Assignment of the porcine GLUL gene to the distal end of chromosome 9q.
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Animals↗
Biomedical subjects
M McClenaghan
Publications and source records attributed to M McClenaghan.
Insertion of a casein kinase recognition sequence induces phosphorylation of ovine beta-lactoglobulin in transgenic mice.
We have shown that the cellular mechanisms of the mammary gland can be used to produce a phosphorylated form of a normally unphosphorylated milk protein. This was achieved by the insertion of a beta-casein DNA sequence coding for a group of mammary gland casein kinase recognition sites into ovine beta-lactoglobulin. Transgenic mice carrying this modified gene were generated and lactating females were shown to produce a novel beta-lactoglobulin in their milk. The infrared spectrum, reactivity to antiphosphoserine antibody and reduction of electrophoretic mobility on treatment with alkaline phosphatase showed that the novel protein recovered from the milk whey (serum) was phosphorylated and molecular mass determination by mass spectrometry was consistent with the phosphorylation of one or two residues. A similar level of phosphorylation was measured by quantitative infrared spectroscopy. Centrifugation of the milk to pellet the casein micelles showed that most of the phosphorylated beta-lactoglobulin was in the whey and hence not incorporated into casein micelles.
Amino Acid Sequence↗
Variegated gene expression in mice.
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Animals↗
Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.
Mice carrying an ovine beta-lactoglobulin (BLG) transgene secrete BLG protein into their milk. To explore transgene expression stability, we studied expression levels in three BLG transgenic mouse lines. Unexpectedly, two lines exhibited variable levels of transgene expression. Copy number within lines appeared to be stable and there was no evidence of transgene rearrangement. In the most variable line, BLG production levels were stable within individual mice in two successive lactations. Backcrossing demonstrated that genetic background did not contribute significantly to variable expression. Tissue in situ hybridization revealed mosaicism of transgene expression within individual mammary glands from the two variable lines; in low expressors, discrete patches of cells expressing the transgene were observed. Transgene protein concentrations in milk reflected the proportion of epithelial cells expressing BLG mRNA. Furthermore, chromosomal in situ hybridization revealed that transgene arrays in both lines are situated close to the centromere. We propose that mosaicism of transgene expression is a consequence of the chromosomal location and/or the nature of the primary transgene integration event.
Animals↗
Bovine beta-casein expressed in transgenic mouse milk is phosphorylated and incorporated into micelles.
Transgenic mice carrying the bovine beta-casein gene linked to the promoter sequence of the sheep beta-lactoglobulin milk protein gene were produced. Four of seven lines expressed the transgene protein with mice from the two highest expressing lines producing 4 to 5 mg ml(-1) of this protein in their milk. The foreign protein was associated with the casein micelles and did not segregate into the whey fraction on centrifugation of milk samples. Following purification, the protein was characterized by amino acid analysis, gel electrophoresis, capillary zone electrophoresis, and trypsinolysis. The results showed that the transgene protein was authentic, phosphorylated bovine beta-casein A(1).
Amino Acids↗
Secretory proteins compete for production in the mammary gland of transgenic mice.
To explore the possibility that genes might compete for expression, we have studied transgenic mice producing high levels of the sheep milk protein, beta-lactoglobulin (BLG), in the mammary gland. Mice carrying one or more transgene loci expressed BLG in milk at levels ranging from 7 to 33 mg/ml. The effects of BLG synthesis on the levels of endogenous milk gene expression were examined. No significant increase in total milk protein concentration was recorded even in mice expressing the largest amounts of BLG. Measurement of individual milk proteins showed that transgene protein was manufactured at the expense of host protein synthesized in the gland. Whey acidic protein production was more suppressed than casein production. Suppression of endogenous proteins was matched by a reduction in the corresponding steady-state mRNA levels; in double-transgenic mice, which expressed the largest amounts of BLG, beta-casein and whey acidic protein mRNA populations were reduced to 75 and 56% of control levels respectively. We demonstrate that an exogenous gene competes effectively for expression with endogenous genes. Possible mechanisms of competition are discussed.
Animals↗
Enhancing the efficiency of transgene expression.
