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PubMed1992

Growth response and androgen receptor expression in seminal vesicles from aging transgenic mice expressing human or bovine growth hormone genes.

Previous work has shown that expression of human (h) GH in transgenic mice is associated with significant age-related enlargement of seminal vesicles. To further explore this aberrant growth activity, we have characterized seminal vesicles from various GH transgenic lines and examined their androgen receptor (AR) content and distribution. Six groups of animals were initially studied: young adult (3-5 months) control mice, old (greater than 12 months) control mice, young adult hGH transgenic mice, old hGH transgenics, young adult bovine (b) GH transgenics, and old bGH transgenic mice. Young transgenic mice (hGH and bGH) possessed seminal vesicles with similar relative weights, DNA and protein contents, and AR levels as nontransgenic littermates. Histologically, the glands appeared similar. With aging, the hGH transgenic seminal vesicles exhibited massive stromal hyperplasia, whereas the glands from controls and bGH transgenic mice did not show this response. Seminal vesicles from old hGH mice presented with a marked increase in cell number (DNA content) and a marked decrease in cell size and/or glandular secretions (protein/DNA ratio) compared to those from old controls and young hGH transgenic mice. Tissue AR content was markedly reduced in old hyperplastic hGH seminal vesicles compared to that in seminal vesicles from young hGH transgenics, old controls, and old bGH transgenic mice. Immunohistochemistry indicated the absence of AR in the proliferating stromal cells, whereas acinar epithelial cells showed similar or moderately reduced AR staining intensity compared to control seminal vesicles. To examine whether the above results may be due to insertional mutagenesis rather than hGH itself, two additional GH transgenic lines were examined. Aged transgenic mice expressing bGH with an alternate promoter possessed seminal vesicle weights that were not different from those of old controls, whereas aged transgenic mice expressing an hGH. V gene (variant gene, placental origin) possessed significantly larger vesicles than the controls, which further suggests that vesicular hyperplasia is specifically related to hGH. To assess androgen responsiveness, aged control and hGH transgenic mice were castrated and examined after 15 days. While control seminal vesicles significantly decreased in size, glands from transgenic mice did not. Regressive changes were observed in the remaining epithelium of hGH transgenic mice; however, stromal tissue exhibited no response to androgen withdrawal. The present results suggest that the aging-associated seminal vesicle hyperplasia in hGH transgenic mice is a result of a massive increase in stromal tissue that is low or devoid of AR, suggesting a loss of direct androgen regulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1996

Vasoactive intestinal polypeptide mRNA and peptide levels are decreased in the anterior pituitary of the human growth hormone-releasing hormone transgenic mouse.

We recently reported that galanin gene expression is markedly increased in the hyperplastic anterior pituitary gland of the human growth hormone-releasing hormone (hGHRH) transgenic mouse. To determine if another pituitary peptide hormone with putative growth-promoting activity is similarly affected, or if this effect is specific to the peptide galanin, we examined vasoactive intestinal polypeptide (VIP) gene expression in the hypothalamic-pituitary axis of male hGHRH transgenic and non-transgenic mice. The objectives were to: 1) assess VIP peptide concentrations, 2) estimate relative differences in VIP mRNA levels, 3) determine the effects of acute treatment with 17beta-estradiol on VIP peptide and mRNA levels, and 4) quantify the density of immunoreactive VIP pituitary cells by immunohistochemistry. Four to five month old male hGHRH transgenic mice and their non-transgenic siblings were identified by PCR. Immunoreactive VIP concentrations were decreased by 50% in the anterior pituitary glands of hGHRH transgenic mice as compared to non-transgenic siblings. In contrast, no differences in immunoreactive VIP concentrations were observed in the hypothalamus or frontal cerebral cortex of transgenic and non-transgenic mice. Treatment with 17beta-estradiol significantly increased VIP concentrations in the anterior pituitary gland of both transgenic and non-transgenic mice; however, VIP peptide concentrations in the anterior pituitary glands of hGHRH transgenic mice remained 50% lower. Relative differences in VIP mRNA levels were estimated by RT-PCR, and were found to be 2.5-fold higher in the anterior pituitary glands of non-transgenic mice. In contrast, no differences in VIP mRNA levels in the cerebral cortex were detected between transgenic and non-transgenic mice. Treatment with 17beta-estradiol increased VIP mRNA levels in the anterior pituitary, but not in the cerebral cortex. In concert with the changes in VIP peptide and mRNA, the density of immunoreactive VIP pituitary cells was decreased approximately 50% in hGHRH transgenic mice. In conclusion, unlike galanin gene expression, VIP peptide and mRNA levels are significantly decreased in the anterior pituitary gland of hGHRH transgenic mice. Moreover, these changes appear to be tissue-specific and are likely due, in part, to the decrease in the density of VIP-containing pituitary cells in the hyperplastic pituitary. Although the pituitary cell type(s) synthesizing VIP remains unclear, these data suggest that VIP in the anterior pituitary is not stimulating pituitary tumor development in hGHRH transgenic mice.

