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Biomedical subjects

R J Wall

Publications and source records attributed to R J Wall.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Induction of lactogenesis in transgenic virgin pigs: evidence for gene and integration site-specific hormonal regulation.

Five month-old transgenic female pigs from three lines carrying the mouse whey acidic protein (WAP) gene and nontransgenic female littermates were implanted with slow-release estrogen and progesterone pellets. Histological analysis of biopsies taken at the time of implantation and 4 weeks later revealed that mammary alveolar development had occurred upon hormonal stimulation in vivo. beta-Casein and beta-lactoglobulin mRNA was found in all induced animals, and WAP mRNA was detected in two of the three transgenic pigs. Differential hormonal regulation between the transgenes in the three lines and also between endogenous milk protein genes was observed in induced mammary tissue cultured in vitro. In the presence of insulin, hydrocortisone, and PRL, beta-casein and WAP mRNA levels increased in all transgenic pigs. In contrast, beta-lactoglobulin mRNA had reached or exceeded lactational levels in response to the in vivo induction, and no further increase was observed in vitro. This suggests that the regulation of the beta-lactoglobulin gene is distinct from that of beta-casein and WAP. Differences were also observed during pregnancy; whereas beta-lactoglobulin gene expression was induced in early pregnancy, a time when PRL levels are low, WAP mRNA levels increased sharply around parturition. Finally, the observation that hormonal regulation of WAP transgenes greatly differed between the three lines suggests that chromatin surrounding the integration site can modify the response of transcription elements.

Animals↗
PubMed1991

Expression of a whey acidic protein transgene during mammary development. Evidence for different mechanisms of regulation during pregnancy and lactation.

Expression of the mouse whey acidic protein (WAP) gene is specific to the mammary gland, is induced several thousand-fold during pregnancy, and is under the control of steroid and peptide hormones. To study developmental regulation of the mouse WAP gene, a 7.2-kilobase (kb) WAP transgene, including 2.6 kb of 5'- and 1.6 kb of 3'-flanking sequences, was introduced into mice. Of the 13 lines of mice examined, 6 expressed the transgenes during lactation at levels between 3 and 54% of the endogenous gene. Although expression was dependent on the site of integration, the transgenes within a given locus were expressed in a copy number-dependent manner and were coordinately regulated. The WAP transgenes were expressed specifically in the mammary gland, but showed a deregulated pattern of expression during mammary development. In all six lines of mice, induction of the WAP transgenes during pregnancy preceded that of the endogenous gene. During lactation, expression in two lines increased coordinately with the endogenous gene, and in three other lines of mice, transgene expression decreased to a basal level. These data indicate that the 7.2-kb gene contains some but not all of the elements necessary for correct developmental regulation. At a functional level it appears as if a repressor element, which inactivates the endogenous gene until late pregnancy, and an element necessary for induction during lactation are absent from the transgene. Complementary results from developmental and hormone induction studies suggest that WAP gene expression during pregnancy and lactation is mediated by different mechanisms.

Animals↗
PubMed1991

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

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. that it is possible to produce high levels of a foreign protein in

Animals↗
PubMed1991

Over-expression of an endogenous milk protein gene in transgenic mice is associated with impaired mammary alveolar development and a milchlos phenotype.

The whey acidic protein (WAP) gene is expressed in mammary epithelial cells at late pregnancy and throughout lactation. We have generated transgenic mice in which a mouse WAP transgene is expressed precociously in pregnancy. From 13 founder mice bearing WAP transgenes, two female founders and the daughters from a male founder failed to lactate and nurture their offspring. We named this phenotype milchlos. Mammary tissue from postpartum milchlos mice was underdeveloped, contained too few alveoli and resembled the glands of non-transgenic mid-pregnant mice. The hypothesis that alveolar development in milchlos mice was functionally arrested in a prelactational state is consistent with low levels of alpha-lactalbumin mRNA, and an unidentified keratin RNA in mammary tissue from postpartum mice. Defects in alveolar function in milchlos mice were detected at mid-pregnancy; in non-transgenic mice, WAP was secreted into the alveolar lumen but remained preferentially in the cytoplasm of the alveolar epithelial cells in the milchlos mice. Since deregulated WAP expression resulted in impaired mammary development, it is possible that WAP plays a regulatory role in the terminal differentiation and development of mammary alveolar cells.

Animals↗
PubMed1991

Production of the mouse whey acidic protein in transgenic pigs during lactation.

