Immunology Today, vol. 7, No. 5, 1986
-revlew$
Transgenicmice: 'new wave' immunogenetics It is now possible to investigate the function of cloned genes in vivo by injecting the genes into mouse embryos and tracing the pattern of expression of the genes' products. In this review Christophe Benoist and his colleagues discuss the technique of gene transfer, its power and limitations, and its particular application in the study of immune function. During the past few years, the genes for,many of the protein products of lymphocytes have been cloned: the immunoglobulins (Igs), the T-cell antigen receptor and adjunct molecules, various lymphokines, and class I, II and Ill molecules of the major histocompatibility complex (MHC). Rapidly accumulated sequence information has provided a preliminary understanding of the structure of these proteins as well as some hints about their function. Other important clues to their mode of action derive from experiments in which cloned DNA, wild-type or mutated, has been transfected into tissue culture cells and various functional parameters assayed. However, these in-vitro experiments can give only a limited perspective of immune function. Since the immune response involves the whole organism, the introduction of manipulated genes into a whole organism is required for a fuller understanding of their role(s) within the immune system. This goal has recently become realizable1. Several groups have injected cloned genes into fertilized mouse eggs, reimplanted the embryos into foster mothers, and analysed the resultant progeny. In a certain percentage of the offspring, termed 'transgenic' mice, the injected DNA is incorporated into the genome and can be transmitted to future generations in a Mendelian fashion. In many, although not all, transgenic mice, the incorporated gene is expressed accurately and with tissue - or developmental stage - specificity. The list of injected genes has increased rapidly over the past year and now includes globins 2 4 , elastase5 , growth 9 hormone 6~, transferrin, myosin 10 , e-feto-protein 11 , oncogenesl2 17. Here, we will focus attention on those transgenic mice which have incorporated genes whose products function in the immune system.
How (and how well) does the technique work? The ability to introduce genes into the germline of mice depends initially on skillful egg manipulation and micro-injection by the experimenter. Female mice are hormonally super~vulated and then mated. About 12 h after fertilization one-cell eggs are flushed from the oviduct, treated with hyaluronidase to remove cumulus cells, and placed in a drop of cytocholasin B, to make them more adherent. Eggs are held in place by a blunt holding pipette while an injecting .needle is inserted into one of the two pronuclei, preferably the male. As the solution of DNA is discharged, the pronucleus swells,
138
Unite 184 de BiologieMol~culaireet de 5~nie G#n#tiquede I'INSERM, Laboratoirede G#n~tiqueMol#culairedesEucaryotesdu CNRS,Institut de ChimieBiologique,Facultede M#decine, 67085StrasbourgCedex, France. ~) 1986, Elsevier Science Publishers B.V., Amsterdam
0167 - 4919/86/$02.00
ChristopheBenoist,Pierre Gerlinger, Marianne LeMeur and DianeMathis indicating successful injection. About 30 intact injected eggs are reimplanted in the oviduct of a pseudopregnant mother and some give rise to viable offspring. To test for the presence of the injected gene, a portion of each animal's tail is excised and the DNA is extracted, dotted onto a filter, and hybridized with the appropriate probe. Under optimum conditions, 10-20% of the reimplanted eggs give rise to pups and 10-30% of these are transgenic. Some factors known to affect these percentages are experimental dexterity, DNA concentration, DNA form, and choice of mouse strain 18. The importance of strain choice was highlighted in a recent experiment that directly compared the efficiency of DNA integration after injection into (C57 BI/6 x SJL) hybrids versus inbred C57 BI/6 mice. Several parameters were affected (e.g. number of eggs per donor, percentage of eggs surviving injection) resulting in an overall 8-fold higher efficiency for the hybrids. The ability to develop stable lines from the original transgenic mouse depends on at least three variables: the embryonic stage at which the injected DNA was incorporated into the chromosome, the number of integration loci, and the actual sites of integration. If incorporation occurs in the one-cell stage embryo, all cells of the transgenic mouse will contain the injected gene; however, if integration takes place after the onset of cleavage, the mouse will be a mosaic. In mosaic mice, germ-line cells may not contain the foreign gene and hence it will not be transmitted to progeny. The second factor reflects simple Mendelian genetics. Transgenic mice often have many copies per cell of the injected gene, which usually reside at a single locus as a tandem, head-to-tail array. When the transgenic mouse is backcrossed with a normal mouse, the foreign DNA is inherited by about 50% of the offspring. However, examples exist of multiple integration sites in a single mouse, and in such cases, more than half the pups inherit the foreign gene. Thirdly, the actual site of integration can affect inheritance in rare cases. In particular, incorporation of foreign DNA into the X or Y chromosome may cause sterility due to insertional mutagenesis. These three variables are essentially uncontrollable with techniques available at present. It is also largely impossible to predict whether the foreign DNA will be expressed in a tissue - and developmental stage specific fashion. Certain genes, like elastase5, work very well in transgenic mice, exhibiting highly specific patterns of expression; others, the classic example being 6-globin, have proved to be more problematic (Ref. 2-4 and references therein). Factors that influence correct expression are multiple and poorly understood. The number of integrated copies may have some influence but, as yet, there has emerged no clear relationship between copy number and levels of trans-
Immunology Today, vol. 7, No. 5, 1986
cription. It is quite possible that any such effect varies according to the gene, depending on cellular concentrations of positive or negative regulatory molecules. Certainly, the actual site of integration can be important, though some genes seem to be less influenced by this position effect than others. One might surmize in such cases that the presence of strong tissue-specific transcription factors can override the position effect. Finally, DNA sequences adjacent but extraneous to the injected gene can greatly influence the pattern of expression in transgenics. Contiguous prokaryotic vector sequences can inhibit or completely abolish transcription from some genes (e.g. globin 2-4, c~-fetoprotein11), while others appear to be not or much less affected (e.g. Ref. 19). Perhaps more comprehensible, coinjected eukaryotic sequences that bear regulatory elements can also modify transcription patterns2°but, surprisingly, the effect is not always predictable 21. Expression may occur reproducibly in a tissue where neither the gene of interest nor the extrinsic promoter are normally active. To conclude, it is not possible with existing protocols to control either the pattern of inheritance or the specificity of transcription of a foreign gene in transgenic mice. Nevertheless, transgenics have already proven a valuable system in which to answer questions of interest to immunologists.