Animals↗
PubMed1999

Active transgenes in zebrafish are enriched in acetylated histone H4 and dynamically associate with RNA Pol II and splicing complexes.

We have investigated the functional organization of active and silent integrated luciferase transgenes in zebrafish, with the aim of accounting for the variegation of transgene expression in this species. We demonstrate the enrichment of transcriptionally active transgenes in acetylated histone H4 and the dynamic association of the transgenes with splicing factor SC35 and RNA Pol II. Analysis of interphase nuclei and extended chromatin fibers by immunofluorescence and in situ hybridization reveals a co-localization of transgenes with acetylated H4 in luciferase-expressing animals only. Enrichment of expressed transgenes in acetylated H4 is further demonstrated by their co-precipitation from chromatin using anti-acetylated H4 antibodies. Little correlation exists, however, between the level of histone acetylation and the degree of transgene expression. In transgene-expressing zebrafish, most transgenes co-localize with Pol II and SC35, whereas no such association occurs in non-expressing individuals. Inhibition of Pol II abolishes transgene expression and disrupts association of transgenes with SC35, although inactivated transgenes remains enriched in acetylated histones. Exposure of embryos to the histone deacetylation inhibitor TSA induces expression of most silent transgenes. Chromatin containing activated transgenes becomes enriched in acetylated histones and the transgenes recruit SC35 and Pol II. The results demonstrate a correlation between H4 acetylation and transgene activity, and argue that active transgenes dynamically recruit splicing factors and Pol II. The data also suggest that dissociation of splicing factors from transgenes upon Pol II inhibition is not a consequence of changes in H4 acetylation.

Acetylation↗
PubMed1998

Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA.

Integration of transgenic DNA into the plant genome was investigated in 13 transgenic oat (Avena sativa L.) lines produced using microprojectile bombardment with one or two cotransformed plasmids. In all transformation events, the transgenic DNA integrated into the plant genome consisted of intact transgene copies that were accompanied by multiple, rearranged, and/or truncated transgene fragments. All fragments of transgenic DNA cosegregated, indicating that they were integrated at single gene loci. Analysis of the structure of the transgenic loci indicated that the transgenic DNA was interspersed by the host genomic DNA. The number of insertions of transgenic DNA within the transgene loci varied from 2 to 12 among the 13 lines. Restriction endonucleases that do not cleave the introduced plasmids produced restriction fragments ranging from 3.6 to about 60 kb in length hybridizing to a probe comprising the introduced plasmids. Although the size of the interspersing host DNA within the transgene locus is unknown, the sizes of the transgene-hybridizing restriction fragments indicated that the entire transgene locus must be at least from 35-280 kb. The observation that all transgenic lines analyzed exhibited genomic interspersion of multiple clustered transgenes suggests a predominating integration mechanism. We propose that transgene integration at multiple clustered DNA replication forks could account for the observed interspersion of transgenic DNA with host genomic DNA within transgenic loci.

Avena↗
PubMed1995

Field trial analysis of nitrate reductase co-suppression: a comparative study of 38 combinations of transgene loci.