Two strategies for enhancing gene expression in transgenic animals are described with particular reference to targeting expression to the mammary gland. Gene constructs in which the protein-encoding DNA sequences are contained within a genomic segment (comprising most or all of the natural introns of the corresponding gene) are shown to be expressed more efficiently than their intronless counterparts. Secondly, co-integrating an otherwise poorly expressed transgene in the vicinity of an actively expressed transgene can dramatically improve its efficiency of expression.
Animals↗
Position-independent expression of the ovine beta-lactoglobulin gene in transgenic mice.
The major milk whey protein of sheep, beta-lactoglobulin (BLG), is expressed specifically in the mammary gland in a developmentally regulated pattern. To identify the cis-acting DNA regions involved in the regulation of BLG expression, resected gene constructs were analysed in transgenic mice. BLG transgenes which contain at least the proximal 406 bp of the 5' flanking region were expressed in all mice analysed, at levels related to transgene copy number, and thus were expressed in a position-independent manner. Expression was restricted to the mammary gland, except in a few lines where low-level expression was also detected in the salivary gland. In these mice, BLG transgenes were expressed during pregnancy and lactation in the appropriate temporal pattern. Further resection of the 5' proximal region to -146 bp resulted in a dramatically reduced frequency of expression, without affecting tissue specificity, while a construct which retained only 79 bp of 5' flanking region was not expressed. Chromatin analysis of isolated sheep nuclei showed that the promoter resides within a DNAaseI-hypersensitive region in the mammary gland but not in the liver. A BLG transgene displayed a similar tissue-specific pattern of DNAaseI hypersensitivity in mice. These data demonstrate an essential role of the proximal DNAaseI-hypersensitive sequences for position-independent expression of the BLG gene.
Animals↗
Mammary development and milk secretion in transgenic mice expressing the sheep beta-lactoglobulin gene.
Mammary development and milk secretion were studied in transgenic mice which exhibited mammary tissue-specific expression of the sheep beta-lactoglobulin gene, and secreted significant quantities of the foreign protein in milk. Mammary development was unaffected by transgenesis. Tissue DNA content and the activities of several key enzyme markers of cell differentiation were similar in transgenic mice and non-transgenic controls. Milk yield, whether estimated by pup weight gain or measured by a 3H2O-dilution method, was unchanged by foreign gene expression. Gross milk composition, including milk protein concentration, was also similar in transgenic and non-transgenic animals, even though beta-lactoglobulin accounted for 29% of total milk protein. Therefore the foreign gene product was synthesized at the expense of endogenous milk proteins. However, transgenic mammary tissue in vitro exhibited a significantly higher rate of total protein synthesis than did control tissue. This suggested that a factor limiting milk protein synthesis or secretion in transgenic mice in vivo may have been removed by short-term explant culture of mammary tissue. The results emphasize that the use of transgenesis for manipulating milk composition may depend not only on high-level mammary-specific expression of the foreign gene, but also on the biosynthetic capacity of the mammary gland itself.
Aging↗
Production of pharmaceutical proteins in milk.
There is every reason to expect that it will be possible within the next few years to begin to use farm animals to produce large quantities of some of the human proteins that are needed for the treatment of disease. Revolutionary new opportunities for the production of novel proteins in milk have been created by the development of methods for gene transfer. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue specific hormonally induced expression in the mammary gland. Studies with three genes, ovine beta-lactoglobulin, rat beta-casein and whey acidic protein of rat and mouse, suggest that they may all meet this requirement. Fragments of the ovine beta-lactoglobulin, murine whey acidic protein and rabbit beta-casein genes have directed production of novel proteins in the milk of transgenic mice, sheep, rabbits and pigs. The proteins were biologically active and usually co-migrated with authentic proteins. In early experiments, protein concentration was low, but our recent observations suggest that fusion genes containing genomic clones direct production of concentrations of protein that are suitable for commercial exploitation. In the longer term, two approaches may offer the potential of more reliable expression. Control elements capable of directing expression that is independent of site of insertion of the gene, but dependent on the number of copies of the gene, have been identified for a small number of genes. The availability of such elements for the milk protein genes would increase the reliability of gene expression considerably. Alternatively, targeted mutation of genes may allow the insertion of coding sequences within an existing gene so avoiding position effects.