Ig gene transgenic mice Igs are synthesized only in lymphoid tissue. This specificity depends on the regulation of two chromosomal events: DNA rearrangement and RNA transcription (see Ref. 22 for review). In most somatic cells and in the germline, the Ig coding sequences are dispersed over vast lengths of DNA; discrete chromosome segments must be brought into juxtaposition to create a functional gene. These rearrangements are precisely programmed throughout B-cell differentiation (although certain events occur in some T cells). First, the heavy (H) chain gene is assembled by joining DH to JH-Cu and then VH to DH--JH--CH. Subsequently, functional light (L) chain genes are constructed by joining VL to JL--CL. Concomitant with, and to some extent a function of, the assembly of a correctly rearranged gene, the Ig promoter becomes transcriptionally competent. Levels of lg synthesis increase after appropriate antigen stimulation, reaching enormous levels in terminally differentiated plasma cells. Transgenic mice may provide a unique opportunity to examine some of the mechanisms involved in regulating Ig gene rearrangement and transcription. To date three types of Ig transgenics have been produced: 1. A functional, rearranged K gene from the MOPC 21 myeloma has been injected into (C57 BI/6 x SJL)F2 hybrids 23-26. 2. A rearranged 1~ heavy chain gene coding for a nitrophenyl (NP) hapten specificity normally absent from C57 BI/6 mice was injected into that strain 27- 28 . 3. Rearranged K and I~ genes coding for a trinitrophenyl (TNP) hapten specificity and carried on the same plasmid molecule were injected into Swiss albino mice 29. In all three cases, transgenic lines were developed that had highly raised serum concentrations of antibody encoded by the injected gene. Of prime importance was to determine whether the foreign Ig genes are subject to correct transcriptional regulation. For both K and I~, significant mRNA accumu-
.re¢iewslation was limited essentially to lymphoid tissue, although low-level transcripts were detected in a few other organs 24'27. A marked cell-type specificity was also evident: the foreign K gene was active almost exclusively in B cells (as is the endogenous analogue)24; F transcripts were made primarily in B cells, but also in some T cells (as are endogenous I~ transcripts) 27. Lastly, injected Ig genes could exhibit regulated expression during B-cell differentiation. Just like its unrearranged endogenous counterpart, the rearranged foreign K gene was not active in pre-B cells (unless it was present at high copy number) z6. In short, the transcriptional fidelity of foreign Ig genes is among the best yet observed for an injected DNA. There seems to be little line to line variation with a given construct, suggesting that chromosome position effects are dampened. This pronounced efficacity could well be the result of a strong tissue-specific enhancer, but other promoter components might play a role (see Ref. 22 for references). No doubt the appropriate enhancerless and hybrid gene constructs have already been injected in order to study this question. Various aspects of the control of DNA rearrangement in lymphocytes can be studied to advantage in transgenic mice injected with pre-rearranged Ig genes. The mechanism of 'allelic exclusion' has been the first topic to be so addressed. B cells have a monospecific receptor for antigen -i.e. each Ig-producing cell has only one heavy chain and one light chain allele that is functional. The others are 'excluded' from Ig synthesis, either because they are not rearranged or because they are aberrantly rearranged. Until now it has not been possible to decide between two mechanisms for allelic exclusion: the stochastic model postulated that because of the high probability of aberrant rearrangement, only one functional allele per cell was likely to be constructed; the feedback model proposed that the creation of a functional Ig somehow inhibited subsequent rearrangement at allelic loci. Transgenic mouse studies have provided support for the feedback model of allelic exclusion for the Ig heavy chain. H chain genes are not found in the germline configuration in Abelson murine leukemia virus transformed pre-B lines from normal mice. However, when derived from transgenic mice injected with a prerearranged i~ gene, 40% of the pre-B lines had endogenous H chain DNA of germline pattern 28. Thus, the presence of a functional i~ gene seemed to repress H chain rearrangement in pre-B cells. Endogenous H chain genes in the germline configuration were likewise observed in more mature splenic B-cell hybridomas from I~ transgenic mice 28'29. Somewhat enigmatically, the percentage of B-cell lines exhibiting non-rearranged H chain loci (10%) appeared lower than the percentage of pre-B lines (40%) 28. The significance of this observation is uncertain, but the discrepancy may represent a selection for cells in which the pre-rearranged injected gene is poorly expressed, allowing for rearrangement of endogenous H chain loci and thus expansion of the antibody repertoire. It may be worth noting that Ig gene expression in transgenics is subject to influences not operative in other systems, such as selective amplification of a subset of B-cell clones and T-cell suppression. Somewhat contradictory results have been reported about light chain attelic exclusion. The pre-rearranged MOPC 21 K gene clearly curtailed L chain rearrangements in splenic B-cell hybridomas from transgenic
139
-re ,iews mice 25. Analysis of secreted and intracellular Ig protein from individual hybridomas revealed that the crucial event was the accumulation of complete Ig complexes consisting of associated H and L chains. When the MOPC 21 K chain participated in an H: L chain complex, the endogenous K chain genes remained in the germline configuration. In contrast, no inhibition of rearrangement was detected in splenic B-cell hybridomas from transgenics injected simultaneously with H and L chain genes coding for a TNP hapten specificity 29. It should be noted, however, that in these mice K protein levels were only Vlo those of the corresponding i~ chain. Allelic exclusion of L chain genes could require a critical concentration of functional Ig complex or a balanced ratio of H to L chain protein, tt is also possible that when injected on a plasmid that also contains an H chain gene (and the SV40 enhancer), the K genes are not subject to correct regulation during differentiation, and allelic exclusion is precluded as a consequence. Because it has proved so difficult to establish in-vitro B-cell differentiation systems, the transgenic mouse should be invaluable for future studies of Ig expression. More precise information about the mechanism(s)- of allelic exclusion may come from experiments on transgenic mice injected with appropriately engineered Ig genes. The processes involved in somatic mutation and heavy chain class switching may be similarly amenable to analysis.
