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Biomedical subjects

R J Wall

Publications and source records attributed to R J Wall.

At least 37 records · Page 2Linked to original sources
PubMed1997

Transfer of an ovine metallothionein-ovine growth hormone fusion gene into swine.

An ovine metallothionein-1a (oMT1a)-ovine growth hormone (oGH) fusion gene was microinjected into 400 pig zygotes, the zygotes were transferred into recipient females, and 15 founder transgenic pigs were born. Of 12 transgenic pigs assayed, five expressed high levels of oGH (> 900 ng/mL plasma), one expressed low levels of oGH (10 to 30 ng/mL), and six did not express oGH. Dietary supplementation with 2,000 ppm of zinc for 6 d induced a 20-fold increase in plasma oGH in the transgenic pig with low expression but did not induce expression in the six transgenic pigs with no constitutive oGH expression. The average daily gain of five transgenic pigs with elevated oGH was similar to that of non-transgenic littermates during a 9-wk feeding trial (P = .52). The liver, kidney, adrenal, and thyroid weights were all significantly heavier for the oGH-expressing transgenic pigs than for non-transgenic littermates. Total carcass fat, longissimus muscle fat, subcutaneous backfat thickness, and loin eye area were lower and carcass protein and water content and beta R fiber area of longissimus muscle were higher in the transgenic pigs with elevated oGH than in their littermate controls (P < .05 for each). The data indicate that even though the oMT1a promoter was more inducible by zinc than was previously reported for the mouse MT promoter in swine, the former provided a higher level of oGH expression than the mouse MT promoter.

Animals↗
PubMed1997

Transgenic dairy cattle: genetic engineering on a large scale.

Amid the explosion of fundamental knowledge generated from transgenic animal models, a small group of scientists has been producing transgenic livestock with goals of improving animal production efficiency and generating new products. The ability to modify mammary-specific genes provides an opportunity to pursue several distinctly different avenues of research. The objective of the emerging gene "pharming" industry is to produce pharmaceuticals for treating human diseases. It is argued that mammary glands are an ideal site for producing complex bioactive proteins that can be cost effectively harvested and purified. Consequently, during the past decade, approximately a dozen companies have been created to capture the US market for pharmaceuticals produced from transgenic bioreactors estimated at $3 billion annually. Several products produced in this way are now in human clinical trials. Another research direction, which has been widely discussed but has received less attention in the laboratory, is genetic engineering of the bovine mammary gland to alter the composition of milk destined for human consumption. Proposals include increasing or altering endogenous proteins, decreasing fat, and altering milk composition to resemble that of human milk. Initial studies using transgenic mice to investigate the feasibility of enhancing manufacturing properties of milk have been encouraging. The potential profitability of gene "pharming" seems clear, as do the benefits of transgenic cows producing milk that has been optimized for food products. To take full advantage of enhanced milk, it may be desirable to restructure the method by which dairy producers are compensated. However, the cost of producing functional transgenic cattle will remain a severe limitation to realizing the potential of transgenic cattle until inefficiencies of transgenic technology are overcome. These inefficiencies include low rates of gene integration, poor embryo survival, and unpredictable transgene behavior.

Animals↗
PubMed1996

Time-sensitive reversal of hyperplasia in transgenic mice expressing SV40 T antigen.

The role of viral oncoprotein expression in the maintenance of cellular transformation was examined as a function of time through controlled expression of simian virus 40 T antigen (TAg). Expression of TAg in the submandibular gland of transgenic mice from the time of birth induced cellular transformation and extensive ductal hyperplasia by 4 months of age. The hyperplasia was reversed when TAg expression was silenced for 3 weeks. When TAg expression was silenced after 7 months, however, the hyperplasia persisted even though TAg was absent. Although the polyploidy of ductal cells could be reversed at 4 months of age, cells at 7 months of age remained polyploid even in the absence of TAg. These results support a model of time-dependent multistep tumorigenesis, in which virally transformed cells eventually lose their dependence on the viral oncoprotein for maintenance of the transformed state.

Animals↗
PubMed1996

Effects of transferred ova per recipient and dual use of donors as recipients on production of transgenic swine.

The purpose of the study was to determine whether the number of transferred ova per recipient influenced the efficiency of producing transgenic pigs and whether donor gilts were as effective as unmated gilts as recipients of microinjected ova. Eight genes were microinjected into 4,232 ova that were transferred into 169 recipients over a 5-yr period. Although the farrowing rate and litter size was highest for recipients receiving 31 to 41 ova per recipient, the percentage of transferred ova developing into piglets was highest for recipients receiving 13 to 20 ova (P = 0.021 for all recipients and P = 0.011 for pregnant recipients). Based on these data we conclude transferring more than 20 ova per recipient may incur some loss due to uterine crowding. Gilts used as recipients of microinjected ova immediately after their own ova were flushed from their oviducts had the same farrowing rate, litter size, and ovum development efficiency as unmated gilts that were only used as recipients. However, donor-recipients that ovulated 21 or more ova had smaller litters (P = 0.009) and were less efficient in producing pigs (P = 0.024) and transgenic pigs (P = 0.054) from transferred ova than donor-recipients that ovulated 20 or fewer ova. Dual use of donors as recipients was an effective method of reducing the number of recipients in a transgenic pig project by nearly one-half.

