Regulating gene expression in transgenic animals Catherine A. Kappel, Simon Xin-Min Zhang, Charles J. Bieberich and Gilbert Jay Jerome H. Holland Laboratory, Rockville, M a r y l a n d , USA Regardless of the field of application, the raison d'etre of transgenic animals is to study gene regulation and function. With increasing frequency, mammalian genes are being isolated with no concomitant knowledge of their function. The human genome mapping initiative will undoubtedly produce a cornucopia of such genes. While the merit of taking a transgenic route to study genes of unknown function is axiomatic, the choices of strategies for gene regulation in vivo may not be fully appreciated. This review will address two main points: first, the targeted and regulated expression of genes, and second, the structural and functional ablation of genes. Current Opinion in Biotechnology 1992, 3:548-553
Introduction For the first hundred years after the birth of Mendelian genetics, genes were identified almost exclusively by observing some phenotype that arose as a consequence of inheritance of that gene, either in its normal or mutated form. Since the advent of molecular genetics, genes are often identified well before any clue to their functions is elucidated. This so-called reverse genetics approach is fast becoming a mainstay of mammalian genetics. Before the introduction of transgenic animal technology, DNA transfection of tissue-culture cells was the primary m e t h o d for assessing gene expression and function. While tissue-culture systems have provided invaluable insight into gene regulation, their limitations are multifarious. Only a few cell types can be cultured and cells in culture rarely reflect the natural state of cells in the animal. Ceils are often immortalized, mutated, selected, or in an unusual state of differentiation and it is difficult to reproduce cell-cell or cell-extracellular matrix interactions in culture.
restricted [1]. This discrepancy in expression pattern of a gene in cell culture and in vivo has also b e e n seen for non-viral genes. MyoD expression is restricted to skeletal muscle in transgenic mice, but the same gene can be expressed in epithelial, fibroblast, and myoblast cell lines in culture [2",3]. Transgenic animal studies have also revealed new twists in gene expression that could not have been predicted from tissue culture. This is illustrated by the conserved X and Y boxes in the promoter region of the class II major histocompatibility complex (MHC) genes. Sequence conservation of the X and Y boxes between animal species implied their importance in controlling gene expression, but only transgenic analysis was able to demonstrate that these regions are separately responsible for expression in the cortex (X box) and medulla (Y box) of the thymus [4].
Targeted and regulated expression of genes 1
Transgenic approaches can both complement and enhance tissue culture studies in the analysis of new genes. Early on it became clear, however, that w h e n studies are extended to whole animals, potential differences between gene expression in cultured cells and gene expression in whole animals have to be considered - - what was d o g m a in cell culture is not necessarily so in animals. For instance, strong universal promoters in tissue culture were shown to be much less promiscuous in animals. The simian virus (SV) 40 early promoter w o r k e d well in virtually all tissue-culture cells, but expression in transgenic mice was very
The area of research that has derived the most benefit from transgenic technology is undeniably the study of the regulation of gene expression: w h e n is a gene expressed, where is it expressed, and what are the consequences of its expression? An increasingly diverse array of regulatory elements that influence gene expression is being characterized in transgenic mice. Most of these w o u l d have b e e n impossible to define in cultured cells. For example, the Pcp-2 gene is expressed only in Purkinje ceils, a subset of neurons [5,6], and the mitochondrial uncoupling protein is expressed only in b r o w n fat cells, a subset of adipocytes [7"]. Others target a wide
Abbreviations LCR--Iocus control region; LTR---long terminal repeat; MHC--major histocompatibility complex; MMTV--mouse mammary tumor virus; Mo-MLV--Moloney murine leukemia virus; MT-~metallothionein; SV simian virus; TIMP--tissue inhibitor of metalloproteinase.