140
MHC gene transgenic mice Many of the genes residing within the MHC have recently been cloned and sequenced. Subsequently, several were reintroduced into cells in culture in order to dissect structure/function relationships. Although important information may be gleaned from this type of experiment, there are limitations on the types of questions that can be posed. For example, the influence of particular MHC genes on the induction of self tolerance, the composition of the T-cell repertoire, responder versus non-responder distinctions and the maturation of the immune response must be analysed by other means. Transgenic mice may prove ideal in this regard. An excellent model system for evaluating MHC Class II gene function in transgenic mice is provided by strains of the H-2 b and H-25 haplotype 3°,31. These mice do not express their endogenous Ec~ gene because it has a 627 base pair deletion that removes the promoter region and first exon. They do not, then, exhibit an Ec~:El3 cell surface complex and consequently are unable to mount an immune response to certain foreign antiqens, e.g. GLPhe and pigeon cytochrome C. Can H-2 b or H-2 s haplotype mice be endowed with a new immune response pattern by developing transgenic lines harboring a functional E~ gene? Three groups have attempted to answer this question by injecting EeLk into (C57 BI/6 x SJL)F2 mice 19, E~d into C57 BI/6 32 or E~ d into (C57 BI/6 x SJL)F2 mice 33. As a prelude to immunological studies an assessment of the transcriptional fidelity of the injected Ee gene was made. In normal mice,.E~ is synthesized predominantly in lymphoid tissue: in B cells, in thymic epithelial and dendritic cells, and in macrophages stimulated by gamma interferon (~y-IFN) (see Ref. 34 for review): Class It molecules are also occasionally detected on specialized cells in non-lymphoid tissue, such as brain astrocytes. The foreign Ec, gene in transgenic mice exhibited a transcriptional
Immunology Today, voL ~ No.& 1986
specificity which mimicked quite closely the endogenous gene norm. That is, Ec~ mRNA accumulated in spleen and thymic tissue, but was usually present at low or undetectable levels in heart, kidney, liver, muscle, brain and lung, except when these tissues were contaminated by blood19'32'33. Cell=type specificity of expression was demonstrated by three methods: first, two-dimensional cytofluorometric analysis showed that essentially all cells which made Ae, AI3 and El3 also synthesized Eo~19. Second, two color fluorescent staining of tissue sections revealed that Ac~ and Ee protein occurred coincidentally in the thymus (C. Benoist eta/., unpublished results). Third, separation of splenic B and T cells by 'panning' resulted in a parallel depletion of foreign Ec~ gene and endogenous Ac~ gene transcripts in the T-cell fraction (D. Mathis et al., unpublished results). As expected, transcription from the injected gene could be induced by ~/-IFN in peritoneal macrophages 19,33. Surprisingly, though, macrophages from one transgenic line showed constitutive expression of cell surface Ee:El3 which was not further increased by ~/-IFN treatment 32. The authors attributed this aberration to the high copy number of the injected Ec~ gene, but it remains puzzling that El3 synthesis was also constitutive. The remarkable specificity of transcription from the injected gene provoked expectations that the Ec~ transgenics would be endowed with new immune response capabilities. Indeed, spleen cells from Ec~transgenics, but not from parental H-2 bx5 or H-2 b controls, could act as antigen-presenting cells for two TH hybridomas specific for a herpes simplex glycopeptide in the context of I-E 33. Secondly, after priming with GLPhe, the E~ transgenics yielded lymph node cells that proliferated in response to this polypeptide antigen, whereas lymph nodes from the H-2 ° haplotype controls were unreactive 32. Finally, as the ultimate test of immune function, the blood of E~ transgenics was titred for anti-GLPhe antibodies after a secondary antigen challenge 19. These mice made a healthy response to GLPhe injection, while H-2 bxs haplotype controls were not stimulated. Thus, by all criteria tested, the foreign Ec~ gene product in transgenic mice was immunologically functional. Similar success has been achieved with MHC Class I gene transgenic mice (Ref. 35, and D. Singer, pers. commun.). The porcine MHC gene PD1 was injected into C57 BIll0 SCN embryos and a transgenic line with several integrated copies developed. In general, the transcription pattern in transgenic mice closely paralleled that in swine. That is, PD1 synthesis was highest