Rabbit β-Casein Promoter Directs Secretion of Human Interleukin-2 into the Milk of Transgenic Rabbits

Bühler, Th.; Bruyère, Th.; Went, Dirk F.; Stranzinger, G.; Bürki, Kurt

doi:10.1038/nbt0290-140

Cited by 105 publications

(32 citation statements)

References 23 publications

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“…4), which is rich in sebaceous glands. (24), the rat WAP (25) and /-casein (26) genes, and several hybrid genes containing mammary regulatory elements (27)(28)(29)(30) (31). However, the concentrations of the encoded proteins factor IX and a1-antitrypsin were only 25 pg/liter and 5 mg/liter, respectively (31).…”

Section: Resultsmentioning

confidence: 99%

High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine.

Wall

Pursel

Shamay

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

133

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The whey acidic protein (WAP) is a major milk protein in mice, rats, and rabbits but has not been found in milk of livestock including swine. To determine whether mammary gland regulatory elements from the WAP gene function across species boundaries and whether it is possible to qualitatively alter milk protein composition, we introduced the mouse WAP gene into the genome of swine. Three lines of transgenic swine were analyzed, and mouse WAP was detected in milk from all lactating females at concentrations of about 1 g/liter; these levels are similar to those found in mouse milk.Expression of the corresponding RNA was specific to the mammary gland. Our results suggest that the molecular basis of mammary-specific gene expression is conserved between swine and mouse. In addition the WAP gene must share, with other milk protein genes, elements that target gene expression to the mammary gland. Mouse WAP accounted for about 3% of the total milk proteins in transgenic pigs, thus demonstrating that it is possible to produce high levels of a foreign protein in milk of farm animals.Milk protein genes are transcribed in the mammary gland of lactating animals, and the encoded proteins are secreted in large quantities into milk. The whey acidic protein (WAP) is an abundant milk protein in mice (1, 2) but has not been found in swine or other livestock. Expression of the WAP gene is confined to the mammary gland (2, 3) and is under the control of steroid and peptide hormones as well as other developmental signals during pregnancy (4-6).By targeting synthesis of foreign proteins to the mammary gland of transgenic animals, it should be possible to produce valuable proteins on a large scale in milk (7,8 Swine were chosen for these studies because they offer both economy in animal resources and time when compared to ruminantia as a transgenic animal model and because the questions being addressed did not require harvesting large quantities of milk that would be more easily obtained from dairy animals such as cows, goats, or sheep. The two primary constraints in any large animal transgenic project are the number of fertilized ova obtainable and the number of embryo recipients available. On average it is possible to recover 2-3 times more injectable ova per donor gilt than can be collected from a cow, doe, or ewe. The efficiency of producing expressing transgenic pigs or sheep per injected ovum is about 0.3% (calculated from refs. 11 and 12). Though a live-born-expressing transgenic calf has not been reported, a larger number of ova will probably be required to produce an expressing transgenic cow (13). Furthermore, because swine are polytocous, a recipient sow can carry 5 times as many fetuses as a cow, doe, or ewe. Additionally, the generation interval of swine is -11 months, whereas that of goats is between 11 and 21 months and that of cattle at least 24 months. Considering all of these factors, the use of swine rather than cows, goats, or sheep requires one-sixth the number of animals, with results obtainable in less th...

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Section: Resultsmentioning

confidence: 99%

High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine.