Co-suppression of host genes and 35S transgenes encoding nitrate reductase was previously reported in transgenic tobacco plants (Nicotiana tabacum cv. Paraguay or Burley) using either a full-length cDNA or fragments devoid of the 3' and/or 5' UTR. Co-suppression was previously shown to affect a limited fraction of the progeny of one transgenic tobacco line homozygous for a single transgene locus, and the phenomenon occurred at each generation. In this work, 38 combinations of transgene loci derived from 13 independent transgenic lines homozygous for a single transgene locus were field-tested under two different conditions in an attempt to determine the corresponding frequencies of co-suppression, i.e. the percentage of plants showing co-suppression. Each of the 13 homozygous lines exhibited a different frequency of co-suppression, ranging from 0% to 57%. High frequencies were found to be associated with transgene loc carrying a high number of copy of the transgene, suggesting a transgene dose effect. Combinations carrying 2 non-allelic transgene loci in a hemizygous state exhibited frequencies of co-suppression between those of each of the 2 transgene loci in a homozygous state, while combinations carrying 2 non-allelic transgene loci in a homozygous state exhibited frequencies of co-suppression higher than the sum of those of the 2 transgene loci alone in a homozygous state, clearly confirming a transgene dose effect. Co-suppression frequencies were increased when the plants were grown initially in vitro, suggesting some environmental effect. The roles of transgene copy number, number of transgene loci and environmental factors are discussed in the light of a threshold hypothesis.

Chromosome Mapping↗
PubMed1990

Early deletion and late positive selection of T cells expressing a male-specific receptor in T-cell receptor transgenic mice.

The ontogeny of T cells in T-cell receptor (TCR) transgenic mice, which express a transgenic alpha beta heterodimer, specific for the male (H-Y) antigen in association with H-2Db, was determined. The transgenic alpha chain was expressed on about 10% of the fetal thymocytes on day 14 of gestation. About 50% of day-15 fetal thymocytes expressed both alpha and beta transchains and virtually all fetal thymocytes expressed the transgenic alpha beta heterodimer by day 17. The early expression of the transgenic TCR on CD4-8- thymocytes prevented the development of gamma delta cells, and led to accelerated growth of thymocytes and an earlier expression of CD4 and CD8 molecules. Up to day 17, no significant differences in T-cell development could be detected between female and male thymuses. By day 18 of gestation, the male transgenic thymus contained more CD4-8- thymocytes than the female transgenic thymus. The preponderance of CD4-8- thymocytes in the male transgenic thymus increased until birth and was a consequence of the deletion of the CD4+8+ thymocytes and their CD4-8+ precursors. By the time of birth, the male transgenic thymus contained half the number of cells as the female transgenic thymus. The deletion of autospecific precursor cells in the male transgenic mouse began only at day 18 of gestation, despite the fact that the ligand could already be detected by day 16. The preferential accumulation of CD4-8+ T cells, which expressed a high density of the transgenic TCR, occurred only after birth and was obvious in 6-week-old female thymus. These data support the hypothesis that the positive selection of T cells expressing this transgenic heterodimer may involve two steps, i.e., the commitment of CD4+8+ thymocytes to the CD4-8+ lineage following the interaction of the transgenic TCR with restricting major histocompatibility molecules, followed by a slow conversion of CD4+8+ thymocytes into CD4-8+ T cells. In normal mice, the precursors of CD4+8+ and single positive thymocytes have the CD4-8- CD3-J11d+ (or M1/69+) phenotype. Because of the early expression of the transgenic alpha beta heterodimer, this population was not detected in adult transgenic mice. All CD4-8- M1/69+ cells expressed the transgenic receptor associated with CD3 and could be readily grown in media containing T-cell lectins and interleukin 2.

Animals↗
PubMed2003

[The viral resistance of transgenic tobacco plants containing untranslatable PVYN coat protein gene].