in lymphoid tissues and moderate in others, except for the brain where it was essentially undetectable (a few differences did occur, however). The pig MHC molecule could act as a transplantation antigen since skin from the transgenic line was rejected by the C57 BIll0 parental strain. Not surprisingly, then, the porcine antigen could be recognized by cytotoxic T lymphocytes (CTL). The strength of the CTL response was significantly less than in an allo-reaction, and the number of anti-PDI precursors was ten-fold lower than the number of precursors with allo-specificity, although this was still higher than expected for antigen-specific MHC-restricted CTLs. In preliminary studies of the T-cell repertoire in PDI transgenic mice, the interesting observation was made that the xeno-specific CTLs exhibit unexpectedly high crossreactivities with some murine Kb mutants. No explanation for this phenomenon comes from scanning the
ImmunologyToday, voL 7, No. 5, 1986
protein sequences, and its significance remains a mystery. In summary, transgenic mice harboring foreign MHC genes exhibit new immunological reactivities. No doubt these animals will be useful for studying the immune response in toto. In addition, the marked transcriptional fidelity of the injected genes may prompt experiments designed to pinpoint promoter control elements. Perspectives
Transgenic mice will no doubt have an important future in immunological research. The study of gene expression in lymphoid cells will be greatly facilitated, the benefits being analogous to those already realized with genes such as globin and elastase. However, there does exist a set of questions which is more specifically immunological. For some of these, transgenic mice will extend or improve on pre-existing experimental systems that include congenic mice and irradiation chimeras; for other questions entirely new approaches may be envisioned. We list below several types of experiment in which transgenic mice might be used to advantage. The list is not exhaustive and continues to evolve. Identifying the function of certain proteins: The injection of allelic variants, mutant genes, or 'antisense' transcripts will help in elucidating the role of a number of gene products. Consequently, cell-surface molecules such as the Qa and Lyt series might attain a more noble status than mere 'differentiation antigens'. Creating new mouse lines: Congenic lines have been important experimental material for immunologists of varied interests. In a sense, transgenic animals represent 'perfect congenics': cloned genetic material is introduced into a perfectly defined background. Thus, problems like those presently arising over the interpretation of I-J should be avoidable 36 37. Verifying genetically mapped loci: The injection of large chromosomal segments might help to reconcile molecular biologists and immunologists in cases where molecular mapping has so far failed to clarify the basis of known immunological phenomenology. I-J and the T locus are obvious candidates 37'38. Establishing new cell lines: The injection of oncogenes under the control of various promoters has enabled the development of mouse lines harboring tissues that can be immortalized in culture 13'17. The hope is that this phenomenon will also be true of the immune system, allowing one to establish, relatively painlessly, lines of desired cell type and antigen specificity. Provoking aberrant gene expression: An interesting type of transgenic mouse might result from completely aberrant gene expression. What happens to an la ÷, slg ÷ liver cell? What happens to a mouse with heart cells expressing an MHC molecule of a haplotype different from that of its thymus? Thus novel approaches may emerge to study tolerance and suppression. Creating murine disease models: New disease-prone lines can be generated; alternatively gene therapy can be attempted on pre-existing models. In this respect, an important first step has already been accomplished by curing the C57 BI/6 mouse of its susceptibility to the major murine pathogen, pigeon cytochrome C 19"32'33,
We thank Drs D. Baltimore, R. Brinster and D. Singer for sending preprints of papers.
References
