Transgenic animals production of foreign proteins in milk Lothar Hennighausen, Leonard Ruiz* and Robert Walr National Institutes of Health, Bethesda, MD 20892, *Land O'Lakes Inc., 4001 Lexington Ave, Arden Hills, MN 55440, ~ US Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA Current Opinion in Biotechnology 1990, 1:74--78
Introduction Purification of pharmacologically active proteins such as coagulation Factors VIII and IX from human blood is expensive, and the possibility of contamination with pathogenic human viruses still persists. The isolation of genes encoding a number of medically important proteins has permitted cheaper production of these highunit-value proteins in heterologous systems. An appealing expression system for such proteins is the transgenic animal. By introducing foreign genes into the germline of animals it is possible to generate animals that have altered phenotypes [1,2]. The selective attachment of specific DNA control elements to the foreign genes makes it possible to target expression of the proteins they encode to different tissues or body fluids of transgenic animals. It is advantageous to be able to harvest proteins from body fluids rather than from solid tissue because fluids are renewable. Several body fluids come to mind as a suitable source of foreign proteins in transgenic animals: blood, urine, saliva and milk. It is possible to direct protein secretion into blood by targeting gene expression either to the liver or to lymphocytes. This system is probably not viable because the volume of blood that can be harvested is limited and high levels of circulating biologically active proteins may prove detrimental to the animal's health. Similarly, large-scale harvesting of saliva may be problematic. Urine is an abundant fluid but is protein poor. Therefore, synthesis of foreign proteins in the mammary gland with secretion into the milk appears to be a more realistic approach. Isolation of milk protein genes that are expressed at high levels in the mammary glands has provided an experimental way to manipulate the trait of milk composition in transgenic animals. It may be possible to increase the concentration of caseins, thereby improving cheese production. Furthermore, by targeting the expression of valuable non-milk proteins to the mammary gland, this organ could serve as a 'bioreactor' for producing phar-
macologically active proteins on a large scale. Transgenic animals could become an economically viable alternative to existing tissue culture systems. This review highlights the progress made in the expression of foreign genes in the mammary gland of transgenic animals and discusses some of the economic aspects of the mammary gland bioreactor.
Milk protein genes Milk proteins are synthesized by the mammary gland and secreted into milk in gram quantities per liter. Several milk protein genes have been isolated [3-6,7 °] and it is found that their expression is essentially limited to functional mammary epithelial cells [8,9,10"]. Studies in transgenic animals have shown that promoter sequences from the milk protein genes encoding the mouse whey acidic protein (WAP) [8,11] and rat 13-casein [12] contain mammary regulatory elements and confer mammary specificity to genes they are ligated to, and which are normally not expressed in the mammary gland. However, these fusion genes are expressed at levels well below those of milk protein genes. This suggests the presence of additional, and not yet identified, control elements outside the sequences that act as promoters. Evidence for the existence of such elements comes from studies in which WAP genomic sequences with 5' and 3' flanking sequences have been introduced into transgenic mice, some of which express the transgene at levels similar to the endogenous WAP genes [10-,13,14] (Maschio et al., submitted). In contrast, a sequence containing the rat [3-casein gene with its 5' and 3' flanking sequences was expressed in trans o genic mice at less than 1% of its endogenous level [9], suggesting that some of its regulatory elements are located outside the cloned promoter region. Additional studies have shown that the regulatory elements that target gene expression to the mammary gland are functional across species boundaries, suggesting that they are derived from a common ancestor. Thus, the
Abbreviation
WAP--whey acidic protein. 74
~ Current Biology Ltd ISSN 0958-1669
Transgenic animals Hennighausen, Ruiz, Wall WAP gene. In contrast, none of the hybrid genes with • a 13-casein gene promoter has been expressed at high levels (Table 1) [12] suggesting that the WAP, but not the promoter proximal region of the 13-casein gene, contains control elements for high-level expression. A hybrid gene containing bovine ¢~Sl-casein promoter and structural gene sequences was expressed at about 10% of the endogenous level in one transgenic mouse (Table 1) [18,, P1 o]. Because only one mouse was analyzed and a similar aSl-casein promoter from rabbit was non-functional (Houdebine, personal communication), it is not clear that the cxSl-casein promoter contains the regulatory elements that are necessary for integration-site-independent high-level expression.
ovine 13-1actoglobulin gene can be expressed in transgenic mice [13] and the mouse WAP gene in transgenic pigs (Wall and Hennighausen, unpublished) despite the absence of corresponding endogenous genes in these species.