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Regulating gene expression in transgenic animals Kappel, Zhang, Bieberich and lay spectrum of cell types, such as the retinoic acid-binding protein control region that targets the majority of embryonic tissues between gestation days 10 and 12 [8"], and the apolipoprotein E / C / g e n e s which primarily target liver, brain, and skin [9"]. Enhancers directing tissue specificity in animals are not always located in the promoter region of a gene. At times they are found within introns [10"] or in the 3' flanking region [11]. Silencers can turn off gene expression in specific tissues and at specific developmental stages. For example, a downstream sequence is responsible for turning off apolipoprotein E gene expression in the kidney but at the same time it induces expression in the sMn of transgenic mice [9"]. In addition to promoters, enhancers, and silencers, other types of cis regulatory elements have b e e n defined. For example, elements referred to as the locus control region (LCR), located far upstream from the transcriptional promoter, have b e e n identified in a variety of genes including the ~-globin cluster and the CD2 gene [12,13]. Transgenic analysis has also revealed that the order of genes in a cluster m a y be important in transcription. During development, genes in the ~globin cluster are developmentally turned on and off in a 5' to 3' direction. If the order of the genes is changed, developmental transcriptional timing is also altered [14"]. Several genes studied in transgenic animals are expressed at different stages of differentiation and development. The regulatory element of the gene for the tissue inhibitor of metalloproteinase (TIMP) is expressed in a defined differentiation state: in cells in the inner root sheath of hair follicles during the growth phase and not in other stages of follicle d e v e l o p m e n t [15"]. Phosphoenolpyrurate carboxykinase is expressed selectively in the embryonic intestine and then switches to the liver a n d kidneys post-partum [16"]. One application that requires tissue-specific and developmental-specific gene expression involves targeting genes to lactating m a m m a r y glands, with the ultimate goal of producing large quantities of genetically engineered proteins in the milk [11,17"',18]. There are several potential problems that m a y prevent the successful use of m a m m a r y glands to produce high levels of proteins. First, signal sequences may be necessary for secretion, which may simply entail designing a gene with a leader peptide. Second, any specific enzymes involved in the correct processing of a precursor protein must be produced in these cells. If the appropriate processing enzymes are not normally present in m a m mary cells, it is possible to derive a double transgenic animal carrying the gene of interest and the processing enzyme required. The p r o p e r assembly of a heterodimer is yet another consideration; for example, the ratio of the two subunits m a y b e important for correct assembly [17"']. If one is interested in studying a gene, the expression of which induces a deleterious p h e n o t y p e such as perinatal lethality, then it is critical for the investigator to be able to control the activity of that gene. This can
be achieved in transgenic animals with an inducible promoter. Two such promoters that have been used extensively are the m o u s e m a m m a r y tumor virus (MMTV) long terminal repeat (LTR) [19] and the metallothionein (MT) promoter [20]. Their relatively high basal level of expression, however, has limited their use. While earlier studies focused on hormonal induction, m o r e recent studies have identified c/s-acting elements from several genes that respond to other physiologic and environmental stimuli. For example, hypoxia induces the erythropoietin gene [21"], reduced temperature induces a mitochondrial uncoupling protein in b r o w n fat [7"], and aromatic hydrocarbons induce a cytochrome P450 transgene [22"]. Unfortunately, there are only a limited n u m b e r of inducible promoters presently available, which limits the n u m b e r of cell types that can be targeted. To overcome the limitations to induction, instead of dep e n d i n g on the cis element, specificity can be provided b y an exogenous trans-activating factor. This effectively broadens the range of target cell types where gene expression can be induced to include all those for which specific control elements have b e e n defined. Technically this entails placing a trans-acting factor under the control of a regulatory element that targets expression to the cell type of choice. This can b e achieved by creating two transgenic lines: o n e carries a gene for a trans-acting factor (for example, yeast GAL4 [23"']) and the second line carries the corresponding cis-acting elements (for example the yeast UAS regulatory sequence) driving the gene to be defined. A similar approach using a viral regulatory element and trans activator has also b e e n e m p l o y e d [24]. While the investigator has the ability to target transgene expression to a large extent, there are inherent cellular mechanisms that may alter the pattern of gene expression. For example, DNA imprinting, resulting from differential CpG methylation, m a y affect transgene expression, depending on the sex of the parent from which the gene was inherited [25",26q. Alternatively, a detrimental transgene may undergo somatic deletion [27"].
Structural and functional ablation of genes G e n e expression in animals can not only b e altered b y addition of a gene, but also b y the elimination of a gene. Two types of gene ablation are possible: stmctural and functional. Structural ablation is achieved b y introducing a mutation into a gene in an embryonic stem cell line, selecting for the mutation, and generating animals with the mutation [28,29]. To date, the knockout approach has b e e n applied predominantly to three groups of genes: those related to the immune system, the proto-oncogenes, and the h o m e o b o x genes. The immune-related knockouts include ~2-microglobulin [30], MHC class II [31"], interleukin-2 [32"], inter-
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Expressionsystems leukin-4 [33"], and CD8 [34"]. As all of these genes are c o m p o n e n t s of critical immunological pathways, severe i m m u n o - i n c o m p e t e n c e is expected following ablation. This was not the case, however, in any of the mice expressing a k n o c k o u t gene, although a variety of immunological defects were observed in each case. For example, mice that do not express CD8 are unable to m o u n t a class I restricted cytotoxic T-lymphocyte response, but their helper T-cell-dependent B-cell response is normal [34"]. The proto-oncogene knockouts include p 5 3 [35], src [36"] , a b l [37,38], and m y b [39"]. The lack of a drastic p h e n o t y p e in the src knockouts is interesting. Csrc is a m e m b e r of a family of protein tyrosine kinases. Early lethality w o u l d b e expected because src is expressed b y embryonic day 10 and in a variety of critical tissues, including bone, the brain, and platelets. However, h o m o z y g o u s s r c /- mice can live for at least 5 months. No detrimental effects were noted in the brain or in the platelets where the expression of src is highest, though these mice are deficient in osteoclast function. It appears that many of the predicted functions of a gene are not supported b y the results obtained from knockout experiments. It is possible that m a n y biochemical pathways are m o r e plastic during development than was previously anticipated. Developmental plasticity can be defined as the ability of the embryo to use an alternative gene w h e n the preferred gene is unavailable. For example, src is a m e m b e r of a protein tyrosine kinase gene family, and its function m a y be replaced b y another member. It will be interesting to see the levels of activity of other tyrosine kinases in the platelets and the nervous system of these s r c / - mice. As a consequence of the potential developmental plasticity of an organism, the function of a gene cannot be fully elucidated by structural ablation. An alternative a p p r o a c h is to down-regulate the expression of a gene by functional ablation. In this case, any low level gene expression may obviate the need for an alternative pathway. As a consequence, the down-regulation of gene expression m a y suffice to define the gene function. Functional ablation reduces the level of expression without altering the gene. To date, this has only b e e n achieved in transgenic mice using the antisense approach. A gene can b e turned off selectively in a particular tissue or at a particular time during development b y expression of an antisense RNA driven b y the promoter of choice. This has b e e n achieved with myelin basic protein [40], Moloney murine leukemia virus (Mo-MLV) [41"], and glucocorticoid receptor [42-']. Antisense Mo-MLV prevented leukemia from developing in transgenic mice infected with Mo-MLV. Mice producing an antisense glucocorticoid receptor s h o w e d a reduction of approximately 50% in the level of e n d o g e n o u s sense glucocorticoid receptor mRNA. As anticipated, these mice s h o w e d abnormal hypothalamic-pituitaryadrenal axis function.