Journal Article↗
PubMed1996

Severe position effects imposed on a 1 kb mouse whey acidic protein gene promoter are overcome by heterologous matrix attachment regions.

Matrix attachment regions (MARs) have been shown to participate in the insulation of transcription elements from surrounding chromatin in tissue culture cells and transgenic mice. A whey acidic protein (WAP) transgene containing 1 kb promoter sequence was active in mammary tissue from 1 out of 17 lines of mice, demonstrating that the transcription elements were highly susceptible to position effects. To test whether MARs could insulate this WAP gene promoter and thereby restore transcription, we ligated MARs from the chicken lysozyme gene to the WAP transgene. Seven of the nine lines generated exhibited WAP transgene activity, expression was confined to mammary tissue, and correct regulation was observed in three of the four lines analyzed. This study provides strong additional evidence that the MAR fragments from the chicken lysozyme gene have the capacity to insulate transgenes from severe position effects.

Animals↗
PubMed1996

Synthesis and secretion of the mouse whey acidic protein in transgenic sheep.

The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.

Animals↗
PubMed1995

Conditional gene expression in secretory tissues and skin of transgenic mice using the MMTV-LTR and the tetracycline responsive system.

Molecular mechanisms of development and disease can be studied in transgenic animals. Controlling the spatial and temporal expression patterns of transgenes, however, is a prerequisite for the elucidation of gene function in the whole organism. Previously we reported that mice carrying a tetR/VP16 hybrid gene (tTA), under the control of the human cytomegalovirus immediate early 1 (HCMV-IE1) gene promoter, can be used to temporally activate the expression of transgenes under the control of a promoter containing tetop sequences. We now show that the MMTV-LTR can be used to target expression of tTA to the epithelial cells of secretory organs and skin in transgenic mice. Notably, nearly uniform expression of a tetop-lacZ transgene was found in seminal vesicle, salivary gland, and Leydig cells of mice carrying also the MMTV-tTA transgene. More heterogeneous patterns of gene expression were observed in mammary epithelial cells and basal cells of the epidermis. Different MMTV-tTA lines had comparable tissue expression patterns. Transcriptional activation mediated by tTA was up to several hundredfold, and it was abrogated after the administration of tetracycline. The MMTV-tTA mice established in this work will be useful for experiments examining the roles of biological factors at defined developmental stages in the epithelial cells of salivary gland, seminal vesicle, mammary gland, and skin and the Leydig cells of testes. In addition, in combination with the CRE/lox recombination system, these mice wil be useful to achieve gene deletions at defined time points in these organs.

3T3 Cells↗
PubMed1995

Expression of genomic and cDNA transgenes after co-integration in transgenic mice.

In general, genomic transgenes are expressed efficiently in mice, while their cDNA-based transgenes are frequently silent. Clark et al. (1992) have shown that silent cDNA transgenes under the control of the sheep beta-lactoglobulin promoter can be activated after co-injecting them with a genomic sheep beta-lactoglobulin transgene. We have tested the general utility of this concept using mouse whey acidic protein (WAP) transgenes. Here we show that WAP cDNA transgenes are virtually silent in transgenic mice. In contrast, WAP transgenes containing all introns are expressed in approximately 50% of the lines at levels ranging from 1% to more than 100% of the endogenous RNA (McKnight et al., 1992). When a WAP-genomic transgene was co-injected with a WAP-cDNA, basal activation of the cDNA was possible. However, the activity of the WAP-cDNA transgene did not exceed 1% of the WAP-genomic transgene. This suggests that a permissive integration site capable of supporting basal level transcription can be established, but further events are required for full activation of the transgene.

Animals↗
PubMed1995

An Ets site in the whey acidic protein gene promoter mediates transcriptional activation in the mammary gland of pregnant mice but is dispensable during lactation.