Production of foreign proteins in milk of transgenic animals The demand for a number of therapeutically important proteins cannot be matched by current production and isolation procedures; the mammary gland may solve this dearth by producing the proteins on a large scale and at a reasonable cost. To produce a pharmacologically active protein in the milk of transgenic animals the DNA sequence encoding this protein has to be linked to manunary-specific regulatory elements and the resulting hybrid gene has to be introduced into the germline of transgenic animals. Although several cloned milk protein gene promoters have been shown to direct the expression of heterologous genes in the mammary gland (Table 1) [7,15°,16°,17°,18°,19,], there appear to be quantitative differences. Hybrid genes containing promoters of the mouse (Table 1) and rabbit (Houdebine, personal communication) WAP genes can be expressed at levels up to 100% of those found for the endogenous
The protein coding sequence, or structural gene component of the hybrid gene, can be introduced as a cDNA or as a genomic sequence that contains introns and exons. As introns may be necessary for regulated gene expression in transgenic animals [20], many investigators be lieve that it is advisable, whenever possible, to use the genomic form rather than the cDNA form of the structural gene. For example, hybrid genes containing the ovine 13lactoglobulin gene promoter and a cDNA encoding human al-antitrypsin are expressed at low levels, whereas those containing genomic sequences encoding al-antitrypsin are expressed at levels similar to those of the endogenous 13-1actoglobulin gene [19 °]. However, in some
l Table 1. Expression of foreign proteins in milk. Transgenic animals Transgene
Encoded protein
Number
Expressors
Concentration (pg/ml)
Reference
WAP-hGH
Growth hormone
11
4
4-400
Reddy eta/., submitted
WAP-hGH
Growth hormone
8
4
1000-2000
G[Jnzburg et al., submitted
WAP-PS2 I
PS2
7
0
WAP-tPA
Plasminogen activator
6
4
0.4-100
WAP-CD4
CD4 receptor
7
5
0.4
[16.1
WAP-PS2 II
PS2
1
1
40
[17-]
WAP-hPC
Protein C
3
3
0.2
Valander et al., unpublished
~CAS-hUK
Urokinase
1
1
1000
[18-]
~CAS-hlL2
Interleukin 2
4
4
0.001-0.01
[7°]
~LAC-FIX
Clotting Factor IX
2
2
0.01
[15 .]
[3LAC-0~I-AT I
0d-antitrypsin
2
2
5
[15"]
~-~I-AT II
czl-antitrypsin
13
7
6-7000
[19 "]
[17"1 [8,10
]
75
76
Expressionsystems hybrid genes containing the WAP gene promoter, expression of structural genes containing introns does not appear to be different from cDNA expression. The WAP PS2I gene (Table 1) containing genomic sequences of PS2 (a breast cancer protein) as the structural part of the hybrid gene construct did not give rise to expression in seven lines of transgenic mice [17-]. In contrast, the WAP-PSII gene containing the PS2 cDNA was expressed at significant levels [17-]. So far, the importance of introns for high-level expression in transgenic animals has not been studied extensively and remains uncertain. Moreover, in some cases it will be necessary to use structural genes that are devoid of introns simply because the introns may contain regulatory elements targeting expression to non-mammary tissue which, in turn, could compromise the animal's health. Post-translational modification and stability of human proteins secreted into milk has not been studied in detail. From the limited studies available [15,], it appears that Factor IX and tissue plasminogen activator secreted into milk have biological activity but may not be as stable as the native proteins isolated from blood and may not be post-translationally modified in the same way. Although the caseins and other milk proteins are produced specifically in the mammary gland, transgenic animals carrying hybrid genes with a milk protein gene promoter may not show such clear specificity. Novel genetic regulatory elements may be generated through a combination of promoter and structural DNA sequences [1,2]. This appears to have happened in a hybrid gene containing the mouse WAP gene promoter and genomic sequences encoding human growth hormone (Table 1); the hybrid gene was ectopically expressed in Bergman glial cells (GOnzburg et al., submitted).
Expression levels At least 12 hybrid genes have been introduced into transgenic animals with the aim of producing a human protein in milk (Table 1) [19"]. The DNA control elements guiding expression of these genes to the mammary gland were derived from the genes for mouse WAP [3], bovine 0tSl-casein [18.], rabbit IB-casein [20] and ovine 13-1actoglobulin [6,19,]. In most cases, expression of the transgene was found in the mammary gland but the levels of the encoded proteins secreted into milk differed widely among lines of animals and were generally much lower than those of the endogenous milk proteins. The notable exception was a hybrid gene containing the promoter/upstream region of the ovine 13-1actoglobulin gene and a promoterless minigene encoding human czl-antitrypsin [19"]. Some ,of the mice carrying this fusion gene secreted czl-antitrypsin into milk at concentrations of more than 5mg/ml [19o]. The large differences in expression of a particular transgene between individual lines of animals are probably a result of position effects; surrounding sequences at different integration sites of transgenes may modulate their ex-
pression levels. This is a well described phenomenon and has been observed in basically all transgenic animal systems [1,2]. Recent finding regarding [3-globin gene regulation may help to overcome the inconsistent expression of transgenes. Grosveld et al. [22] have identified sequences at the 5' end of the globin locus that can function as a dominant control region or locus-activating region and confer integration-site independent high-level gene expression in erythroid cells of transgenic mice. Such elements may also exist in the vicinity of milk protein genes, and considerable efforts should be made to identify and isolate them. In addition, elements conferring post-transcriptional regulation, which have not yet been identified, may be required for the high-level expression of native milk protein genes. A further complication in assessing expression levels could be poor translation of the transgenic mRNAs and/or instability of the human proteins in milk. In the case of tissue plasminogen activator [8,11] and the CD4 receptor [16.] the amount of both biologically active and immunologicaUy reactive protein in milk correlated well with the RNA levels, suggesting that the proteins were stable and the RNAs were translated eiticiently. In contrast, in transgenic animals producing Factor IX [15"] and PS2 [17"] the protein levels were about 100- and 30-fold lower than predicted from the RNA levels in the mammary gland. This suggests that these two proteins may be unstable in milk or poorly secreted and/or that the mRNAs are poorly translated.