Animals↗
Comparison of isolates of Chlamydia psittaci of ovine, avian and feline origin by analysis of polypeptide profiles from purified elementary bodies.
The single species Chlamydia psittaci is a diverse grouping which contains several different types of chlamydial strain for which there is no generally accepted typing method. The results obtained when profiles of polypeptides from purified elementary bodies are compared are consistent with type designations obtained using other criteria. However, the method still requires large scale culture and extensive purification of the chlamydial cells.
Animals↗
Developmental regulation of the sheep beta-lactoglobulin gene in the mammary gland of transgenic mice.
beta-Lactoglobulin (BLG) is the most abundant whey protein in sheep milk but it is not present in mouse milk. We have previously shown that transgenic mice carrying the BLG gene express it specifically in the mammary gland and secrete BLG into milk at high concentrations. Here we demonstrate that BLG transcription is correctly initiated in mice and that BLG synthesis is restricted to the secretory epithelial cells of the mammary gland. We have also determined the temporal pattern of milk protein gene expression and find that the BLG transgene is regulated coordinately with mouse beta-casein and that the patterns of regulation of BLG in mouse and sheep share some similarities.
Animals↗
Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice.
We are studying the tissue-specific expression of the sheep milk-whey protein gene, beta-lactoglobulin. We have used sequences derived from this gene to target the expression of biomedical proteins into milk with the intention to exploit this technology in transgenic sheep as a means of protein production. In the present study, a series of beta-lactoglobulin hybrid genes and beta-lactoglobulin minigenes were evaluated for expression in the mammary gland of transgenic mice. In particular, we have assessed whether there is a requirement for introns for efficient transgene expression in the mammary gland, since the coding sequences of many candidate proteins are available only as cDNAs. The results suggest that the inclusion of natural introns in constructs can enhance the efficiency of transgene expression. Thus, a hybrid construct comprising 4.3 kb of the immediate 5' flanking sequences of beta-lactoglobulin fused to a genomic minigene encoding human alpha-antitrypsin (alpha 1AT) was expressed much more efficiently than an alpha 1AT-cDNA construct containing the same beta-lactoglobulin segment. Similarly, the intact beta-lactoglobulin gene was expressed more efficiently than the corresponding intronless beta-lactoglobulin minigene. This effect was not seen in transient expression experiments in baby hamster kidney cells when beta-lactoglobulin-alpha 1AT constructs were driven by SV40 enhancer sequences. The effect cannot be explained by a simple requirement for splicing, since the inclusion of the first beta-lactoglobulin intron into cDNA constructs encoding human alpha 1AT or beta-lactoglobulin itself failed to enhance the efficiency of transgene expression. It is concluded that sequence elements within introns may interact with the upstream 5' flanking sequences of beta-lactoglobulin and enable the latter to function efficiently in the mammary gland of transgenic mice.
Animals↗
Characterisation of the alleles encoding ovine beta-lactoglobulins A and B.
beta-Lactoglobulin (BLG) is the major whey protein in the milk of ruminants and is produced in the mammary gland during pregnancy and lactation. Here, we compare the nucleotide sequences of two BLG-encoding clones isolated from a sheep genomic library. The two clones are very similar differing by only 1 bp in their coding regions, giving rise to a Tyr/His difference in the gene product, and suggesting that the two clones correspond to A and B allelic variants of BLG. The isoelectric points (pI) of BLGs A and B were estimated as 5.7 and 6.0, respectively. Transgenic mice carrying a particular clone secrete BLG of the expected pI into their milk. Restriction fragment length polymorphism analysis of a small sheep population demonstrated the existence of at least four BLG haplotypes.
Alleles↗
High-level expression of biologically active human alpha 1-antitrypsin in the milk of transgenic mice.