The whey acidic protein (WAP) gene is specifically expressed in mammary tissue, and its transcription is induced several thousand-fold during pregnancy and remains high throughout lactation. A purine-rich sequence (PRS) located around -110 of the WAP gene promoter is conserved between mice, rats, and rabbits, suggesting that it features a regulatory element. This PRS contains an invariant GGAA/T core motif characteristic of the binding site for Ets transcription factors. Electromobility shift assays demonstrate that Ets1 binds specifically to the PRS. Experiments in transgenic mice further demonstrate that this PRS/Ets site plays a critical role in the activation of WAP transgenes during pregnancy, but that its presence is not required for high expression throughout lactation. Transgenes with an intact PRS/Ets site are expressed at high levels at day 13 of pregnancy, with little further increase during late pregnancy and lactation. In contrast, WAP transgenes with a mutation in the PRS/Ets site, which abrogates the binding of Ets1, are not expressed at midpregnancy, but their transcriptional activity is not affected during lactation. These results demonstrate that Ets-signaling pathways can function as stage-specific transcriptional activators of milk protein genes in the developing mammary gland. In addition, this work extends earlier findings that gene activation during pregnancy and lactation is mediated, in part, by different mechanisms.

Animals↗
PubMed1994

Development of transgenic sheep that express the visna virus envelope gene.

The ovine lentiviruses cause encephalitis, pneumonia, and arthritis in sheep worldwide. Visna virus is a prototype of this family and the pathogenesis and molecular biology of the virus has been well characterized. The envelope proteins of visna virus are responsible for binding of virus to host cells and for causing cell fusion. The surface glycoprotein also elicits cellular and humoral immune responses to the virus, the former being thought to be responsible for eliminating infected cells as well as causing inflammatory lesions. In this study, transgenic sheep were constructed that expressed the envelope genes of visna virus under the control of the visna LTR to investigate the role of the env gene in the pathogenesis of lentiviral disease in its natural host. Three transgenic lambs were identified that contain the env transgene and express the envelope glycoproteins. These transgenic animals have remained healthy and expression of the viral gene has had no obvious deleterious effect. Expression of the visna envelope protein was demonstrated by cell fusion mediated by the envelope gene as well as by immunoprecipitation of the envelope proteins with monoclonal antibodies and immunofluorescence analyses of Env protein in cells. The target cell for visna virus replication in infected animals is the monocyte/macrophage. In natural infection, the level of viral gene expression in these cells increases with cell maturation. In the transgenic sheep, monocytes did not express the envelope glycoproteins until they differentiated into macrophages in vitro. Expression of the env mRNA in macrophages was quantitated by an RNase protection assay. In addition to expression in macrophages, the transgene was expressed by fibroblasts isolated from skin of the transgenic sheep. Expression of both the Env and Rev proteins was detected by immunoprecipitation and immunofluorescence. Two of the three lambs responded immunologically to the expression of the transgene by producing binding antibodies to the envelope glycoproteins. Thus, these transgenic sheep provide a model to study whether a lentivirus glycoprotein will prevent infection or modulate disease in its natural host after virus challenge.

Animals↗
PubMed1994

Whey acidic protein extrinsically expressed from the mouse mammary tumor virus long terminal repeat results in hyperplasia of the coagulation gland epithelium and impaired mammary development.

The whey acidic protein (WAP) is a milk protein that contains a cysteine-rich motif. This characteristic WAP signature has also been found in some protease inhibitors and certain proteins involved in tissue modeling. WAP is specifically synthesized in mammary tissue from late pregnant and lactating animals, and precocious synthesis results in impaired lobuloalveolar development of the gland in some transgenic lines. To determine whether growth modulatory effects of WAP are confined to mammary tissue, we expressed the WAP gene under the control of the mouse mammary tumor virus long terminal repeat in transgenic mice. The transgene was expressed at high levels in organs with exocrine function, such as mammary and salivary glands, prostate, seminal vesicle, and the coagulation gland. In addition to impaired mammary development, we observed hyperplasia and dysplasia of the coagulation gland epithelium. These findings suggest that WAP or a member of the WAP signature family can, in certain tissue contexts, function as an epithelial growth regulator. It appears from the present study that growth regulatory effects of WAP are restricted in the mouse to the mammary and coagulation gland epithelium.

Animals↗
PubMed1993

A 5' control region of the human epsilon-globin gene is sufficient for embryonic specificity in transgenic mice.