Although it has not yet b e e n tested in transgenic mice, an alternative approach to eliminating expression in transgenic mice is the use of ribozymes. Rib o z y m e s are nucleic acid enzymes with exquisite nucleic-acid cleavage-site specificity. One w o u l d anticipate ribozymes to function m o r e efficiently than antisense RNA, as ribozymes act enzymatically and antisense acts stoichiometrically. It remains to b e seen, however, whether ribozymes will function in transgenic animals [43].
Reverse genetics in action: misexpression and ablation of homeobox genes The explosion of research in the molecular biology of mammalian embryonic d e v e l o p m e n t over the last decade has b e e n fueled largely by the suggestion that well conserved genes may regulate similar processes in evolutionarily disparate organisms. For example, the h o m e o b o x genes, a g r o u p of well defined segment identity regulators in insects, were postulated to control aspects of mammalian segmentation. Reverse genetic analysis in transgenic mice has provided solid evidence in support of this theory and has p a v e d the w a y for an in-depth characterization of the hierarchy of gene regulation that is central to embryonic development. The first experiment to provide strong data linking h o m e o b o x genes to the segmental p h e n o t y p e in mammals used a promiscuous promoter, the ~-actin promoter, to alter the pattern of expression of a single h o m e o b o x gene ( H o x 1.1) during embryogenesis [44,45]. The results were dramatic. The transgenic mice, b o r n with multiple craniofacial abnormalities and, most interestingly, malformation of bones in the skull and in the cervical vertebrae, died shortly after birth. These results provided the first clear demonstration that the m u c h ballyhooed h o m e o b o x genes were intimately involved in developmental processes in mammals. More importantly, they prompted researchers to ask whether a regulatory hierarchy exists a m o n g the m o u s e h o m e o b o x genes sirhilar to the insect h o m e o b o x hierarchy. Using the other major transgenic approach, g e n e ablation, three groups have created null mutants for homeo b o x genes [46",47,48]. The most striking observation with regard to altered segmental p h e n o t y p e in the mouse resulted from a knockout of the H o x 3.1 gene [46"q. Mice that are h o m o z y g o u s for the null mutation exhibit clear transformations of the axial skeleton, including an extra pair of ribs on the first lumbar vertebra. The alterations in vertebral phenotype represented 'anteriorizations' and were remarkably well predicted b y mutations of homologous genes in Drosophila. These results, taken together with the results of misexpression of H o x 1.1 [45], unequivocally demonstrate the pivotal role that the h o m e o b o x genes play in establishing the identity of segmental structures in mammals.
Regulating gene expression in transgenic animals Kappel, Zhang, Bieberich and Jay
Conclusion
tissues in the developing embryo, but by day 18 of gestation expression was only observed in the intestine.
Transgenic animal technology is n o w well established as a critical m e t h o d for analyzing gene expression and function. The approach continues to evolve, however, to include n e w strategies that offer a broad array of regulatory regimes. An appropriate choice of cis-acting elements can give the investigator increasingly precise temporal and spatial control over the expression of genes added to the genome. In addition, transgenes can be either inducible or expressed constituitively. Homologous recombination can n o w eliminate a gene entirely or make a subtle change in its coding or regulatory sequences [49]. Each strategy provides different insights and clearly a combination of these approaches will be required to dissect the function of most genes.
9.
1991, 266:8651-8654. Positive and negative tissue specific cis-regulatory elements from apollpoprotein E / C / w e r e identified in a series of eleven constructs in transgenic mice. 10.