Livestock The transgenic mouse serves as a valuable model system for evaluating the feasibility of producing high levels of pharmacologically active proteins in milk. However, to scale up production to a practical level those pharmaceuticals should be expressed in the mammary gland of livestock. So far, three transgenes encoding pharmacologically active proteins have been tested in animals other than mice; interleukin 2 in rabbits [7"] and Factor IX and an czl-antitrypsin in sheep [15"]. The expression levels reported in those studies, however, are still too low to be of economic value. The lead time needed to generate transgenic livestock, in particular cows, for use as bioreactors in the production of biopharmaceuticals would be at least 3 years. However, this represents only a small portion of the total time needed to take those products through clinical trials and finally to the marketplace; the first biopharmaceutical products to be introduced into the marketplace from a transgenic cow are probably at least a decade away, partly because of regulatory requirements.
Increasing milk protein content Products from transgenic cows engineered to produce higher levels of endogenous milk proteins could reach
Transgenic animals Hennighausen, Ruiz, Wall the market soon. The yield of cheese from milk is dependent on the casein content of milk. Casein represents 78% of the total protein in milk, the remainder being whey proteins. The USA dairy industry produces approximately five billion pounds of cheese per year. Increasing the 061-casein content of milk from transgenic cows would result in increased cheese yield (Fig. 1). A 20% increase in the czSl-casein content, for example, would represent $190 million per year to the dairy industry.
Cheese I0,5
pression. Specifically, it will be necessary to isolate genetic elements that enable the transgene to be expressed in a manner that is largely independent of the site at which it is integrated, or to develop methods of site-specific integration. That is to say that not all of the elements that promote and enhance expression of any single milk protein are yet fully known and under control. The vision of a herd of grazing farm animals replacing an industrial complex remains an inviting one and efforts should be increased to generate the mammary bioreactor.
yield A n n o t a t e d references
10.25 10
• ••
Of interest Of outstanding interest
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2.
PALMITERRD, BRINSTERRL: Germ-line transformation of mice. A n n Rev Genet 1986, 20:465-499.
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CAMPBELLSM, ROSEN JM, HENNIGHAUSEN LG, STRECH-JURKU, SIPPEL RE: Comparison of the w h e y acidic protein g e n e s of t h e rat and mouse. Nucl Acid Res 1984, 12:8685-8697.
4.
YU-LEEL, RICHTERMANN L, COUCH CH, STEWARTAF, MACKINLEY AG, ROSEN JM: Evolution of t h e casein multigene family: conserved s e q u e n c e s in t h e 5' flanking and e x o n regions. Nucl Acid Res 1986, 14:18891902.
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JONESWK, YU-LEE L, CLIFT SM, BROWN TL, ROSENJM: The rat casein multigene family. J Biol Chem 1985, 260:7042-7050.
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ALl S, CLARK AJ: Characterization of t h e gene encoding ovine [3-1actoglobulin: similarity to the genes for retinolbinding protein and other secretory proteins. J Mol Biol 1988, 199:415-426.
9.75
9.5
0
I
I
I
10
20
30
40
% o~Sl-casein increase
Fig. 1. The effects on cheese yield of increasing czSl-casein concentration where yield is defined as kg 35% moisture cheese per lOOkg milk.
Efforts have been made to increase the content of specific milk proteins in milk of transgenic mice. Animals carrying the genes for ovine ~-lactoglobulln [12], bovine 0tlactalbumin [10.], guinea pig c~-lactalbumin (Maschio et al., submitted), mouse WAP (Burdon et al., unpublished) and rat WAP secrete the encoded proteins into milk at the same levels as their endogenous counterparts, i.e. about 1-4 g/liter milk. Efforts to increase casein concentration by introducing the rat [3-casein gene into mice have so far been unsuccessful [9], probably because of the absence of key regulatory elements in the proximity of this casein gene. However, it should be possible to overexpress caseins by fusing their structural sequences with whey protein gene regulatory dements.
Outlook Although it is now possible to produce pharmacologically active proteins in the milk of transgenic animals, the mammary gland is not yet economically viable as a bioreactor. The primary limitation is that present systems have not yet reached the stage at which the DNA transferred to the egg will yield a sufficiently high level of mammary-specific expression independent of its integration site. Overcoming this limitation requires a better understanding of the genomic regulatory elements that serve to guide ex-
7. •
BOHLERTA, BRUYERE T, WENT DF, STRANZINGER G, BORKa K: Rabbit ~-casein p r o m o t e r directs secretion of hum a n interleukin-2 into t h e milk of transgenic rabbits. Bit/Technology 1990, 8:140~143. A 2 kb promoter fragment from the rabbit ~-casein gene was ligated to genomic sequences encoding h u m a n interleukin-2 and the hybrid gene was expressed in four transgenic rabbits. Interleukin-2 was secrete(] into milk in quantities of 50-400 ng/ml, which is at least 104-fold lower than the endogenous G-casein. 8.
PITnUS C'XX/,HENNIGHAUSENL, LEE E, WESTPHALH, NICOLS E, V1TALE J, GORDON K: A milk protein gene p r o m o t e r directs t h e expression of h u m a n tissue plasminogen activator eDNA to t h e m a m m a r y gland in transgenic mice. Proc Natl Acad Sci USA 1988, 85:5874-5878.
9.
LEE K-F, DEMAYO FJ, ATLEE SH, ROSEN JM: Tissue-specific expression of t h e rat ~-casein g e n e in transgenic mice. Nucl Acid Res 1988, 16:1027-1041.