The mouse whey acidic protein (WAP) gene was introduced into the genome of pigs and its expression was analyzed in the mammary gland. Mouse WAP was detected in milk of lactating females from five lines at levels between .5 and 1.5 g/liter, thereby representing as much as 2% of the total milk proteins. The corresponding mRNA was expressed in mammary tissue at levels similar to those of pig beta-lactoglobulin and beta-casein. The pattern of WAP secretion in three pigs over a period of 6 wk was quantitatively similar to that of pig beta-lactoglobulin. From the eight transgenic pigs analyzed, three successfully completed one lactational period, but five pigs stopped lactating a few days after parturition. Our results show that it is possible to produce large quantities of a foreign protein in milk of pigs over a full lactational period. However, expression of WAP can compromise the mammary gland and render it nonfunctional.

Animals↗
PubMed1990

Developmentally regulated and erythroid-specific expression of the human embryonic beta-globin gene in transgenic mice.

Transgenic mice have proven to be an effective expression system for studying developmental control of the human fetal and adult beta-globin genes. In the current work we are interested in developing the transgenic mouse system for the study of the human embryonic beta-globin gene, epsilon. An epsilon-globin gene construction (HSII,I epsilon) containing the human epsilon-globin gene with 0.2 kb of 3' flanking sequence and 13.7 kb of extended 5' flanking region including the erythroid-specific DNase I super-hypersensitive sites HSI and HSII was made. This construction was injected into fertilized mouse ova, and its expression was analyzed in peripheral blood, brain, and liver samples of 13.5 day transgenic fetuses. Fetuses carrying intact copies of the transgene expressed human epsilon-globin mRNA in their peripheral blood. Levels of expression of human epsilon-globin mRNA in these transgenic mice ranged from 2% to 26% per gene copy of the endogenous mouse embryonic epsilon y-globin mRNA level. Furthermore, the human epsilon-globin transgene was expressed specifically in peripheral blood but not in brain or in liver which is an adult erythroid tissue at this stage. Thus, the HSII,I, epsilon transgene was expressed in an erythroid-specific and embryonic stage-specific manner in the transgenic mice. A human epsilon-globin gene construction that did not contain the distal upstream flanking region which includes the HSI and HSII sites, was not expressed in the embryos of transgenic mice. These data indicate that the human epsilon-globin gene with 5' flanking region extending to include DNase I super-hypersensitive sites HSI and HSII is sufficient for the developmentally specific activation of the human epsilon-globin gene in erythroid tissue of transgenic mice.

Animals↗
PubMed1990

Effects of alfentanil on induction and recovery from propofol anaesthesia in day surgery.

The influence of alfentanil on induction and recovery characteristics of propofol was examined in women presenting as day cases for minor gynaecological surgery. Alfentanil reduced the incidence of pain on injection and the induction and maintenance requirements of propofol. This was at the expense of more prolonged apnoea. Immediate recovery characteristics were similar in each group although changes in Critical Flicker Fusion Threshold as a measure of psychomotor impairment were more pronounced after alfentanil. Such differences were insignificant at three hours by which time Critical Flicker Fusion Threshold had returned to baseline and patients were ready for discharge home.

Adult↗
PubMed1988

Identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene.

Endogenous and foreign DNA sequences can be detected in an extremely small number of cells via sequence amplification in vitro. The polymerase chain reaction (PCR) technique applied in multiple cycles allows the amplification of specific short regions of the genome to levels that can be detected by DNA blotting techniques. Cow and mouse blastocysts were analyzed by PCR for the presence of an endogenous singlecopy gene or an integrated foreign gene. The endogenous single-copy gene encoding the beta chain of bovine luteinizing hormone was detectable in cow blastocysts and in purified bovine genomic DNA representing as few as 25 cells. To determine whether exogenous genes (transgenes) can be detected in preimplantation embryos, transgenic male mice hemizygous for the prokaryotic gene encoding neomycin resistance were bred to nontransgenic females, and the resulting blastocysts were analyzed. The neo gene was detected in approximately half of the embryos. The capability to identify specific gene sequences in a limited number of embryonic cells affords investigators the opportunity to study genetics in early development.

Animals↗
PubMed1988

Lipid interactions with in vitro development of mammalian zygotes.