Wall

Pursel

Shamay

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

133

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“…However, assigning a clear biological significance to a far distal CoRE in the aS1-casein promoter is in keeping with previous experiences gained, when casein promoters were used to drive mammary gland-specific expression of transgenes. It was found that short rat b-casein promoter constructs (!3 kbp) express the transgene only at a low level in the mammary gland (Lee et al 1988, Bü hler et al 1990, while high level and lactationally stimulated expression was observed using 3 . 8 kbp (but not 1 .…”

Section: The Far Upstream Enhancer Is Functionally Significant In Vivomentioning

confidence: 99%

DNA-remethylation around a STAT5-binding enhancer in the αS1-casein promoter is associated with abrupt shutdown of αS1-casein synthesis during acute mastitis

Vanselow

Yang²,

Herrmann³

et al. 2006

Journal of Molecular Endocrinology

109

114

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Prolactin stimulates the expression of milk genes during lactation through the activation of STAT5 transcription factors, which subsequently bind to their cognate target sequence on the promoters. Demethylation of 5methylCpG dinucleotides permits the tissue-specific accessibility of transcription factor-binding sites during development, but remethylation has not been shown to contribute to acute suppression of gene expression. We characterize functionally a novel STAT5-binding lactational enhancer in the far upstream promoter (wK10 kbp) of the bovine aS1-casein-encoding gene. This promoter area is hypomethylated in the lactating udder only. Remethylation of this area accompanies an experimentally elicited acute shutdown of casein synthesis in fully lactating cows, whose udder quarters have experimentally been infected with a pathogenic E. coli strain. Within 24 h after infection, the relevant promoter area was remethylated from 10% of the DNA molecules in the uninfected control quarters to w50% in the infected quarters, the typical values for fully lactating and not lactating udders respectively. Increased methylation resulted in tighter chromatin packing. Concomitantly, the aS1-casein mRNA concentration dropped to w50% while the protein synthesis was shut down to w2 . 5% in the infected quarters, alone. The methylation status of the promoter from a not lactationally regulated gene was unaltered, and the distal aS1-casein promoter was not remethylated in udder quarters with subclinical Staphylococcus aureus infections featuring sustained casein synthesis. Hence, infection-related remethylation of the aS1-casein promoter and chromatin remodelling serves as an acute, spatially restricted regulatory mechanism, which might insulate the promoter against the systemically unchanged high levels of circulating prolactin. This provides a rare example for an acute regulatory significance of CpG methylation.

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“…Among these proteins, the human C1 inhibitor and alpha-glucosidase reached the phase III clinical trials (http:// www.pharming.com/, Van den Hout, 2004). Buhler et al, 1990 Human growth hormone Mouse whey acidic protein-genomic DNA 0.05 g/l. hGH in the serum.…”

Section: Transgenic Rabbits As Bioreactors Pharmaceutical Proteins Prmentioning

confidence: 99%

Application of rabbits in biomedical research: a review.

Bösze¹,

Houdebine²

2010

World rabbit sci.

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The first transgenic rabbits were obtained two decades ago by pronuclear microinjection. Several characteristics of rabbit made it the first and classical model for the study of lipoproteins and atherosclerosis. Rabbit models include normal cholesterol-fed rabbits, spontaneous mutants for lipid metabolism and transgenic rabbits. Though most molecular investigations of the cardiovascular system have used transgenic mice, the small rodents do not accurately reflect crucial facets of human cardiovascular physiology, therefore a number of different transgenic rabbit models of hypertrophic cardiomyopathy were created. Transgenic rabbits have been found to be suitable bioreactors for the production of pharmaceutical proteins filling an important niche between the laboratory mouse and larger farm mammals. It is the smallest animal that can be used to produce recombinant proteins in its milk or serum both on an experimental and a commercial scale. The rabbit appears particularly flexible for the preparation of human antibodies, and recombinant human proteins for replacement therapies have also been produced in rabbit milk. A specific biotechnology of the rabbit is emerging. The scientific community which uses rabbits as experimental animals or as a tool to produce biotech products, as well as those involved in breeding, are invited to focus their efforts on this species.

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Rabbit β-Casein Promoter Directs Secretion of Human Interleukin-2 into the Milk of Transgenic Rabbits

Cited by 105 publications

References 23 publications

High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine.

High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine.

DNA-remethylation around a STAT5-binding enhancer in the αS1-casein promoter is associated with abrupt shutdown of αS1-casein synthesis during acute mastitis

Application of rabbits in biomedical research: a review.

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