VILOTrEJ-L, SOULIER S, STINNAKREM-G, MASSOUD M, MERCIER J-C: Efficient tissue-specific e x p r e s s i o n of bovine ~-lactalb u m i n in transgenic mice. Eur J Biochem 1989, 186:43-48. Transgenic mice carrying the bovine ct-lact-albumin gene including 750bp and 336bp of 5' and 3' flanking regions, respectively, were generated. Mammary specificity was obtained and some mice expressed the transgene at levels close to those found for the endogenous counterpart in bovine. 10. •
11.
GORDON K, LEE E, VITALE JA, SMITH AE, WESTPHAL H, t]£NNIGHAUSEN L: Production of h u m a n tissue plasminogen activator in transgenic m o u s e milk. Bit/Technology 1987, 5:1183-1187.
77
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Expressionsystems 12.
LEE KF, ATIEE SH, ROSEN JM: Differential regulation of rat beta-casein chloramphenicol acetyltransferase fusion g e n e expression in transgenic mice. Mol Cell Bio11989, 9:560-565.
13.
SIMONSJP, MCCLENAGHANM, CLARKAJ: Alteration of t h e quality of milk by expression of s h e e p ]3-1actoglobulin in transgenic mouse. Nature 1987, 328:530-532.
14.
BAYNAEM, ROSEN JM: Tissue specific high-level expression of the rat w h e n acidic protein g e n e in transgenic mice. Nucl Acids Res 1990, 18:2977-2985
15. •
CLARKAJ, BESSOS H, BISHOP JO, BROWN P, HARRIS S, LATHE R, MCCLENAGHANM, PROWSE C, SIMONS JP, WHITELAW CBA, WILMUT I: Expression of h u m a n anti-hemophilic Factor IX in t h e milk of transgenic sheep. Bio/Technology 1989, 7:487-492. A hybrid gene containing the entire ovine 13-1actoglobulin gene with a cDNA sequence encoding h u m a n Factor IX inserted into the 5' untranslated region of the first exon was expressed in two transgenic ewes. Factor IX was secreted into milk at a concentration of 25 ng/ml. Yu S-H, DEEN KC, LEE E, ITIENNIGHAUSEN L, SWEET RW, ROSENBERGM, WESTPHAL H: Functional h u m a n CD4 protein p r o d u c e d in milk of transgenic mice. Mol Biol Med 1989, 6:255-261. A hybrid gene containing 1.8 kb of the promoter region of the m o u s e WAP gene and a cDNA encoding the soluble form of the human CD4 Tcell receptor was expressed in the mammary glands of transgenic mice. Low levels of the active CD4 protein were found in the milk of some of the animals.
A fusion gene containing 20 kb of the bovine c61-casein promoter, genomic sequences encoding human urokinase and exonic, intronic and 3' flanking sequences of the bovine c61-casein gene was expressed in the mammary gland of one transgenic mouse. The expression level was about 20% of the endogenous gene. 19.
ARCHIBALDAL, MCCLENAGHANM, HORNSEYV, SIMONSJP, CLARK AJ: High-level expression of biologicaUy active h u m a n atlantitrypsin in t h e milk of transgenic mice. Proc Natl Acad Sci USA, in press. A fusion gene consisting of the ovine ~-lactoglobulin gene promoter and genomic sequences encoding h u m a n ~l-antitrypsin was expressed in transgenic mice. Out of 13 independent mouse lines, five expressed the gene in the mammary gland, five in the salivary glands and two in both these tissues. Mice from four of these lines produced at least 5 m g / m l of an antitrypsin in their milk. The protein from transgenic m o u s e milk was similar in size to h u m a n plasma derived al-antitrypsin and had similar activity. •
20.
BRINSTERRL, ALLENJM, BEHRINGER RR, GELINAS RE, PALMITER RD: Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA 1988, 85:83~840.
21.
DEVINOY E, HUBERT C, JOt/VET G, THEPOT D, CLERQUE N, DESALEUX M, DION M, SERVELYJ-L, HOUDEBINE L-M: Recent data o n the structure of rabbit milk protein g e n e s and o n the m e c h a n i s m of t h e h o r m o n a l control of their expression. Reprod Nutr Develop 1988, 28:1145-1164.
22.
GROSVELD F, VAN ASSENDELFT GB, GREAVES DR, KOLLIAS G: Position-dependent, high-level expression of the h u m a n 9globin g e n e in transgenic mice. Cell 1987, 51:975-985.
16. •
17. •
TOMASETrO C, WOLF C, RIO M-C, MEHTALI M, LEMEuR M, GERLINGERP, CHAMBON P, LATHE Pc Breast cancer protein PS2 synthesis in m a m m a r y gland of transgenic mice and secretion into milk. Mol Endocrinol 1989, 3:1579-1584. Two hybrid genes were generated and expressed in transgenic animals. The transgene containing the m o u s e WAP gene promoter linked to genomic sequence encoding the protein PS2 was not expressed in the seven lines tested. O n e animal with a transgene consisting of the WAP genomic sequence with the PS2 cDNA integrated into the 5' untranslated region of the first exon was expressed at a level of about 2% of the endogenous WAP gene. 18. •
MEADEH, GATES L, LACY E, LONBERG N: Bovine c~Sl-casein gene s e q u e n c e s direct high level expression of h u m a n urokinase in m o u s e milk. Bio/Technology 1990, 8:443-446.
Annotated patents • •• P1. .