PALMITERRD, SANDGKEN EP, AVARBOCK MR, ALLEN DD, BRINSTER RL: He~erologous Introns Can E n h a n c e Exp r e s s i o n o f Transgenes i n Mice. Proc Natl A c a d Sci USA 1991, 88:478-482. The authors investigate whether a heterologous intron can improve transgene transcription. They use an extensive series of transgenes with a variety of intron/gene/promoter combinations, including growth hormone with and without its natural introns. In some combinations introns improve transcription. 11.
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ZIJLSTRAM, BIX M, SIMISTER NE, LORING JM, RAULET DH, JAENISCH R: [~2-Mieroglobulin Deficient Mice Lack CD48 + C y t o l y t i c T Cells. N at ur e 1990, 344:742-746.
39.
MUCENSKI~IL, MCLAIN K, KIER AB, SWERDLOWSH, SCHREINER CM, MILLER TA, PIETRYGA DW, SCOTT WJ JR, POTTER SS: A Functional C-myb Gene is Required for Normal Murine Fetal Hepatic Hematopoiesis. Cell 1991, 65:677-689. Mice h o m o z y g o u s for a c-myb b mutation perturbed h e m a t o poiesis in the liver resulting in embryonic lethality. 40.
KATSUmM, SATO M, KIMURAM, YOKOYAMAM, KOBAYASHIK, NOMURA T: Conversion o f Normal Behavior to Shiverer b y Myelin Basic Protein Antisense cDNA i n Transgenic Mice. Science 1988, 241:593-595.
HAN L, YUN JS, WAGNER TE: I n h i b i t i o n o f Moloney Murine Virus-induced L e u k e m i a i n Transgenic Mice Expressing Antisense RNA Complementary to the R e t r o v t r a l P a c k a g i n g S e q u e n c e s . Proc Natl A c a d Sci U S A 1991, 88:4313-4317. Mo-MLV i n d u c e d leukemia was prevented in transgenic mice expressing an antisense m e s s a g e specific for the proviral packaging sequences. This was achieved with two promoters: the Mo-MLV LTR a n d the cytomegalovirus immediate early region. 41.
R e g u l a t i n g g e n e e x p r e s s i o n in t r a n s g e n i c a n i m a l s PEPINM-C, POTHIER F, BARDENN; I m p a i r e d T y p e lI Glucoc o r t i c o i d R e c e p t o r F u n c t i o n i n Mice B e a r i n g A n t i s e n s e RNA T r a n s g e n e . Nature 1992, 355:725-728. Predicted alterations in the hypothalamus-pituitary-adrenal axis were observed in mice expressing a n antisense glucocorticoid receptor. Homozygous a n d heterozygous mice had similar phenotypes, including increased fat deposition. 42. •.
43.
COTTEN M: T h e I n Vivo A p p l i c a t i o n Trends in Biotech 1990, 8:174.
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BALLINGR, M ~ R G, GRUSS P, KESSEL M: C r a n i o f a c i a l Abnormalities Induced by Ectopic Expression of the H o m e o b o x G e n e H o x - l . 1 i n T r a n s g e n i e Mice. Cell 1989, 58:337-347.
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null mutant mice. Transformation of the axial skeleton w a s observed, including eight vertebrostemal ribs and a n extra pair of ribs. This is the first demonstration of a loss-of-function homeotic mutation of vertebrae in mice. 47.
LUFKIN T, DIERICH A, LEMEURM, MARK M, CHAMBON P: Disr u p t i o n o f H o x 1.6 H o m e o b o x G e n e R e s u l t s i n D e f e c t s i n a R e g i o n C o r r e s p o n d i n g t o Its R o s t r a l D o m a i n o f E x p r e s s i o n . Cell 1991, 66:1105-1119.
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CHISAKA O, CAPECCHI MR: R e g i o n a l l y R e s t r i c t e d Developmental D e f e c t s R e s u l t i n g f r o m Targeted D i s r u p t i o n o f t h e M o u s e H o m e o b o x G e n e H o x - l . 5 . N at ure 1991, 350:473-479.
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HASTY P, RAMIREZ-SOLISR, KRUMLAUFR, BRADLEYA: I n t r o d u c t i o n o f a Subtle M u t a t i o n i n t o t h e H o x - 2 . 6 L o t u s i n E m b r y o n i c S t e m Cells. N a t u r e 1991, 350:243-246.
of Ribozymes.
KESSEL M, BALLING R, GRUSS P: V a r i a t i o n s o f C e r v i c a l
Vertebrae a f t e r E x p r e s s i o n o f a H o x - l . 1 T r a n s g e n e i n Mice. Cell 1990, 61:301-308. LE MOUELLIC H, LALLEMAND Y, BRULET P: H o m e o s i s i n the Mouse Induced by a Null Mutation in the Hox3.1 Gene. Cell 1992, 69:251-264. This paper describes the replacement of the H o x 3.1 coding seq u e n c e with the lacZ gene by h o m o l o g o u s recombination to create
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CA Kappel, SX-M Zhang, CJ Bieberich a n d G Jay, Laboratory of Virology, The Jerome H. Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855, USA.