1 Brinster, R.L. and Palmiter, R.D. (I 986) Harvey Lectures80 (in press) Alan R. LissInc. New Jersey 2 Townes, T.M., Lingrel, J.B., Chen, H.Y. etal. (1985) EMBOJ. 4, 1715 1723 3 Chada, K., Magram, J., Raphael, K. etaL (1985)Nature (London) 314, 377-380 4 Magram, J., Chada, K. and Costantini, F. (1985)Nature (London) 315, 338-340 5 Swift, G.H., Hammer, R.E., McDonald, R.J.etaL (1984) Cell 38, 639-646 6 Palmiter, R.D., Brinster, R.L., Hammer, R.E.etaL (1982) Nature (London) 300, 611-615 7 Hammer, R.E., Palmiter, R.D. and Brinster, R.L. (1984) Nature (London) 311,65-67 80rnitz, D.M., Palmiter, R.D., Hammer, R.E.etal. (1985) Nature (London) 313, 600-602 9 McKnight, G.S., Hammer, R.E., Kuenzel, E.A. etaL (1983) Cell 34, 335-341 10 Shani, M. (1985) Nature (London) 314, 283-286 11 Krumlauf, R., Hammer, R.E.,Tilghman, S.M. etal. (1985) Mol. Cell. BioL 5, 1639-1648 12 Stewart, T.A. Pattengale, P.K. and Leder, P. (1984) Ce1138, 627-637 13 Brinster, R.L., Chen, H.Y., Messing, A. etaL (1984) Ce1137, 367-379 14 Palmiter, R.D., Chen, H.Y., Messing, A. et al. (1985)Nature (London) 316, 457-460 15 Messing, A., Chen, H.Y., Palmiter, R.D. et aL (1985)Nature (London) 316, 461-463 16 Hanahan, D. (1985) Nature (London) 315, 115 122 17 Small, J.A., Blair, D.G., Showalter, S.D. etal. (1985) Mol. Cell. Biol. 5, 642±648 18 Brinster, R.L., Chen, H.Y., Trumbauer, ME. etaL (1985) Proc Natl Acad. Sci. USA 82, 4438-4442 lg LeMeur, M., Gerlinger, P., Benoist, C. etal. (1985)Nature (London) 316, 38-42 20 Townes, T.M., Chen, H.Y., Lingrel, J.B. etaL (1985) Mol. Cell. BioL 5, 1977-1983 21 Swanson, L.W., Simmons, D.M., Arriza, J. etal. (1985) Nature (London) 317, 363 366 22 Calame, K.L. (1985)Ann. Rev. ImmunoL 3, 159-198 23 Brinster, R.L., Ritchie, K.A., Hammer, R.E.etal. (1983) Nature (London) 306, 332-336 24 Storb, U., O'Brien, R.L., McMullen, M.D. etaL (1984)Nature (London) 310, 238-241 25 Ritchie, K.A., Brinster, R.L. and Storb, U. (1984)Nature (London) 312, 517-520 26 Storb, U., Denis, K., Brinster, R.L. etal. (1985)Nature (London) 316, 356-358 27 Grosschedl, R., Weaver, D., Baltimore, D. etal. (1984)Cell 38, 647-658 28 Weaver, D., Costantini, F., Imanishi~Kari,T. etaL (1985) Cell 42, 117-127 29 Rusconi,S. and K~hler, G. (1985) Nature (London) 314, 330-334 30 Mathis, D.J., Benoist, C., Williams, V.E. etal. (1983) Proc. NatlAcad. Sci. USA 80, 273-277 31 Dembic, Z., Ayane, M., Klein, J. etal. (1985) EMBOJ. 4, 127-131 32 Yamamura, K.I., Kikutani, H., Folsom, V. etal. (1985)Nature (London) 316, 67-69 33 Pinkert, C.A., Widera, G., Cowing, C. etaL (1985)EMBOJ. 4, 2225-2230 34 Mengle-Gaw, L. and McDevitt, H.O. (1985)Ann. Rev. ImmunoL 3,367-396 35 Frels,W.I., Bluestone, J.A., Hodes, R.J.eta/. (1985)Science 228, 577-580 36 Hayes,C.E., Klyczek, K.K., Krum, D.P. etaL (1984)Science 223, 559 563 37 Murphy, D.B. (1985)J. Immunol. 135, 1-5 38 Frischauf,A.M. (1985) Trends Genetics 1,100-103
141
-revlew$
Transgenicmice: 'new wave' immunogenetics It is now possible to investigate the function of cloned genes in vivo by injecting the genes into mouse embryos and tracing the pattern of expression of the genes' products. In this review Christophe Benoist and his colleagues discuss the technique of gene transfer, its power and limitations, and its particular application in the study of immune function. During the past few years, the genes for,many of the protein products of lymphocytes have been cloned: the immunoglobulins (Igs), the T-cell antigen receptor and adjunct molecules, various lymphokines, and class I, II and Ill molecules of the major histocompatibility complex (MHC). Rapidly accumulated sequence information has provided a preliminary understanding of the structure of these proteins as well as some hints about their function. Other important clues to their mode of action derive from experiments in which cloned DNA, wild-type or mutated, has been transfected into tissue culture cells and various functional parameters assayed. However, these in-vitro experiments can give only a limited perspective of immune function. Since the immune response involves the whole organism, the introduction of manipulated genes into a whole organism is required for a fuller understanding of their role(s) within the immune system. This goal has recently become realizable1. Several groups have injected cloned genes into fertilized mouse eggs, reimplanted the embryos into foster mothers, and analysed the resultant progeny. In a certain percentage of the offspring, termed 'transgenic' mice, the injected DNA is incorporated into the genome and can be transmitted to future generations in a Mendelian fashion. In many, although not all, transgenic mice, the incorporated gene is expressed accurately and with tissue - or developmental stage - specificity. The list of injected genes has increased rapidly over the past year and now includes globins 2 4 , elastase5 , growth 9 hormone 6~, transferrin, myosin 10 , e-feto-protein 11 , oncogenesl2 17. Here, we will focus attention on those transgenic mice which have incorporated genes whose products function in the immune system.