Of interest Of outstanding interest
BIOGENNV: Mammalian milk containing recombinant protein also, n e w DNA s e q u e n c e s containing a g e n e u n d e r control of milk specific p r o m o t e r and transgenic animals. 23/6/87 87US-065994. 27/12/89 EP-347431 A. The transgenic mouse o n which this patent is based is described in [18"].
Introduction Purification of pharmacologically active proteins such as coagulation Factors VIII and IX from human blood is expensive, and the possibility of contamination with pathogenic human viruses still persists. The isolation of genes encoding a number of medically important proteins has permitted cheaper production of these highunit-value proteins in heterologous systems. An appealing expression system for such proteins is the transgenic animal. By introducing foreign genes into the germline of animals it is possible to generate animals that have altered phenotypes [1,2]. The selective attachment of specific DNA control elements to the foreign genes makes it possible to target expression of the proteins they encode to different tissues or body fluids of transgenic animals. It is advantageous to be able to harvest proteins from body fluids rather than from solid tissue because fluids are renewable. Several body fluids come to mind as a suitable source of foreign proteins in transgenic animals: blood, urine, saliva and milk. It is possible to direct protein secretion into blood by targeting gene expression either to the liver or to lymphocytes. This system is probably not viable because the volume of blood that can be harvested is limited and high levels of circulating biologically active proteins may prove detrimental to the animal's health. Similarly, large-scale harvesting of saliva may be problematic. Urine is an abundant fluid but is protein poor. Therefore, synthesis of foreign proteins in the mammary gland with secretion into the milk appears to be a more realistic approach. Isolation of milk protein genes that are expressed at high levels in the mammary glands has provided an experimental way to manipulate the trait of milk composition in transgenic animals. It may be possible to increase the concentration of caseins, thereby improving cheese production. Furthermore, by targeting the expression of valuable non-milk proteins to the mammary gland, this organ could serve as a 'bioreactor' for producing phar-
macologically active proteins on a large scale. Transgenic animals could become an economically viable alternative to existing tissue culture systems. This review highlights the progress made in the expression of foreign genes in the mammary gland of transgenic animals and discusses some of the economic aspects of the mammary gland bioreactor.
Milk protein genes Milk proteins are synthesized by the mammary gland and secreted into milk in gram quantities per liter. Several milk protein genes have been isolated [3-6,7 °] and it is found that their expression is essentially limited to functional mammary epithelial cells [8,9,10"]. Studies in transgenic animals have shown that promoter sequences from the milk protein genes encoding the mouse whey acidic protein (WAP) [8,11] and rat 13-casein [12] contain mammary regulatory elements and confer mammary specificity to genes they are ligated to, and which are normally not expressed in the mammary gland. However, these fusion genes are expressed at levels well below those of milk protein genes. This suggests the presence of additional, and not yet identified, control elements outside the sequences that act as promoters. Evidence for the existence of such elements comes from studies in which WAP genomic sequences with 5' and 3' flanking sequences have been introduced into transgenic mice, some of which express the transgene at levels similar to the endogenous WAP genes [10-,13,14] (Maschio et al., submitted). In contrast, a sequence containing the rat [3-casein gene with its 5' and 3' flanking sequences was expressed in trans o genic mice at less than 1% of its endogenous level [9], suggesting that some of its regulatory elements are located outside the cloned promoter region. Additional studies have shown that the regulatory elements that target gene expression to the mammary gland are functional across species boundaries, suggesting that they are derived from a common ancestor. Thus, the
Abbreviation
WAP--whey acidic protein. 74
~ Current Biology Ltd ISSN 0958-1669
Transgenic animals Hennighausen, Ruiz, Wall WAP gene. In contrast, none of the hybrid genes with • a 13-casein gene promoter has been expressed at high levels (Table 1) [12] suggesting that the WAP, but not the promoter proximal region of the 13-casein gene, contains control elements for high-level expression. A hybrid gene containing bovine ¢~Sl-casein promoter and structural gene sequences was expressed at about 10% of the endogenous level in one transgenic mouse (Table 1) [18,, P1 o]. Because only one mouse was analyzed and a similar aSl-casein promoter from rabbit was non-functional (Houdebine, personal communication), it is not clear that the cxSl-casein promoter contains the regulatory elements that are necessary for integration-site-independent high-level expression.
ovine 13-1actoglobulin gene can be expressed in transgenic mice [13] and the mouse WAP gene in transgenic pigs (Wall and Hennighausen, unpublished) despite the absence of corresponding endogenous genes in these species.