When introduced as part of DNA constructions containing the human beta-globin locus control region (LCR), the human embryonic beta-globin gene, epsilon, is expressed in primitive but not definitive erythroid cells of recipient transgenic mice. In contrast to this pattern, the human fetal beta-globin gene, gamma, has been shown to be expressed in both primitive and definitive erythroid cells of transgenic mice when introduced in similar LCR-containing constructions. To begin to identify the minimal sequence(s) necessary for the epsilon expression pattern, we have fused a DNA fragment that contains the human epsilon-globin gene promoter region, and 13.7-kilobase (kb) of contiguous upstream flanking sequence containing super-hypersensitive (HS) sites 5'HS-2 and 5'HS-1 of the globin LCR, to the structural portion and near 3'-flanking region of the human gamma-globin gene. This construction, and one containing an intact human gamma-globin gene with the same 3'-flanking sequence and 383 base pairs of 5'-flanking sequence linked to LCR DNA from -0.86 to -13.7 kb upstream of epsilon, were each microinjected to produce transgenic mice. While the construction containing the intact gamma-globin gene is transcriptionally active in primitive and definitive erythroid cells of the transgenic mice, the fusion construction, in which the gamma-globin gene promoter and promoter proximal region is essentially replaced by that of epsilon, is not active in definitive erythroid cells and expresses with the same pattern as an intact epsilon gene. These results indicate that the promoter and near 5'-flanking region of epsilon, when linked to the LCR, is sufficient for embryonic-specific expression in transgenic mice. The level of expression of the fusion construction in primitive erythroid cells of transgenic mice is similar to that previously observed for the intact epsilon gene when identically cloned. This suggests that the epsilon 5'-region of the fusion construction also contains all the sequence necessary for the LCR-dependent activation of epsilon in transgenic mice.

Animals↗
PubMed1992

Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. We have tested whether MAR sequences from the chicken lysozyme locus, the so-called A-elements, can confer position-independent regulation to a whey acidic protein (WAP) transgene in mammary tissue of mice. In the absence of MARs, expression of WAP transgenes was observed in 50% of the lines, and regulation during pregnancy, during lactation, and upon hormonal induction did not mimic that of the endogenous WAP gene and varied with the integration site. In contrast, all 11 lines in which WAP transgenes were juxtaposed to MAR elements showed expression. Accurate position-independent hormonal and developmental regulation was seen in four out of the five lines analyzed. These results indicate that MARs can establish independent genetic domains in transgenic mice.

Animals↗
PubMed1992

Making transgenic livestock: genetic engineering on a large scale.

The feasibility of introducing foreign genes into the genomes of cattle, goats, pigs, and sheep has only recently been demonstrated. Studies have thus far focused on improving growth efficiency or directing expression of pharmaceutical proteins to the mammary glands of these species. The general strategy for producing transgenic livestock and mice is similar. In addition to the obvious difference in scale between mice and livestock experiments, there are noteworthy obstacles that significantly reduce the efficiency of producing transgenic livestock. Low embryo viability, low transgene integration rates, and high animal costs contribute to project costs that can easily exceed hundreds of thousands of dollars. A better understanding of the mechanisms that govern transgene integration should lead to improved efficiencies. But, the full potential of the transgenic livestock system will not be fully realized until: 1) gene constructs can be designed that function in a reproducible, predictable manner; and 2) the genetic control of physiological processes are more clearly elucidated. Newly emerging approaches may resolve at least some of these issues within the next decade.

Animals↗
PubMed1992

Fate of microinjected genes in preimplantation mouse embryos.

The state of genes microinjected into mouse embryos was followed from the one-cell to the blastocyst stage using the polymerase chain reaction (PCR). Microinjected DNA was detected in all one-, two-, and four-cell injected embryos and in 44% of morula and 26% of blastocysts. Head-to-tail ligation of microinjected genes, a common feature of stably integrated transgene arrays, was detected in all embryos after injection of microinjected genes and occurred irrespective of the structure at the ends of the injected genes. Sensitivity of microinjected DNA to a methylation-dependent restriction endonuclease Dpn I was lost in all embryos by the two-cell stage (24 hr), indicating a change in DNA methylation, independent of transgene integration. Dissociation of blastomeres prior to compaction revealed a mosaic distribution of the microinjected DNA within the embryo and supports the notion that injected genes form a limited number of arrays, which segregate independently until they integrate into the genome or are degraded.

Animals↗
PubMed1992

Expression of the whey acidic protein in transgenic pigs impairs mammary development.

The whey acidic protein has been found in milk of mice, rats, rabbits and camels, and its gene is expressed specifically in mammary tissue at late pregnancy and throughout lactation. A characteristic of whey acidic protein is the 'four-disulfide-core' signature which is also present in proteins involved in organ development. We have generated six lines of transgenic pigs which carry a mouse whey acidic protein transgene and express it at high levels in their mammary glands. Transgenic sows from three lines could not produce sufficient quantities of milk to support normal development of healthy offspring. This phenotype appears to be similar, if not identical, to the milchlos phenotype exhibited by mice expressing whey acidic protein transgenes. Mammary tissue from post-partum milchlos sows had an immature histological appearance, which was distinct from that observed during normal development or involution. Expression of the whey acidic protein transgene was found in mammary tissue from sexually immature pigs from milchlos lines, but not in sows from lines that appeared to lactate normally. We suggest that precocious synthesis of whey acidic protein impairs mammary development and function. Impaired mammary development due to inappropriate timing of whey acidic protein expression is consistent with the notion that proteins with the 'four-disulfide-core' signature participate in tissue formation.

Aging↗