How (and how well) does the technique work? The ability to introduce genes into the germline of mice depends initially on skillful egg manipulation and micro-injection by the experimenter. Female mice are hormonally super~vulated and then mated. About 12 h after fertilization one-cell eggs are flushed from the oviduct, treated with hyaluronidase to remove cumulus cells, and placed in a drop of cytocholasin B, to make them more adherent. Eggs are held in place by a blunt holding pipette while an injecting .needle is inserted into one of the two pronuclei, preferably the male. As the solution of DNA is discharged, the pronucleus swells,
138
Unite 184 de BiologieMol~culaireet de 5~nie G#n#tiquede I'INSERM, Laboratoirede G#n~tiqueMol#culairedesEucaryotesdu CNRS,Institut de ChimieBiologique,Facultede M#decine, 67085StrasbourgCedex, France. ~) 1986, Elsevier Science Publishers B.V., Amsterdam
0167 - 4919/86/$02.00
ChristopheBenoist,Pierre Gerlinger, Marianne LeMeur and DianeMathis indicating successful injection. About 30 intact injected eggs are reimplanted in the oviduct of a pseudopregnant mother and some give rise to viable offspring. To test for the presence of the injected gene, a portion of each animal's tail is excised and the DNA is extracted, dotted onto a filter, and hybridized with the appropriate probe. Under optimum conditions, 10-20% of the reimplanted eggs give rise to pups and 10-30% of these are transgenic. Some factors known to affect these percentages are experimental dexterity, DNA concentration, DNA form, and choice of mouse strain 18. The importance of strain choice was highlighted in a recent experiment that directly compared the efficiency of DNA integration after injection into (C57 BI/6 x SJL) hybrids versus inbred C57 BI/6 mice. Several parameters were affected (e.g. number of eggs per donor, percentage of eggs surviving injection) resulting in an overall 8-fold higher efficiency for the hybrids. The ability to develop stable lines from the original transgenic mouse depends on at least three variables: the embryonic stage at which the injected DNA was incorporated into the chromosome, the number of integration loci, and the actual sites of integration. If incorporation occurs in the one-cell stage embryo, all cells of the transgenic mouse will contain the injected gene; however, if integration takes place after the onset of cleavage, the mouse will be a mosaic. In mosaic mice, germ-line cells may not contain the foreign gene and hence it will not be transmitted to progeny. The second factor reflects simple Mendelian genetics. Transgenic mice often have many copies per cell of the injected gene, which usually reside at a single locus as a tandem, head-to-tail array. When the transgenic mouse is backcrossed with a normal mouse, the foreign DNA is inherited by about 50% of the offspring. However, examples exist of multiple integration sites in a single mouse, and in such cases, more than half the pups inherit the foreign gene. Thirdly, the actual site of integration can affect inheritance in rare cases. In particular, incorporation of foreign DNA into the X or Y chromosome may cause sterility due to insertional mutagenesis. These three variables are essentially uncontrollable with techniques available at present. It is also largely impossible to predict whether the foreign DNA will be expressed in a tissue - and developmental stage specific fashion. Certain genes, like elastase5, work very well in transgenic mice, exhibiting highly specific patterns of expression; others, the classic example being 6-globin, have proved to be more problematic (Ref. 2-4 and references therein). Factors that influence correct expression are multiple and poorly understood. The number of integrated copies may have some influence but, as yet, there has emerged no clear relationship between copy number and levels of trans-
Immunology Today, vol. 7, No. 5, 1986
cription. It is quite possible that any such effect varies according to the gene, depending on cellular concentrations of positive or negative regulatory molecules. Certainly, the actual site of integration can be important, though some genes seem to be less influenced by this position effect than others. One might surmize in such cases that the presence of strong tissue-specific transcription factors can override the position effect. Finally, DNA sequences adjacent but extraneous to the injected gene can greatly influence the pattern of expression in transgenics. Contiguous prokaryotic vector sequences can inhibit or completely abolish transcription from some genes (e.g. globin 2-4, c~-fetoprotein11), while others appear to be not or much less affected (e.g. Ref. 19). Perhaps more comprehensible, coinjected eukaryotic sequences that bear regulatory elements can also modify transcription patterns2°but, surprisingly, the effect is not always predictable 21. Expression may occur reproducibly in a tissue where neither the gene of interest nor the extrinsic promoter are normally active. To conclude, it is not possible with existing protocols to control either the pattern of inheritance or the specificity of transcription of a foreign gene in transgenic mice. Nevertheless, transgenics have already proven a valuable system in which to answer questions of interest to immunologists.
Ig gene transgenic mice Igs are synthesized only in lymphoid tissue. This specificity depends on the regulation of two chromosomal events: DNA rearrangement and RNA transcription (see Ref. 22 for review). In most somatic cells and in the germline, the Ig coding sequences are dispersed over vast lengths of DNA; discrete chromosome segments must be brought into juxtaposition to create a functional gene. These rearrangements are precisely programmed throughout B-cell differentiation (although certain events occur in some T cells). First, the heavy (H) chain gene is assembled by joining DH to JH-Cu and then VH to DH--JH--CH. Subsequently, functional light (L) chain genes are constructed by joining VL to JL--CL. Concomitant with, and to some extent a function of, the assembly of a correctly rearranged gene, the Ig promoter becomes transcriptionally competent. Levels of lg synthesis increase after appropriate antigen stimulation, reaching enormous levels in terminally differentiated plasma cells. Transgenic mice may provide a unique opportunity to examine some of the mechanisms involved in regulating Ig gene rearrangement and transcription. To date three types of Ig transgenics have been produced: 1. A functional, rearranged K gene from the MOPC 21 myeloma has been injected into (C57 BI/6 x SJL)F2 hybrids 23-26. 2. A rearranged 1~ heavy chain gene coding for a nitrophenyl (NP) hapten specificity normally absent from C57 BI/6 mice was injected into that strain 27- 28 . 3. Rearranged K and I~ genes coding for a trinitrophenyl (TNP) hapten specificity and carried on the same plasmid molecule were injected into Swiss albino mice 29. In all three cases, transgenic lines were developed that had highly raised serum concentrations of antibody encoded by the injected gene. Of prime importance was to determine whether the foreign Ig genes are subject to correct transcriptional regulation. For both K and I~, significant mRNA accumu-