Production of foreign proteins in milk of transgenic animals The demand for a number of therapeutically important proteins cannot be matched by current production and isolation procedures; the mammary gland may solve this dearth by producing the proteins on a large scale and at a reasonable cost. To produce a pharmacologically active protein in the milk of transgenic animals the DNA sequence encoding this protein has to be linked to manunary-specific regulatory elements and the resulting hybrid gene has to be introduced into the germline of transgenic animals. Although several cloned milk protein gene promoters have been shown to direct the expression of heterologous genes in the mammary gland (Table 1) [7,15°,16°,17°,18°,19,], there appear to be quantitative differences. Hybrid genes containing promoters of the mouse (Table 1) and rabbit (Houdebine, personal communication) WAP genes can be expressed at levels up to 100% of those found for the endogenous
The protein coding sequence, or structural gene component of the hybrid gene, can be introduced as a cDNA or as a genomic sequence that contains introns and exons. As introns may be necessary for regulated gene expression in transgenic animals [20], many investigators be lieve that it is advisable, whenever possible, to use the genomic form rather than the cDNA form of the structural gene. For example, hybrid genes containing the ovine 13lactoglobulin gene promoter and a cDNA encoding human al-antitrypsin are expressed at low levels, whereas those containing genomic sequences encoding al-antitrypsin are expressed at levels similar to those of the endogenous 13-1actoglobulin gene [19 °]. However, in some
l Table 1. Expression of foreign proteins in milk. Transgenic animals Transgene
Encoded protein
Number
Expressors
Concentration (pg/ml)
Reference
WAP-hGH
Growth hormone
11
4
4-400
Reddy eta/., submitted
WAP-hGH
Growth hormone
8
4
1000-2000
G[Jnzburg et al., submitted
WAP-PS2 I
PS2
7
0
WAP-tPA
Plasminogen activator
6
4
0.4-100
WAP-CD4
CD4 receptor
7
5
0.4
[16.1
WAP-PS2 II
PS2
1
1
40
[17-]
WAP-hPC
Protein C
3
3
0.2
Valander et al., unpublished
~CAS-hUK
Urokinase
1
1
1000
[18-]
~CAS-hlL2
Interleukin 2
4
4
0.001-0.01
[7°]
~LAC-FIX
Clotting Factor IX
2
2
0.01
[15 .]
[3LAC-0~I-AT I
0d-antitrypsin
2
2
5
[15"]
~-~I-AT II
czl-antitrypsin
13
7
6-7000
[19 "]
[17"1 [8,10
]
75
76
Expressionsystems hybrid genes containing the WAP gene promoter, expression of structural genes containing introns does not appear to be different from cDNA expression. The WAP PS2I gene (Table 1) containing genomic sequences of PS2 (a breast cancer protein) as the structural part of the hybrid gene construct did not give rise to expression in seven lines of transgenic mice [17-]. In contrast, the WAP-PSII gene containing the PS2 cDNA was expressed at significant levels [17-]. So far, the importance of introns for high-level expression in transgenic animals has not been studied extensively and remains uncertain. Moreover, in some cases it will be necessary to use structural genes that are devoid of introns simply because the introns may contain regulatory elements targeting expression to non-mammary tissue which, in turn, could compromise the animal's health. Post-translational modification and stability of human proteins secreted into milk has not been studied in detail. From the limited studies available [15,], it appears that Factor IX and tissue plasminogen activator secreted into milk have biological activity but may not be as stable as the native proteins isolated from blood and may not be post-translationally modified in the same way. Although the caseins and other milk proteins are produced specifically in the mammary gland, transgenic animals carrying hybrid genes with a milk protein gene promoter may not show such clear specificity. Novel genetic regulatory elements may be generated through a combination of promoter and structural DNA sequences [1,2]. This appears to have happened in a hybrid gene containing the mouse WAP gene promoter and genomic sequences encoding human growth hormone (Table 1); the hybrid gene was ectopically expressed in Bergman glial cells (GOnzburg et al., submitted).
Expression levels At least 12 hybrid genes have been introduced into transgenic animals with the aim of producing a human protein in milk (Table 1) [19"]. The DNA control elements guiding expression of these genes to the mammary gland were derived from the genes for mouse WAP [3], bovine 0tSl-casein [18.], rabbit IB-casein [20] and ovine 13-1actoglobulin [6,19,]. In most cases, expression of the transgene was found in the mammary gland but the levels of the encoded proteins secreted into milk differed widely among lines of animals and were generally much lower than those of the endogenous milk proteins. The notable exception was a hybrid gene containing the promoter/upstream region of the ovine 13-1actoglobulin gene and a promoterless minigene encoding human czl-antitrypsin [19"]. Some ,of the mice carrying this fusion gene secreted czl-antitrypsin into milk at concentrations of more than 5mg/ml [19o]. The large differences in expression of a particular transgene between individual lines of animals are probably a result of position effects; surrounding sequences at different integration sites of transgenes may modulate their ex-
pression levels. This is a well described phenomenon and has been observed in basically all transgenic animal systems [1,2]. Recent finding regarding [3-globin gene regulation may help to overcome the inconsistent expression of transgenes. Grosveld et al. [22] have identified sequences at the 5' end of the globin locus that can function as a dominant control region or locus-activating region and confer integration-site independent high-level gene expression in erythroid cells of transgenic mice. Such elements may also exist in the vicinity of milk protein genes, and considerable efforts should be made to identify and isolate them. In addition, elements conferring post-transcriptional regulation, which have not yet been identified, may be required for the high-level expression of native milk protein genes. A further complication in assessing expression levels could be poor translation of the transgenic mRNAs and/or instability of the human proteins in milk. In the case of tissue plasminogen activator [8,11] and the CD4 receptor [16.] the amount of both biologically active and immunologicaUy reactive protein in milk correlated well with the RNA levels, suggesting that the proteins were stable and the RNAs were translated eiticiently. In contrast, in transgenic animals producing Factor IX [15"] and PS2 [17"] the protein levels were about 100- and 30-fold lower than predicted from the RNA levels in the mammary gland. This suggests that these two proteins may be unstable in milk or poorly secreted and/or that the mRNAs are poorly translated.