Reduced circulating levels of alpha 1-antitrypsin (alpha 1 AT) are associated with certain alpha 1 AT genotypes and increased susceptibility to emphysema. Unfortunately, the amounts of alpha 1 AT that would be required for replacement therapy are beyond the capacity of plasma fractionation and mammalian cell culture systems. Thus, we have examined the potential of transgenic animals as an alternative means of producing human alpha 1 AT. A hybrid gene constructed by using sequences from the ovine milk protein gene beta-lactoglobulin fused to an alpha 1 AT "minigene" was used to generate transgenic mice. Of 13 independent transgenic mice and mouse lines, 5 expressed the hybrid gene in the mammary gland, 5 in the salivary glands, and 2 in both these tissues. Human alpha 1 AT was secreted into the milk of each of the 7 mice and mouse lines that expressed the hybrid gene in the mammary gland. Four of these mammary-expressing transgenic mice and mouse lines produced concentrations of at least 0.5 mg of alpha 1 AT per ml in their milk; one line (AATB 35) produced 7 mg of this protein per ml. alpha 1 AT from transgenic mouse milk was similar in size to human plasma-derived alpha 1 AT and showed a similar capacity to inhibit trypsin. Expression at equivalent levels in transgenic sheep or cattle would yield sufficient alpha 1 AT for therapeutic purposes.
Animals↗
Gene expression in the mammary gland.
We have demonstrated that the ovine genomic clone SS1 can be used to generate transgenic mice that produce significant quantities of BLG protein in milk. The smallest BLG construct so far examined that retains the ability to direct BLG to mouse milk encompasses approximately 7.3 kb of genomic DNA, of which about 0.8 kb is derived from the promoter region. Gene expression is tissue-specific and regulated in a temporal and developmental fashion that is similar to that reported for sheep. We conclude, therefore, that the cis-acting sequences determining mammary expression of the ovine BLG gene are correctly interpreted in mice, despite the absence of an equivalent gene in this species, and that conclusions drawn from future work on BLG expression in the mammary gland of transgenic mice will be broadly applicable in sheep and other ruminant species. Work is currently in progress to define other sequences within the promoter of BLG that are required for regulated expression in transgenic mice. These and other studies into the DNA-protein interactions within the promoter which are required for efficient tissue-specific, regulated expression should lead to a greater understanding of milk protein gene expression in the mammary gland. Furthermore, in the current absence of a suitable in-vitro system, the mouse will be most useful for evaluating the expression of further constructs designed to express foreign proteins in milk of domestic ruminants.
Animals↗
Modification of milk composition.
Revolutionary new opportunities for the modification of milk composition have been created by the development of methods for gene transfer and targeted mutation of genes may extend the range of opportunities still further. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue-specific hormonally induced expression in the mammary gland. Fragments of the ovine beta-lactoglobulin gene fused to cDNA for the human therapeutic proteins clotting factor IX and alpha-1 antitrypsin have directed production of these proteins in the milk of transgenic mice and sheep. Factor IX was biologically active and co-migrated with authentic proteins, but was present at too low a concentration for commercial exploitation. Recent observations suggest that fusion genes containing genomic clones direct production of higher concentrations of protein. Mouse whey acidic protein genomic sequences also directed production of low concentrations of human tissue plasminogen activator in the milk of transgenic mice. Targeted expression of this kind may be used for the production of therapeutic and industrial proteins, to increase the concentration or modify the nature of milk proteins, reduce the concentration of lactose, change the composition of fat or direct production of bacteriocidal proteins in milk in order to combat mastitis.
Animals↗
The molecular manipulation of milk composition.
The introduction of cloned genes into the mouse germ line is now routine. Although more difficult technically, gene transfer has been accomplished in farm animals and offers the potential for genetic improvement. In this regard, we have been investigating the use of transgenic animals as production vehicles for high value proteins in milk. We have shown that DNA sequences derived from the gene encoding sheep beta-lactoglobulin mediate efficient and specific expression in the mammary gland. A fusion gene comprising beta-lactoglobulin sequences and those encoding antihemophilic human factor IX has been constructed. This construct has been introduced into sheep; it is expressed in the mammary gland, and the corresponding protein is secreted into milk.
Animal Husbandry↗