In previous study, we have proved that the resistance of the transgenic tobacco plants containing untranslatable PVYN CP gene was mediated by PVYN CP transgene RNAs, and the resistance mechanism was similar to PTGS. In this paper, T0 progeny transgenic lines with different resistant levels were selected to further study the transgene inheritance and resistant stability of transgenic plants. Results showed that T0 progeny susceptible lines, which contained 1-2 transgene copies, displayed a 3:1 segregation ratio in T1 progeny lines; T0 progeny middle resistant lines, which contained 3-4 transgene copies, revealed a 15:1 segergation ratio in T1 progeny lines; T0 progeny highly resistant lines, which contained 5-7 transgene copies, followed 15:1 or 63:1 segregation pattern in T1 progeny lines. Southern blot analysis revealed the resistance in most T1 and T2 progeny transgenic lines was related to copy numbers of the transgene. Northern blot analysis indicated that PVYN CP transgenes were expressed at transcription level in most T1 and T2 progeny transgenic lines, and the transgene mRNA accumulation in cytoplasm varied among transgenic lines. There was an inverse correlation between transgene transcript accumulation and virus resistance. Resistance of transgenic lines was inheritable over at least two generations, and the resistance of T2 progeny transgenic lines, which containing untranslatable PVYN CP gene, was (1) not overcome by PVYN particle or PVYN RNA; (2) independent of inoculum levels; (3) resistant to either aphid or mechanically transmitted PVYN; (4) not dependent on plant development stages; (5) PVY-specific (i.e., broad-spectrum resistance was not observed).

Capsid Proteins↗
PubMed2006

Field released transgenic papaya effect on soil microbial communities and enzyme activities.

Soil properties, microbial communities and enzyme activities were studied in soil amended with replicase (RP)-transgenic or non-transgenic papaya under field conditions. Compared with non-transgenic papaya, significant differences (P<0.05) were observed in total nitrogen in soils grown with transgenic papaya. There were also significant differences (P<0.05) in the total number of colony forming units (CFUs) of bacteria, actinomycetes and fungi between soils amended with RP-transgenic plants and non-transgenic plants. Compared with non-transgenic papaya, the total CFUs of bacteria, actinomycetes and fungi in soil with transgenic papaya increased by 0.43-1.1, 0.21-0.80 and 0.46-0.73 times respectively. Significantly higher (P<0.05) CFUs of bacteria, actinomycetes and fungi resistant to kanamycin (Km) were obtained in soils with RP-transgenic papaya than those with non-transgenic papaya in all concentrations of Km. Higher resistance quotients for Kmr (kanamycin resistant) bacteria, actinomycetes and fungi were found in soil planted with RP-transgenic papaya, and the resistance quotients for Kmr bacteria, actinomycetes and fungi in soils with transgenic papaya increased 1.6-4.46, 0.63-2.5 and 0.75-2.30 times. RP-transgenic papaya and non-transgenic papaya produced significantly different enzyme activities in arylsulfatase (5.4-5.9x), polyphenol oxidase (0.7-1.4x), invertase (0.5-0.79x), cellulase (0.23-0.35x) and phosphodiesterase (0.16-0.2x). The former three soil enzymes appeared to be more sensitive to the transgenic papaya than the others, and could be useful parameters in assessing the effects of transgenic papaya. Transgenic papaya could alter soil chemical properties, enzyme activities and microbial communities.

Actinomyces↗
PubMed2004

Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking.

Transgenes can affect transgenic mice via transgene expression or via the so-called positional effect. DNA sequences can be localized in chromosomes using recently established mouse genomic databases. In this study, we describe a chromosomal mapping method that uses the genomic walking technique to analyze genomic sequences that flank transgenes, in combination with mouse genome database searches. Genomic DNA was collected from two transgenic mouse lines harboring pCAGGS-based transgenes, and adaptor-ligated, enzyme restricted genomic libraries for each mouse line were constructed. Flanking sequences were determined by sequencing amplicons obtained by PCR amplification of genomic libraries with transgene-specific and adaptor primers. The insertion positions of the transgenes were located by BLAST searches of the Ensembl genome database using the flanking sequences of the transgenes, and the transgenes of the two transgenic mouse lines were mapped onto chromosomes 11 and 3. In addition, flanking sequence information was used to construct flanking primers for a zygosity check. The zygosity (homozygous transgenic, hemizygous transgenic and non-transgenic) of animals could be identified by differential band formation in PCR analyses with the flanking primers. These methods should prove useful for genetic quality control of transgenic animals, even though the mode of transgene integration and the specificity of flanking sequences needs to be taken into account.

5' Flanking Region↗
PubMed1992

An X-linked human collagen transgene escapes X inactivation in a subset of cells.