.re¢iewslation was limited essentially to lymphoid tissue, although low-level transcripts were detected in a few other organs 24'27. A marked cell-type specificity was also evident: the foreign K gene was active almost exclusively in B cells (as is the endogenous analogue)24; F transcripts were made primarily in B cells, but also in some T cells (as are endogenous I~ transcripts) 27. Lastly, injected Ig genes could exhibit regulated expression during B-cell differentiation. Just like its unrearranged endogenous counterpart, the rearranged foreign K gene was not active in pre-B cells (unless it was present at high copy number) z6. In short, the transcriptional fidelity of foreign Ig genes is among the best yet observed for an injected DNA. There seems to be little line to line variation with a given construct, suggesting that chromosome position effects are dampened. This pronounced efficacity could well be the result of a strong tissue-specific enhancer, but other promoter components might play a role (see Ref. 22 for references). No doubt the appropriate enhancerless and hybrid gene constructs have already been injected in order to study this question. Various aspects of the control of DNA rearrangement in lymphocytes can be studied to advantage in transgenic mice injected with pre-rearranged Ig genes. The mechanism of 'allelic exclusion' has been the first topic to be so addressed. B cells have a monospecific receptor for antigen -i.e. each Ig-producing cell has only one heavy chain and one light chain allele that is functional. The others are 'excluded' from Ig synthesis, either because they are not rearranged or because they are aberrantly rearranged. Until now it has not been possible to decide between two mechanisms for allelic exclusion: the stochastic model postulated that because of the high probability of aberrant rearrangement, only one functional allele per cell was likely to be constructed; the feedback model proposed that the creation of a functional Ig somehow inhibited subsequent rearrangement at allelic loci. Transgenic mouse studies have provided support for the feedback model of allelic exclusion for the Ig heavy chain. H chain genes are not found in the germline configuration in Abelson murine leukemia virus transformed pre-B lines from normal mice. However, when derived from transgenic mice injected with a prerearranged i~ gene, 40% of the pre-B lines had endogenous H chain DNA of germline pattern 28. Thus, the presence of a functional i~ gene seemed to repress H chain rearrangement in pre-B cells. Endogenous H chain genes in the germline configuration were likewise observed in more mature splenic B-cell hybridomas from I~ transgenic mice 28'29. Somewhat enigmatically, the percentage of B-cell lines exhibiting non-rearranged H chain loci (10%) appeared lower than the percentage of pre-B lines (40%) 28. The significance of this observation is uncertain, but the discrepancy may represent a selection for cells in which the pre-rearranged injected gene is poorly expressed, allowing for rearrangement of endogenous H chain loci and thus expansion of the antibody repertoire. It may be worth noting that Ig gene expression in transgenics is subject to influences not operative in other systems, such as selective amplification of a subset of B-cell clones and T-cell suppression. Somewhat contradictory results have been reported about light chain attelic exclusion. The pre-rearranged MOPC 21 K gene clearly curtailed L chain rearrangements in splenic B-cell hybridomas from transgenic
139
-re ,iews mice 25. Analysis of secreted and intracellular Ig protein from individual hybridomas revealed that the crucial event was the accumulation of complete Ig complexes consisting of associated H and L chains. When the MOPC 21 K chain participated in an H: L chain complex, the endogenous K chain genes remained in the germline configuration. In contrast, no inhibition of rearrangement was detected in splenic B-cell hybridomas from transgenics injected simultaneously with H and L chain genes coding for a TNP hapten specificity 29. It should be noted, however, that in these mice K protein levels were only Vlo those of the corresponding i~ chain. Allelic exclusion of L chain genes could require a critical concentration of functional Ig complex or a balanced ratio of H to L chain protein, tt is also possible that when injected on a plasmid that also contains an H chain gene (and the SV40 enhancer), the K genes are not subject to correct regulation during differentiation, and allelic exclusion is precluded as a consequence. Because it has proved so difficult to establish in-vitro B-cell differentiation systems, the transgenic mouse should be invaluable for future studies of Ig expression. More precise information about the mechanism(s)- of allelic exclusion may come from experiments on transgenic mice injected with appropriately engineered Ig genes. The processes involved in somatic mutation and heavy chain class switching may be similarly amenable to analysis.
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MHC gene transgenic mice Many of the genes residing within the MHC have recently been cloned and sequenced. Subsequently, several were reintroduced into cells in culture in order to dissect structure/function relationships. Although important information may be gleaned from this type of experiment, there are limitations on the types of questions that can be posed. For example, the influence of particular MHC genes on the induction of self tolerance, the composition of the T-cell repertoire, responder versus non-responder distinctions and the maturation of the immune response must be analysed by other means. Transgenic mice may prove ideal in this regard. An excellent model system for evaluating MHC Class II gene function in transgenic mice is provided by strains of the H-2 b and H-25 haplotype 3°,31. These mice do not express their endogenous Ec~ gene because it has a 627 base pair deletion that removes the promoter region and first exon. They do not, then, exhibit an Ec~:El3 cell surface complex and consequently are unable to mount an immune response to certain foreign antiqens, e.g. GLPhe and pigeon cytochrome C. Can H-2 b or H-2 s haplotype mice be endowed with a new immune response pattern by developing transgenic lines harboring a functional E~ gene? Three groups have attempted to answer this question by injecting EeLk into (C57 BI/6 x SJL)F2 mice 19, E~d into C57 BI/6 32 or E~ d into (C57 BI/6 x SJL)F2 mice 33. As a prelude to immunological studies an assessment of the transcriptional fidelity of the injected Ee gene was made. In normal mice,.E~ is synthesized predominantly in lymphoid tissue: in B cells, in thymic epithelial and dendritic cells, and in macrophages stimulated by gamma interferon (~y-IFN) (see Ref. 34 for review): Class It molecules are also occasionally detected on specialized cells in non-lymphoid tissue, such as brain astrocytes. The foreign Ec, gene in transgenic mice exhibited a transcriptional
