40 transgenic mice showed a very high sensitivity to SEB, a3 the lethality was very high in these mice as compared with all other genotypes evaluated.
To assess the ln vltro dose response of T
cells from various genotypes of mice to SEB, single Wo 9S/14377 1 ~ 1 ~ ~ ~17 ~5~50 cells suspensions were made from spleens harvested from 6-12 week old m$ce by passage of the splenic tissue through wire mesh. The cells were then placed into 96 well microtiter plates at a density of about l X
106/well, and were qllAr~nd.~ in about 200 Ill of Iscove ' 8 modified Dulbecco's medium (IMDM) supplemented with 10 percent heat inactivated fetal calf serum ~FCS; Hyclone Labs), 5011M beta-mercaptoethanol, 0 . 01 percent penicillin, and 0 . 01 percent streptomycin . The cells were then 8t1 lAted with decreasing concentrations of SEB, as indicated in Figure 5. After about 72 hours of incubation at 37DC and 5 percent C02, the cells were pulsed for about 16 hours with about 1 ~LCi of 3E~-thymidine. Cells were then counted for r~ oAct1vity.
The results are shown in Figure 5. Dark circles represent the double knockout, double transgenic, open circles represent the double knockout with hCD4 transgene, dark squares represent C57B46 wild type, and the open pentagonal represents the double knockout.
As can be seen, T cells of the double knockout double trAnA~c-n~ c mice were more responsive to the presence of SEB as compared with the wild type and other mutant genotypes.

Claims (8)

We Claim:

1. A non-human mammal or its progeny lacking expression of endogenous CD4 and CD8, said mammal comprising:
(a) A transgene encoding human CD4 or a biologically active fragment thereof; and (b) A transgene encoding an allele of the HLA
DQ locus or a biologically active fragment thereof.

2. The mammal of claim 1 wherein the allele of the HLA DQ locus is DQw6.

3. The mammal of claim 1 wherein each transgene is operably linked to a promoter.

4. The mammal of claim 3 wherein the promoter is selected from the group consisting of: the human CD2 promoter, the human CD4 promoter, the HLA DQw6 alpha and beta promoters, the mouse CD4 promoter, the mouse p561ck promoter, the mouse IE-alpha promoter, and the mouse H2k promoter.

5. The mammal of claim 4 wherein the transgene encoding human CD4 is operably linked to the human CD2 promoter, and the transgenes encoding HLA DQw6 alpha and beta chains are operably linked to their endogenous DQw6 alpha and beta promoters, respectively.

6. A process for preparing a mammal or its progeny comprising:
(a) suppreasing expression of endogenous CD8 in the mammal;
(b) suppressing expression of endogenous CD4 in the mammal;

(c) inserting a transgene encoding an allele of the HLA DQ locus or a biologically active fragment hereof into the mammal; and (d) inserting a transgene encoding human CD4 or a biologically active fragment thereof into the mammal.

7. The process of claim 6 wherein the allele of the HLA DQ locus is DQw6.

8. A method of evaluating a therapeutic regimen for its anti-sepsis effect, comprising:
(a) administering to the mammal of claim 1 a therapeutic regimen, wherein the mammal is previously, simultaneously, or subsequently exposed to a compound and/or organism that has the capacity to induce sepsis;
and (b) screening the mammal for sepsis or sepsis-like symptoms.