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Introduction For the first hundred years after the birth of Mendelian genetics, genes were identified almost exclusively by observing some phenotype that arose as a consequence of inheritance of that gene, either in its normal or mutated form. Since the advent of molecular genetics, genes are often identified well before any clue to their functions is elucidated. This so-called reverse genetics approach is fast becoming a mainstay of mammalian genetics. Before the introduction of transgenic animal technology, DNA transfection of tissue-culture cells was the primary m e t h o d for assessing gene expression and function. While tissue-culture systems have provided invaluable insight into gene regulation, their limitations are multifarious. Only a few cell types can be cultured and cells in culture rarely reflect the natural state of cells in the animal. Ceils are often immortalized, mutated, selected, or in an unusual state of differentiation and it is difficult to reproduce cell-cell or cell-extracellular matrix interactions in culture.
restricted [1]. This discrepancy in expression pattern of a gene in cell culture and in vivo has also b e e n seen for non-viral genes. MyoD expression is restricted to skeletal muscle in transgenic mice, but the same gene can be expressed in epithelial, fibroblast, and myoblast cell lines in culture [2",3]. Transgenic animal studies have also revealed new twists in gene expression that could not have been predicted from tissue culture. This is illustrated by the conserved X and Y boxes in the promoter region of the class II major histocompatibility complex (MHC) genes. Sequence conservation of the X and Y boxes between animal species implied their importance in controlling gene expression, but only transgenic analysis was able to demonstrate that these regions are separately responsible for expression in the cortex (X box) and medulla (Y box) of the thymus [4].
Targeted and regulated expression of genes 1
Transgenic approaches can both complement and enhance tissue culture studies in the analysis of new genes. Early on it became clear, however, that w h e n studies are extended to whole animals, potential differences between gene expression in cultured cells and gene expression in whole animals have to be considered - - what was d o g m a in cell culture is not necessarily so in animals. For instance, strong universal promoters in tissue culture were shown to be much less promiscuous in animals. The simian virus (SV) 40 early promoter w o r k e d well in virtually all tissue-culture cells, but expression in transgenic mice was very
The area of research that has derived the most benefit from transgenic technology is undeniably the study of the regulation of gene expression: w h e n is a gene expressed, where is it expressed, and what are the consequences of its expression? An increasingly diverse array of regulatory elements that influence gene expression is being characterized in transgenic mice. Most of these w o u l d have b e e n impossible to define in cultured cells. For example, the Pcp-2 gene is expressed only in Purkinje ceils, a subset of neurons [5,6], and the mitochondrial uncoupling protein is expressed only in b r o w n fat cells, a subset of adipocytes [7"]. Others target a wide
Abbreviations LCR--Iocus control region; LTR---long terminal repeat; MHC--major histocompatibility complex; MMTV--mouse mammary tumor virus; Mo-MLV--Moloney murine leukemia virus; MT-~metallothionein; SV simian virus; TIMP--tissue inhibitor of metalloproteinase.
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Regulating gene expression in transgenic animals Kappel, Zhang, Bieberich and lay spectrum of cell types, such as the retinoic acid-binding protein control region that targets the majority of embryonic tissues between gestation days 10 and 12 [8"], and the apolipoprotein E / C / g e n e s which primarily target liver, brain, and skin [9"]. Enhancers directing tissue specificity in animals are not always located in the promoter region of a gene. At times they are found within introns [10"] or in the 3' flanking region [11]. Silencers can turn off gene expression in specific tissues and at specific developmental stages. For example, a downstream sequence is responsible for turning off apolipoprotein E gene expression in the kidney but at the same time it induces expression in the sMn of transgenic mice [9"]. In addition to promoters, enhancers, and silencers, other types of cis regulatory elements have b e e n defined. For example, elements referred to as the locus control region (LCR), located far upstream from the transcriptional promoter, have b e e n identified in a variety of genes including the ~-globin cluster and the CD2 gene [12,13]. Transgenic analysis has also revealed that the order of genes in a cluster m a y be important in transcription. During development, genes in the ~globin cluster are developmentally turned on and off in a 5' to 3' direction. If the order of the genes is changed, developmental transcriptional timing is also altered [14"]. Several genes studied in transgenic animals are expressed at different stages of differentiation and development. The regulatory element of the gene for the tissue inhibitor of metalloproteinase (TIMP) is expressed in a defined differentiation state: in cells in the inner root sheath of hair follicles during the growth phase and not in other stages of follicle d e v e l o p m e n t [15"]. Phosphoenolpyrurate carboxykinase is expressed selectively in the embryonic intestine and then switches to the liver a n d kidneys post-partum [16"]. One application that requires tissue-specific and developmental-specific gene expression involves targeting genes to lactating m a m m a r y glands, with the ultimate goal of producing large quantities of genetically engineered proteins in the milk [11,17"',18]. There are several potential problems that m a y prevent the successful use of m a m m a r y glands to produce high levels of proteins. First, signal sequences may be necessary for secretion, which may simply entail designing a gene with a leader peptide. Second, any specific enzymes involved in the correct processing of a precursor protein must be produced in these cells. If the appropriate processing enzymes are not normally present in m a m mary cells, it is possible to derive a double transgenic animal carrying the gene of interest and the processing enzyme required. The p r o p e r assembly of a heterodimer is yet another consideration; for example, the ratio of the two subunits m a y b e important for correct assembly [17"']. If one is interested in studying a gene, the expression of which induces a deleterious p h e n o t y p e such as perinatal lethality, then it is critical for the investigator to be able to control the activity of that gene. This can