Immunology Today, voL ~ No.& 1986
specificity which mimicked quite closely the endogenous gene norm. That is, Ec~ mRNA accumulated in spleen and thymic tissue, but was usually present at low or undetectable levels in heart, kidney, liver, muscle, brain and lung, except when these tissues were contaminated by blood19'32'33. Cell=type specificity of expression was demonstrated by three methods: first, two-dimensional cytofluorometric analysis showed that essentially all cells which made Ae, AI3 and El3 also synthesized Eo~19. Second, two color fluorescent staining of tissue sections revealed that Ac~ and Ee protein occurred coincidentally in the thymus (C. Benoist eta/., unpublished results). Third, separation of splenic B and T cells by 'panning' resulted in a parallel depletion of foreign Ec~ gene and endogenous Ac~ gene transcripts in the T-cell fraction (D. Mathis et al., unpublished results). As expected, transcription from the injected gene could be induced by ~/-IFN in peritoneal macrophages 19,33. Surprisingly, though, macrophages from one transgenic line showed constitutive expression of cell surface Ee:El3 which was not further increased by ~/-IFN treatment 32. The authors attributed this aberration to the high copy number of the injected Ec~ gene, but it remains puzzling that El3 synthesis was also constitutive. The remarkable specificity of transcription from the injected gene provoked expectations that the Ec~ transgenics would be endowed with new immune response capabilities. Indeed, spleen cells from Ec~transgenics, but not from parental H-2 bx5 or H-2 b controls, could act as antigen-presenting cells for two TH hybridomas specific for a herpes simplex glycopeptide in the context of I-E 33. Secondly, after priming with GLPhe, the E~ transgenics yielded lymph node cells that proliferated in response to this polypeptide antigen, whereas lymph nodes from the H-2 ° haplotype controls were unreactive 32. Finally, as the ultimate test of immune function, the blood of E~ transgenics was titred for anti-GLPhe antibodies after a secondary antigen challenge 19. These mice made a healthy response to GLPhe injection, while H-2 bxs haplotype controls were not stimulated. Thus, by all criteria tested, the foreign Ec~ gene product in transgenic mice was immunologically functional. Similar success has been achieved with MHC Class I gene transgenic mice (Ref. 35, and D. Singer, pers. commun.). The porcine MHC gene PD1 was injected into C57 BIll0 SCN embryos and a transgenic line with several integrated copies developed. In general, the transcription pattern in transgenic mice closely paralleled that in swine. That is, PD1 synthesis was highest in lymphoid tissues and moderate in others, except for the brain where it was essentially undetectable (a few differences did occur, however). The pig MHC molecule could act as a transplantation antigen since skin from the transgenic line was rejected by the C57 BIll0 parental strain. Not surprisingly, then, the porcine antigen could be recognized by cytotoxic T lymphocytes (CTL). The strength of the CTL response was significantly less than in an allo-reaction, and the number of anti-PDI precursors was ten-fold lower than the number of precursors with allo-specificity, although this was still higher than expected for antigen-specific MHC-restricted CTLs. In preliminary studies of the T-cell repertoire in PDI transgenic mice, the interesting observation was made that the xeno-specific CTLs exhibit unexpectedly high crossreactivities with some murine Kb mutants. No explanation for this phenomenon comes from scanning the
ImmunologyToday, voL 7, No. 5, 1986
protein sequences, and its significance remains a mystery. In summary, transgenic mice harboring foreign MHC genes exhibit new immunological reactivities. No doubt these animals will be useful for studying the immune response in toto. In addition, the marked transcriptional fidelity of the injected genes may prompt experiments designed to pinpoint promoter control elements. Perspectives
Transgenic mice will no doubt have an important future in immunological research. The study of gene expression in lymphoid cells will be greatly facilitated, the benefits being analogous to those already realized with genes such as globin and elastase. However, there does exist a set of questions which is more specifically immunological. For some of these, transgenic mice will extend or improve on pre-existing experimental systems that include congenic mice and irradiation chimeras; for other questions entirely new approaches may be envisioned. We list below several types of experiment in which transgenic mice might be used to advantage. The list is not exhaustive and continues to evolve. Identifying the function of certain proteins: The injection of allelic variants, mutant genes, or 'antisense' transcripts will help in elucidating the role of a number of gene products. Consequently, cell-surface molecules such as the Qa and Lyt series might attain a more noble status than mere 'differentiation antigens'. Creating new mouse lines: Congenic lines have been important experimental material for immunologists of varied interests. In a sense, transgenic animals represent 'perfect congenics': cloned genetic material is introduced into a perfectly defined background. Thus, problems like those presently arising over the interpretation of I-J should be avoidable 36 37. Verifying genetically mapped loci: The injection of large chromosomal segments might help to reconcile molecular biologists and immunologists in cases where molecular mapping has so far failed to clarify the basis of known immunological phenomenology. I-J and the T locus are obvious candidates 37'38. Establishing new cell lines: The injection of oncogenes under the control of various promoters has enabled the development of mouse lines harboring tissues that can be immortalized in culture 13'17. The hope is that this phenomenon will also be true of the immune system, allowing one to establish, relatively painlessly, lines of desired cell type and antigen specificity. Provoking aberrant gene expression: An interesting type of transgenic mouse might result from completely aberrant gene expression. What happens to an la ÷, slg ÷ liver cell? What happens to a mouse with heart cells expressing an MHC molecule of a haplotype different from that of its thymus? Thus novel approaches may emerge to study tolerance and suppression. Creating murine disease models: New disease-prone lines can be generated; alternatively gene therapy can be attempted on pre-existing models. In this respect, an important first step has already been accomplished by curing the C57 BI/6 mouse of its susceptibility to the major murine pathogen, pigeon cytochrome C 19"32'33,
We thank Drs D. Baltimore, R. Brinster and D. Singer for sending preprints of papers.
References
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