Rabbit zygotes were tested for their ability to sequester radiolabeled acetate, oleate, and arachidonate in intracellular lipid. Radiolabeled arachidonic acid was concentrated 170 +/- 28-fold (mean +/- SEM) and oleic acid was concentrated 105 +/- 26-fold in zygotic lipids during 6 hr of culture when compared with the initial concentrations in culture medium. Acetate was not concentrated into lipids by cultured zygotes. Both long chain fatty acids were incorporated mainly as triglyceride. Polydimethylsiloxane fluid, used to cover the microdroplets of medium during culture, demonstrated lipophilic properties. This characteristic was utilized to indirectly transfer lipids to culture medium, permitting examination only of lipoidal properties of test extracts on embryonal development. For rabbit zygotes, blood plasma extract was detrimental and whole blood extract was beneficial for embryonal cleavage rates during the first 24 hr of culture. A higher proportion of mouse zygotes developed to blastocysts when cultured in modified Ham's F-10 medium compared to BMOC medium, and this difference was negated by inclusion of a lipid extract prepared from rabbit oviductal fluid in the culture system. Comparison of fatty acid analyses of the lipid extracts with development rates of zygotes suggests that modified rates of embryo development may be associated with ratios of individual fatty acids presented to the culture medium rather than with the presence of any single fatty acid.

Acetates↗
PubMed1988

Developmental competence of domestic cat follicular oocytes after fertilization in vitro.

Empirical evaluation of variables affecting oocyte collection, in vitro fertilization, and embryo transfer resulted in establishing a successful procedure for the artificial production of offspring in the domestic cat. Female cats were treated with pregnant mare's serum gonadotropin (PMSG, 150 IU) followed 72 or 80 h later with 100 or 200 IU human chorionic gonadotropin (hCG). After laparoscopic collection, follicular oocytes were inseminated in vitro with ejaculated, processed spermatozoa, cultured (37 degrees C, 5% CO2), and then examined for evidence of fertilization. Two- to 4-cell stage embryos were transferred to the oviducts of oocyte donors. Oocyte donor cats and naturally mated controls also were subjected to sequential laparoscopic examinations and blood sampling to assess corpora lutea (CL) function. At 24-30 h of culture, fewer (p less than 0.001) degenerate oocytes were observed in cats receiving 100 IU hCG (8.2%) compared to those receiving 200 IU (20.6%), regardless of the PMSG-hCG interval. Overall fertilization (48.1%) and cleavage (45.2%, at 30 h post-insemination) rates were greatest following an 80-h PMSG-hCG interval combined with the 100 IU hCG dose. Five of the 6 cats receiving 6 to 18 embryos became pregnant and produced from 1 to 4 kittens/litter. Gonadotropin-treated females subjected to follicular aspiration produced morphologically normal CL and circulating progesterone patterns that were qualitatively similar (p greater than 0.05) to control cats. These data indicate that domestic cat follicular oocytes are capable of fertilization in vitro, but success is dependent on both the timing and dose of the hCG stimulus. Follicles subjected to aspiration appear capable of forming normal, functional CL and the birth of live young after embryo transfer unequivocally demonstrates, for the first time, the developmental competence of in vitro-fertilized carnivore oocytes.

Animals↗
PubMed1988

Development of centrifuged cow zygotes cultured in rabbit oviducts.

Zygotes from superovulated cows were centrifuged and pronuclei were detected by differential interference-contrast microscopy in 73% of 106 zygotes. Zygotes were then transferred to ligated oviducts of follicular-phase, 1-day pseudopregnant or 7-day pseudopregnant rabbits and recovered 5 days later. Their development did not differ from that of uncentrifuged zygotes transferred to the opposite oviduct: 41% of the embryos recovered from rabbit oviducts contained 17-32 nuclei and an additional 5% contained greater than 32 nuclei. In another experiment, 399 ova from unmated cows were transferred to rabbit oviducts to determine whether centrifugation induced parthenogenetic development. After 7 days, 257 ova were recovered; 16% of the recovered ova had developed parthenogenetically and contained 2-30 nuclei. Neither centrifugation of the ova nor reproductive status of the rabbits influenced the proportion of parthenogenotes found. Parthenogenetic development was also observed in 14 of 71 ova (20%) recovered on Day 7 from uninseminated superovulated cows. In an attempt to increase the probability of detecting treatment differences, centrifuged and control cow zygotes were incubated for 7 (rather than 5) days in opposite oviducts of fourteen 1-day pseudopregnant rabbits. Development was unaffected by centrifugation: 61% of the zygotes recovered had developed beyond the 16-cell stage, with 23, 24 and 15% containing 17-32, 33-64, and greater than 64 nuclei, respectively. Taking into account the percentage of zygotes in which pronuclei can be seen, the recovery rate from rabbit oviducts, and the proportion of embryos that develop to the morula stage or beyond, 26% of the original group of zygotes would be candidates for transfer into recipient cows.

Animals↗