be achieved in transgenic animals with an inducible promoter. Two such promoters that have been used extensively are the m o u s e m a m m a r y tumor virus (MMTV) long terminal repeat (LTR) [19] and the metallothionein (MT) promoter [20]. Their relatively high basal level of expression, however, has limited their use. While earlier studies focused on hormonal induction, m o r e recent studies have identified c/s-acting elements from several genes that respond to other physiologic and environmental stimuli. For example, hypoxia induces the erythropoietin gene [21"], reduced temperature induces a mitochondrial uncoupling protein in b r o w n fat [7"], and aromatic hydrocarbons induce a cytochrome P450 transgene [22"]. Unfortunately, there are only a limited n u m b e r of inducible promoters presently available, which limits the n u m b e r of cell types that can be targeted. To overcome the limitations to induction, instead of dep e n d i n g on the cis element, specificity can be provided b y an exogenous trans-activating factor. This effectively broadens the range of target cell types where gene expression can be induced to include all those for which specific control elements have b e e n defined. Technically this entails placing a trans-acting factor under the control of a regulatory element that targets expression to the cell type of choice. This can b e achieved by creating two transgenic lines: o n e carries a gene for a trans-acting factor (for example, yeast GAL4 [23"']) and the second line carries the corresponding cis-acting elements (for example the yeast UAS regulatory sequence) driving the gene to be defined. A similar approach using a viral regulatory element and trans activator has also b e e n e m p l o y e d [24]. While the investigator has the ability to target transgene expression to a large extent, there are inherent cellular mechanisms that may alter the pattern of gene expression. For example, DNA imprinting, resulting from differential CpG methylation, m a y affect transgene expression, depending on the sex of the parent from which the gene was inherited [25",26q. Alternatively, a detrimental transgene may undergo somatic deletion [27"].
Structural and functional ablation of genes G e n e expression in animals can not only b e altered b y addition of a gene, but also b y the elimination of a gene. Two types of gene ablation are possible: stmctural and functional. Structural ablation is achieved b y introducing a mutation into a gene in an embryonic stem cell line, selecting for the mutation, and generating animals with the mutation [28,29]. To date, the knockout approach has b e e n applied predominantly to three groups of genes: those related to the immune system, the proto-oncogenes, and the h o m e o b o x genes. The immune-related knockouts include ~2-microglobulin [30], MHC class II [31"], interleukin-2 [32"], inter-
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Expressionsystems leukin-4 [33"], and CD8 [34"]. As all of these genes are c o m p o n e n t s of critical immunological pathways, severe i m m u n o - i n c o m p e t e n c e is expected following ablation. This was not the case, however, in any of the mice expressing a k n o c k o u t gene, although a variety of immunological defects were observed in each case. For example, mice that do not express CD8 are unable to m o u n t a class I restricted cytotoxic T-lymphocyte response, but their helper T-cell-dependent B-cell response is normal [34"]. The proto-oncogene knockouts include p 5 3 [35], src [36"] , a b l [37,38], and m y b [39"]. The lack of a drastic p h e n o t y p e in the src knockouts is interesting. Csrc is a m e m b e r of a family of protein tyrosine kinases. Early lethality w o u l d b e expected because src is expressed b y embryonic day 10 and in a variety of critical tissues, including bone, the brain, and platelets. However, h o m o z y g o u s s r c /- mice can live for at least 5 months. No detrimental effects were noted in the brain or in the platelets where the expression of src is highest, though these mice are deficient in osteoclast function. It appears that many of the predicted functions of a gene are not supported b y the results obtained from knockout experiments. It is possible that m a n y biochemical pathways are m o r e plastic during development than was previously anticipated. Developmental plasticity can be defined as the ability of the embryo to use an alternative gene w h e n the preferred gene is unavailable. For example, src is a m e m b e r of a protein tyrosine kinase gene family, and its function m a y be replaced b y another member. It will be interesting to see the levels of activity of other tyrosine kinases in the platelets and the nervous system of these s r c / - mice. As a consequence of the potential developmental plasticity of an organism, the function of a gene cannot be fully elucidated by structural ablation. An alternative a p p r o a c h is to down-regulate the expression of a gene by functional ablation. In this case, any low level gene expression may obviate the need for an alternative pathway. As a consequence, the down-regulation of gene expression m a y suffice to define the gene function. Functional ablation reduces the level of expression without altering the gene. To date, this has only b e e n achieved in transgenic mice using the antisense approach. A gene can b e turned off selectively in a particular tissue or at a particular time during development b y expression of an antisense RNA driven b y the promoter of choice. This has b e e n achieved with myelin basic protein [40], Moloney murine leukemia virus (Mo-MLV) [41"], and glucocorticoid receptor [42-']. Antisense Mo-MLV prevented leukemia from developing in transgenic mice infected with Mo-MLV. Mice producing an antisense glucocorticoid receptor s h o w e d a reduction of approximately 50% in the level of e n d o g e n o u s sense glucocorticoid receptor mRNA. As anticipated, these mice s h o w e d abnormal hypothalamic-pituitaryadrenal axis function.