Livestock The transgenic mouse serves as a valuable model system for evaluating the feasibility of producing high levels of pharmacologically active proteins in milk. However, to scale up production to a practical level those pharmaceuticals should be expressed in the mammary gland of livestock. So far, three transgenes encoding pharmacologically active proteins have been tested in animals other than mice; interleukin 2 in rabbits [7"] and Factor IX and an czl-antitrypsin in sheep [15"]. The expression levels reported in those studies, however, are still too low to be of economic value. The lead time needed to generate transgenic livestock, in particular cows, for use as bioreactors in the production of biopharmaceuticals would be at least 3 years. However, this represents only a small portion of the total time needed to take those products through clinical trials and finally to the marketplace; the first biopharmaceutical products to be introduced into the marketplace from a transgenic cow are probably at least a decade away, partly because of regulatory requirements.
Increasing milk protein content Products from transgenic cows engineered to produce higher levels of endogenous milk proteins could reach
Transgenic animals Hennighausen, Ruiz, Wall the market soon. The yield of cheese from milk is dependent on the casein content of milk. Casein represents 78% of the total protein in milk, the remainder being whey proteins. The USA dairy industry produces approximately five billion pounds of cheese per year. Increasing the 061-casein content of milk from transgenic cows would result in increased cheese yield (Fig. 1). A 20% increase in the czSl-casein content, for example, would represent $190 million per year to the dairy industry.
Cheese I0,5
pression. Specifically, it will be necessary to isolate genetic elements that enable the transgene to be expressed in a manner that is largely independent of the site at which it is integrated, or to develop methods of site-specific integration. That is to say that not all of the elements that promote and enhance expression of any single milk protein are yet fully known and under control. The vision of a herd of grazing farm animals replacing an industrial complex remains an inviting one and efforts should be increased to generate the mammary bioreactor.
yield A n n o t a t e d references
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Of interest Of outstanding interest
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2.
PALMITERRD, BRINSTERRL: Germ-line transformation of mice. A n n Rev Genet 1986, 20:465-499.
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CAMPBELLSM, ROSEN JM, HENNIGHAUSEN LG, STRECH-JURKU, SIPPEL RE: Comparison of the w h e y acidic protein g e n e s of t h e rat and mouse. Nucl Acid Res 1984, 12:8685-8697.
4.
YU-LEEL, RICHTERMANN L, COUCH CH, STEWARTAF, MACKINLEY AG, ROSEN JM: Evolution of t h e casein multigene family: conserved s e q u e n c e s in t h e 5' flanking and e x o n regions. Nucl Acid Res 1986, 14:18891902.
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JONESWK, YU-LEE L, CLIFT SM, BROWN TL, ROSENJM: The rat casein multigene family. J Biol Chem 1985, 260:7042-7050.
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ALl S, CLARK AJ: Characterization of t h e gene encoding ovine [3-1actoglobulin: similarity to the genes for retinolbinding protein and other secretory proteins. J Mol Biol 1988, 199:415-426.
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0
I
I
I
10
20
30
40
% o~Sl-casein increase
Fig. 1. The effects on cheese yield of increasing czSl-casein concentration where yield is defined as kg 35% moisture cheese per lOOkg milk.
Efforts have been made to increase the content of specific milk proteins in milk of transgenic mice. Animals carrying the genes for ovine ~-lactoglobulln [12], bovine 0tlactalbumin [10.], guinea pig c~-lactalbumin (Maschio et al., submitted), mouse WAP (Burdon et al., unpublished) and rat WAP secrete the encoded proteins into milk at the same levels as their endogenous counterparts, i.e. about 1-4 g/liter milk. Efforts to increase casein concentration by introducing the rat [3-casein gene into mice have so far been unsuccessful [9], probably because of the absence of key regulatory elements in the proximity of this casein gene. However, it should be possible to overexpress caseins by fusing their structural sequences with whey protein gene regulatory dements.
Outlook Although it is now possible to produce pharmacologically active proteins in the milk of transgenic animals, the mammary gland is not yet economically viable as a bioreactor. The primary limitation is that present systems have not yet reached the stage at which the DNA transferred to the egg will yield a sufficiently high level of mammary-specific expression independent of its integration site. Overcoming this limitation requires a better understanding of the genomic regulatory elements that serve to guide ex-
7. •
BOHLERTA, BRUYERE T, WENT DF, STRANZINGER G, BORKa K: Rabbit ~-casein p r o m o t e r directs secretion of hum a n interleukin-2 into t h e milk of transgenic rabbits. Bit/Technology 1990, 8:140~143. A 2 kb promoter fragment from the rabbit ~-casein gene was ligated to genomic sequences encoding h u m a n interleukin-2 and the hybrid gene was expressed in four transgenic rabbits. Interleukin-2 was secrete(] into milk in quantities of 50-400 ng/ml, which is at least 104-fold lower than the endogenous G-casein. 8.
PITnUS C'XX/,HENNIGHAUSENL, LEE E, WESTPHALH, NICOLS E, V1TALE J, GORDON K: A milk protein gene p r o m o t e r directs t h e expression of h u m a n tissue plasminogen activator eDNA to t h e m a m m a r y gland in transgenic mice. Proc Natl Acad Sci USA 1988, 85:5874-5878.
9.
LEE K-F, DEMAYO FJ, ATLEE SH, ROSEN JM: Tissue-specific expression of t h e rat ~-casein g e n e in transgenic mice. Nucl Acid Res 1988, 16:1027-1041.
VILOTrEJ-L, SOULIER S, STINNAKREM-G, MASSOUD M, MERCIER J-C: Efficient tissue-specific e x p r e s s i o n of bovine ~-lactalb u m i n in transgenic mice. Eur J Biochem 1989, 186:43-48. Transgenic mice carrying the bovine ct-lact-albumin gene including 750bp and 336bp of 5' and 3' flanking regions, respectively, were generated. Mammary specificity was obtained and some mice expressed the transgene at levels close to those found for the endogenous counterpart in bovine. 10. •
11.
