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J P Simons

Publications and source records attributed to J P Simons.

36 records · Page 2Linked to original sources
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

Rescuing transgene expression by co-integration.

To test whether foreign gene expression can be improved in transgenic mice by manipulating the site of integration, we co-integrated the efficiently expressed sheep beta-lactoglobulin gene with two poorly expressed beta-lactoglobulin-derived hybrid genes encoding human proteins. In each case, we observed a significant improvement in the frequency and level of expression of the hybrid gene. "Rescuing" transgene expression by co-integration may provide a general solution for improving the efficiency of heterologous gene expression in transgenic animals.

Animals↗
PubMed1992

The potential role of molecular genetic manipulation in the improvement of reproductive performance.

Revolutionary opportunities for the modification of animal performance are being created by the development of new methods for embryo manipulation and the application of molecular biology. This paper reviews the potential application of these procedures for the improvement of reproductive performance in livestock. There are three sections: a consideration of the methods of molecular manipulation that are available at present and those that seem likely to become available, a discussion of the modifications to hormonal systems and, finally, an analysis of candidate genes for manipulation of seasonality, number of ovulations, sex ratio and prenatal survival. The analysis points to a number of ways forward. Many of the most promising opportunities will depend upon the isolation of embryonic stem cells or the establishment of alternative methods of site-directed mutation. In most cases, the genes of interest have not yet been cloned and much remains to be learned about the molecular regulation of reproduction. A greater understanding seems likely to reveal the inadequacies of some of the present suggestions, but it is also certain to reveal further opportunities. However, in the longer term, there seems to be a real prospect of modification of at least some of these aspects of reproductive performance by molecular means.

Animals↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

Calcium entry blockers and their effects on glucose metabolism.

Calcium entry blockers have been used for cardiovascular disturbances such as angina pectoris and hypertension. Calcium is, however, involved in the release of several hormones. The process of insulin secretion by the pancreatic beta-cells is dependent on calcium. Thus, calcium-entry blockers may interfere with insulin secretion. This effect has been confirmed in vitro in isolated islets as well as in animal studies. A few case reports describe the deterioration of glycaemic control or development of frank diabetes mellitus during treatment with nifedipine or diltiazem. In general, however, there are no important negative effects of calcium-entry blockers on glucose tolerance, either in non-diabetic persons with hypertension, or in patients with diabetes mellitus. Hence, these drugs appear to be a good choice for use in diabetic patients with cardiovascular diseases.

Calcium Channel Blockers↗
PubMed1990

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↗
PubMed1989

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↗
PubMed1988

A Mup promoter-thymidine kinase reporter gene shows relaxed tissue-specific expression and confers male sterility upon transgenic mice.

A hybrid gene was made by fusing the 2.2-kilobase 5' promoter region of a mouse group 1 major urinary protein (Mup) gene to the coding region of the herpes simplex virus type 1 thymidine kinase gene (HSV tk) and introduced into the genomes of mice by microinjection. Transgenic G0 males were sterile, or when fertile did not transmit the foreign gene, and the transgenic male descendants of G0 females were also sterile. Seven "lines" were established by breeding from G0 females and their transgenic female descendants. Six lines expressed HSV thymidine kinase activity in the liver, and activity correlated perfectly with the presence of HSV tk RNA. In three of four lines examined, expression was lower in female than in male liver, and in these lines the same sex difference was observed in the rate of run-on transcription of the foreign genes in liver nuclei. When females of one of the sexually dimorphic lines were treated with testosterone, the levels of HSV tk RNA and thymidine kinase activity were increased, although not to male levels. In these aspects of liver expression, and also in a lack of expression in seven other tissues, the hybrid gene exhibits many of the characteristics of an endogenous group 1 Mup gene. However, the gene was also expressed (at high levels) in the preputial gland and testis, two tissues in which Mup genes are not expressed. The gene, when introduced into five of the seven lines, carried a copy of the Escherichia coli supF gene attached beyond the 3' end of the HSV tk gene, but this did not affect the overall expression pattern. All of the lines were male sterile and expressed HSV thymidine kinase in the testis, but one line showed no activity in the liver, and another showed none in the preputial gland. Testicular expression is therefore the likely cause of sterility. Data are described which suggest that the causes of misexpression in the preputial gland and testis are different. Since expression in each tissue occurred in several lines, the structure of the hybrid gene must be responsible in each case. Five intensively studied lines showed at least four consistently different patterns of relative expression in preputial gland, testis, male liver, and female liver. These differences do not correlate in any way with the copy number of the foreign gene in the different lines and must be due to some other aspect of line specific integration.

Animals↗
PubMed1987

Transgenic livestock.

Single genes can now be added routinely to the genome of mice by molecular manipulation as simple Mendelian dominants; this complements the normal process of reproduction to give 'transgenic' animals. Success in ruminants is limited to a few examples in sheep and although gene expression has yet to be documented, there is every reason to expect that it will be achieved. The application of this technology to livestock improvement depends on the identification of circumstances in which the phenotype is limited by the deficiency of a single protein. While there is little evidence to indicate that single dominant genes are in general likely to have favourable effects, it is argued that there are likely to be exceptions. These include particular combinations of promoter and structural gene sequences to alter feedback control, for example through a change in tissue specificity, and the alteration of definitive proteins such as those of milk. A mouse model has been established to study the molecular manipulation of sheep milk proteins. The sheep beta lactoglobulin gene has been incorporated and the sheep whey protein is secreted by the mammary gland of transgenic mice. For the future, means to delete or reduce the expression of existing genes are likely to be important, as are more effective means of incorporation such as retroviral based methods and the incorporation of multigene constructs. The resources required to test transgenic livestock will, however, be greater than those required to create them.

Animals↗
PubMed1980

Linkage of adult alpha- and beta-globin genes in X. laevis and gene duplication by tetraploidization.

We have used cloned adult X. laevis alpha- and beta-globin cDNAs to analyze globin genes in X. laevis DNA. We detected alpha 1- and beta 1-globin genes which contain intervening sequences and code for the major adult globins, plus additional diverged alpha 2- and beta 2-globin genes of unknown coding potential. Unlike the case in mammals, the X. laevis alpha 1- and beta 1-globin genes are closely linked and occur in the sequence 5'-alpha 1-9 kb-beta 1-3'. The alpha 2- and beta 2-globin genes are also linked, and analysis of globin genes in X. tropicalis suggests that this duplication of an alpha-beta-globin gene pair in X. laevis is the result of chromosome duplication by tetraploidization. The close linkage of alpha- and beta-globin genes in Xenopus provides evidence that vertebrate alpha- and beta-globin genes evolved by tandem duplication of a single primordial globin gene.

Animals↗
PubMedDate not supplied

Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice.

Milk contains a large amount of protein, most of which consists of a few major species synthesized in the mammary gland. The genes encoding these proteins are single-copy, and expressed during pregnancy and lactation. Although beta-lactoglobulin (BLG) is the major protein in the whey of ruminants, it is not present in rodent milk. We have generated transgenic mice carrying the sheep BLG gene, and show that in such mice, BLG is specifically and abundantly expressed in the mammary gland during lactation. This results in a remarkable alteration of milk composition. These findings suggest that the manipulation of milk composition by gene transfer has considerable potential for the improvement of dairy animals.

Animals↗