Although it has not yet b e e n tested in transgenic mice, an alternative approach to eliminating expression in transgenic mice is the use of ribozymes. Rib o z y m e s are nucleic acid enzymes with exquisite nucleic-acid cleavage-site specificity. One w o u l d anticipate ribozymes to function m o r e efficiently than antisense RNA, as ribozymes act enzymatically and antisense acts stoichiometrically. It remains to b e seen, however, whether ribozymes will function in transgenic animals [43].
Reverse genetics in action: misexpression and ablation of homeobox genes The explosion of research in the molecular biology of mammalian embryonic d e v e l o p m e n t over the last decade has b e e n fueled largely by the suggestion that well conserved genes may regulate similar processes in evolutionarily disparate organisms. For example, the h o m e o b o x genes, a g r o u p of well defined segment identity regulators in insects, were postulated to control aspects of mammalian segmentation. Reverse genetic analysis in transgenic mice has provided solid evidence in support of this theory and has p a v e d the w a y for an in-depth characterization of the hierarchy of gene regulation that is central to embryonic development. The first experiment to provide strong data linking h o m e o b o x genes to the segmental p h e n o t y p e in mammals used a promiscuous promoter, the ~-actin promoter, to alter the pattern of expression of a single h o m e o b o x gene ( H o x 1.1) during embryogenesis [44,45]. The results were dramatic. The transgenic mice, b o r n with multiple craniofacial abnormalities and, most interestingly, malformation of bones in the skull and in the cervical vertebrae, died shortly after birth. These results provided the first clear demonstration that the m u c h ballyhooed h o m e o b o x genes were intimately involved in developmental processes in mammals. More importantly, they prompted researchers to ask whether a regulatory hierarchy exists a m o n g the m o u s e h o m e o b o x genes sirhilar to the insect h o m e o b o x hierarchy. Using the other major transgenic approach, g e n e ablation, three groups have created null mutants for homeo b o x genes [46",47,48]. The most striking observation with regard to altered segmental p h e n o t y p e in the mouse resulted from a knockout of the H o x 3.1 gene [46"q. Mice that are h o m o z y g o u s for the null mutation exhibit clear transformations of the axial skeleton, including an extra pair of ribs on the first lumbar vertebra. The alterations in vertebral phenotype represented 'anteriorizations' and were remarkably well predicted b y mutations of homologous genes in Drosophila. These results, taken together with the results of misexpression of H o x 1.1 [45], unequivocally demonstrate the pivotal role that the h o m e o b o x genes play in establishing the identity of segmental structures in mammals.
Regulating gene expression in transgenic animals Kappel, Zhang, Bieberich and Jay
Conclusion
tissues in the developing embryo, but by day 18 of gestation expression was only observed in the intestine.
Transgenic animal technology is n o w well established as a critical m e t h o d for analyzing gene expression and function. The approach continues to evolve, however, to include n e w strategies that offer a broad array of regulatory regimes. An appropriate choice of cis-acting elements can give the investigator increasingly precise temporal and spatial control over the expression of genes added to the genome. In addition, transgenes can be either inducible or expressed constituitively. Homologous recombination can n o w eliminate a gene entirely or make a subtle change in its coding or regulatory sequences [49]. Each strategy provides different insights and clearly a combination of these approaches will be required to dissect the function of most genes.
9.
1991, 266:8651-8654. Positive and negative tissue specific cis-regulatory elements from apollpoprotein E / C / w e r e identified in a series of eleven constructs in transgenic mice. 10.
PALMITERRD, SANDGKEN EP, AVARBOCK MR, ALLEN DD, BRINSTER RL: He~erologous Introns Can E n h a n c e Exp r e s s i o n o f Transgenes i n Mice. Proc Natl A c a d Sci USA 1991, 88:478-482. The authors investigate whether a heterologous intron can improve transgene transcription. They use an extensive series of transgenes with a variety of intron/gene/promoter combinations, including growth hormone with and without its natural introns. In some combinations introns improve transcription. 11.
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1.
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3.
Expression of a Single Transfected eDNA Converts Fibrobiasts to Myoblasts,_. Cell 1987, 51:987-1000.