GORDON K, LEE E, VITALE JA, SMITH AE, WESTPHAL H, t]£NNIGHAUSEN L: Production of h u m a n tissue plasminogen activator in transgenic m o u s e milk. Bit/Technology 1987, 5:1183-1187.
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Expressionsystems 12.
LEE KF, ATIEE SH, ROSEN JM: Differential regulation of rat beta-casein chloramphenicol acetyltransferase fusion g e n e expression in transgenic mice. Mol Cell Bio11989, 9:560-565.
13.
SIMONSJP, MCCLENAGHANM, CLARKAJ: Alteration of t h e quality of milk by expression of s h e e p ]3-1actoglobulin in transgenic mouse. Nature 1987, 328:530-532.
14.
BAYNAEM, ROSEN JM: Tissue specific high-level expression of the rat w h e n acidic protein g e n e in transgenic mice. Nucl Acids Res 1990, 18:2977-2985
15. •
CLARKAJ, BESSOS H, BISHOP JO, BROWN P, HARRIS S, LATHE R, MCCLENAGHANM, PROWSE C, SIMONS JP, WHITELAW CBA, WILMUT I: Expression of h u m a n anti-hemophilic Factor IX in t h e milk of transgenic sheep. Bio/Technology 1989, 7:487-492. A hybrid gene containing the entire ovine 13-1actoglobulin gene with a cDNA sequence encoding h u m a n Factor IX inserted into the 5' untranslated region of the first exon was expressed in two transgenic ewes. Factor IX was secreted into milk at a concentration of 25 ng/ml. Yu S-H, DEEN KC, LEE E, ITIENNIGHAUSEN L, SWEET RW, ROSENBERGM, WESTPHAL H: Functional h u m a n CD4 protein p r o d u c e d in milk of transgenic mice. Mol Biol Med 1989, 6:255-261. A hybrid gene containing 1.8 kb of the promoter region of the m o u s e WAP gene and a cDNA encoding the soluble form of the human CD4 Tcell receptor was expressed in the mammary glands of transgenic mice. Low levels of the active CD4 protein were found in the milk of some of the animals.
A fusion gene containing 20 kb of the bovine c61-casein promoter, genomic sequences encoding human urokinase and exonic, intronic and 3' flanking sequences of the bovine c61-casein gene was expressed in the mammary gland of one transgenic mouse. The expression level was about 20% of the endogenous gene. 19.
ARCHIBALDAL, MCCLENAGHANM, HORNSEYV, SIMONSJP, CLARK AJ: High-level expression of biologicaUy active h u m a n atlantitrypsin in t h e milk of transgenic mice. Proc Natl Acad Sci USA, in press. A fusion gene consisting of the ovine ~-lactoglobulin gene promoter and genomic sequences encoding h u m a n ~l-antitrypsin was expressed in transgenic mice. Out of 13 independent mouse lines, five expressed the gene in the mammary gland, five in the salivary glands and two in both these tissues. Mice from four of these lines produced at least 5 m g / m l of an antitrypsin in their milk. The protein from transgenic m o u s e milk was similar in size to h u m a n plasma derived al-antitrypsin and had similar activity. •
20.
BRINSTERRL, ALLENJM, BEHRINGER RR, GELINAS RE, PALMITER RD: Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA 1988, 85:83~840.
21.
DEVINOY E, HUBERT C, JOt/VET G, THEPOT D, CLERQUE N, DESALEUX M, DION M, SERVELYJ-L, HOUDEBINE L-M: Recent data o n the structure of rabbit milk protein g e n e s and o n the m e c h a n i s m of t h e h o r m o n a l control of their expression. Reprod Nutr Develop 1988, 28:1145-1164.
22.
GROSVELD F, VAN ASSENDELFT GB, GREAVES DR, KOLLIAS G: Position-dependent, high-level expression of the h u m a n 9globin g e n e in transgenic mice. Cell 1987, 51:975-985.
16. •
17. •
TOMASETrO C, WOLF C, RIO M-C, MEHTALI M, LEMEuR M, GERLINGERP, CHAMBON P, LATHE Pc Breast cancer protein PS2 synthesis in m a m m a r y gland of transgenic mice and secretion into milk. Mol Endocrinol 1989, 3:1579-1584. Two hybrid genes were generated and expressed in transgenic animals. The transgene containing the m o u s e WAP gene promoter linked to genomic sequence encoding the protein PS2 was not expressed in the seven lines tested. O n e animal with a transgene consisting of the WAP genomic sequence with the PS2 cDNA integrated into the 5' untranslated region of the first exon was expressed at a level of about 2% of the endogenous WAP gene. 18. •
MEADEH, GATES L, LACY E, LONBERG N: Bovine c~Sl-casein gene s e q u e n c e s direct high level expression of h u m a n urokinase in m o u s e milk. Bio/Technology 1990, 8:443-446.
Annotated patents • •• P1. .
Of interest Of outstanding interest
BIOGENNV: Mammalian milk containing recombinant protein also, n e w DNA s e q u e n c e s containing a g e n e u n d e r control of milk specific p r o m o t e r and transgenic animals. 23/6/87 87US-065994. 27/12/89 EP-347431 A. The transgenic mouse o n which this patent is based is described in [18"].