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HANSCOMBEO, WHATT D, FRASERP, YANNOUTSOSN, GREAVES D, DILLON N, GROSVELD F: Importance o f Globin Gene Order for Correct D e v e l o p m e n t a l Expression. Genes Dev 1991, 5:1387-1394. Genes in the [~-gtobin gene cluster are ordered in a 5' to 3' direction which reflects their developmental timing for expression. The present study suggests a model in which the gene order in the cluster may play a role in developmentally regulated expression. This may reflect w h y gene order has been conserved in this family as well as other developmentally regulated gene family clusters, such as the h o m e o b o x genes. 15.
KAWABETr, REA TJ, FLENNIKENAM, WILLIAMSBRG, GROPPI VE, BUHL AE: Localization o f TIMP i n Cycling Mouse Hair. Development 1991, 111:877-879. TIMP is believed to be involved in tissue remodeling. Tmnsgenic mice with TIMP regulatory sequences directing lacZ expression demonstrated lacZ activity in the growth phase of the hair cycle but not in other stages, supporting a role for TIMP in tissue remodeling. 16.
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This transgene retains developmental, tissue-specific, and hormonal controls the same as the endogenous gene. The rat phosphoenolpymvate carboxykinase transgene is expressed in fetal intestine but in the adult, expression switches to liver, kidney, and adipose tissues. The rat transgene, as the endogenous mouse gene, is also responsive to glucocorticoid treatment in a tissue-specific manner - glucocorticoid increases expression in the kidney and decreases it in adipose tissue. GREENBERGNM, ANDERSON JW, HSUEH AJW, NISHIMORI K, REEVESJJ, DEAVILADM, WARD DN, ROSENJM: Expression o f Biologically Active Heteroditneric Bovine Follides t i m t d a t i n g H o r m o n e i n Milk o f Transgenic Mice. Proc Natl A c a d Sci USA 1991, 88:8327-8331. Lactating mammary glands can be used as a means to produce biologically active, post-tmnslationally modified, heterodimeric proteins in transgenic animals. A rat ~-casein promoter was used to direct the tz- and [~-chains of follicle-stimulating hormone. The m-chain con17. •.
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19.
STEWART TA, PATI*ENGALE PK, LEDER P: Spontaneous M a m m a r y A d e n o c a r c i n o m a s i n Transgenic Mice that C a r r y a n d E x p r e s s M T V / m y c F u s i o n G e n e s . Cell 1984, 38:627-637.
20.
PALMITER RD, NORSTEDT G, GELINAS RE, HAMMER RE, BRINSTER RL: Metallothionein-Human GH Fusion Genes Stinlulate G r o w t h o f Mice. Science 1983, 222:809--814.
21.
SEMENZA GL, KOURY ST, NEJFELT MK, GEARHART JD, ANTONARAKIS SE: Cell-type-specific a n d H y p o x i a - i n d u c i b l e Expression o f the H u m a n E r y t h r o p o i e t i n G e n e i n T r a n s g e n i c Mice. Proc Natl A c a d Sci USA 1991, 88:8725-8729. A h u m a n erythropoietin transgene retains inducibility a n d cell specificity. Expression is maintained at a basal level until there is a physiological stimulus, anemia or hypoxia. 22.
JONES SN, JONES PG, IBARGUEN H, CASKEY CT, CRAIGEN WJ: Induction o f C y p l a - 1 Dioxin-responsive Enhancer i n Transgenic Mice. Nucl Acids Res 1991, 19:6547-6551. The Cypla-1 g e n e codes for a m e m b e r of the potycyclic aromatic hydrocarbon-inducible P450 family. Transgene expression was observed in a variety of tissues without induction, a n d aromatic hydrocarbon treatment strongly induced expression in the liver. ORNITZDM, MOREADITH RW, LEDER P: B i n a r y System for R e g u l a t i n g Transgene Expression i n Mice: Targeting Int-2 Gene Expression w i t h Yeast GAL4/UAS Control Elements. Proc Natl A c a d Sci USA 1991, 88:698-702. The first transgenic m o u s e line contains a potentially lethal gene int2, cloned b e h i n d the yeast e n h a n c e r UAS w h i c h is only functional in the presence of GAL4. The second transgenic m o u s e line contains yeast GAL4 controlled by the MMTV LTR. Expression of transgenic int-2 is only observed in the p r o g e n y of a cross b e t w e e n the two lines. 23. •.
24.
BYRNE GW, RUDDLE FH: Mttltiplex G e n e R e g u l a t i o n : A Two-tiered Approach to Transgene R e g u l a t i o n i n Transgenic Mice. Proc Natl A c a d Sci USA 1989, 86:5473-5477.
ENGLER P, HAASCH D, PINKERT CA, DOGLIO L, GLYMOUR M, BRINSTER R, STORB U: A S t r a i n - s p e c i f i c Modifier o n Mouse C h r o m o s o m e 4 Controls Methylation of Indep e n d e n t Transgene Loci. Cell 1991, 65:939-947. A methylase w a s m a p p e d to m o u s e c h r o m o s o m e 4. If a transgene is passed t h r o u g h SJL or DBA/2 mice it is undermethylated, but passage through C57BL/6 results in a methylated transgene. This study suggests that t h e genetic b a c k g r o u n d of mice m a y affect expression.
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