Transgenic mice carrying one complete copy of the human alpha 1(I) collagen gene on the X chromosome (HucII mice) were used to study the effect of X inactivation on transgene expression. By chromosomal in situ hybridization, the transgene was mapped to the D/E region close to the Xce locus, which is the controlling element. Quantitative RNA analyses indicated that transgene expression in homozygous and heterozygous females was about 125% and 62%, respectively, of the level found in hemizygous males. Also, females with Searle's translocation carrying the transgene on the inactive X chromosome (Xi) expressed about 18% transgene RNA when compared to hemizygous males. These results were consistent with the transgene being subject to but partially escaping from X inactivation. Two lines of evidence indicated that the transgene escaped X inactivation or was reactivated in a small subset of cells rather than being expressed at a lower level from the Xi in all cells, (i) None of nine single cell clones carrying the transgene on the Xi transcribed transgene RNA. In these clones the transgene was highly methylated in contrast to clones carrying the transgene on the Xa. (ii) In situ hybridization to RNA of cultured cells revealed that about 3% of uncloned cells with the transgene on the Xi expressed transgene RNA at a level comparable to that on the Xa. Our results indicate that the autosomal human collagen gene integrated on the mouse X chromosome is susceptible to X inactivation. Inactivation is, however, not complete as a subset of cells carrying the transgene on Xi expresses the transgene at a level comparable to that when carried on Xa.

Animals↗
PubMed1999

Insulin-sensitive regulation of glucose transport and GLUT4 translocation in skeletal muscle of GLUT1 transgenic mice.

Skeletal muscle glucose transport was examined in transgenic mice overexpressing the glucose transporter GLUT1 using both the isolated incubated-muscle preparation and the hind-limb perfusion technique. In the absence of insulin, 2-deoxy-d-glucose uptake was increased approximately 3-8-fold in isolated fast-twitch muscles of GLUT1 transgenic mice compared with non-transgenic siblings. Similarly, basal glucose transport activity was increased approximately 4-14-fold in perfused fast-twitch muscles of transgenic mice. In non-transgenic mice insulin accelerated glucose transport activity approximately 2-3-fold in isolated muscles and to a much greater extent ( approximately 7-20-fold) in perfused hind-limb preparations. The observed effect of insulin on glucose transport in transgenic muscle was similarly dependent upon the technique used for measurement, as insulin had no effect on isolated fast-twitch muscle from transgenic mice, but significantly enhanced glucose transport in perfused fast-twitch muscle from transgenic mice to approximately 50-75% of the magnitude of the increase observed in non-transgenic mice. Cell-surface glucose transporter content was assessed via 2-N-4-(l-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(d -mannos-4-yloxy)-2-propylamine photolabelling methodology in both isolated and perfused extensor digitorum longus (EDL). Cell-surface GLUT1 was enhanced by as much as 70-fold in both isolated and perfused EDL of transgenic mice. Insulin did not alter cell-surface GLUT1 in either transgenic or non-transgenic mice. Basal levels of cell-surface GLUT4, measured in either isolated or perfused EDL, were similar in transgenic and non-transgenic mice. Interestingly, insulin enhanced cell-surface GLUT4 approximately 2-fold in isolated EDL and approximately 6-fold in perfused EDL of both transgenic and non-transgenic mice. In summary, these results reveal differences between isolated muscle and perfused hind-limb techniques, with the latter method showing a more robust responsiveness to insulin. Furthermore, the results demonstrate that muscle overexpressing GLUT1 has normal insulin-induced GLUT4 translocation and the ability to augment glucose-transport activity above the elevated basal rates.

Affinity Labels↗
PubMed1998

Irregular patterns of transgene silencing in allohexaploid oat.

An irregular pattern of transgene silencing was revealed in expression and inheritance studies conducted over multiple generations following transgene introduction by microprojectile bombardment of allohexaploid cultivated oat (Avena sativa L.). Expression of two transgenes, bar and uidA, delivered on the same plasmid was investigated in 23 transgenic oat lines. Twenty-one transgenic lines, each derived from an independently selected transformed tissue culture, showed expression of both bar and uidA while two lines expressed only bar. The relationship of the transgenic phenotypes to the presence of the transgenes in the study was determined using (1) phenotypic scoring combined with Southern blot analyses of progeny, (2) coexpression of the two transgenic phenotypes since the two transgenes always cosegregated, and (3) reactivation of a transgenic phenotype in self-pollinated progenies of transgenic plants that did not exhibit a transgenic phenotype. Transgene silencing was observed in 19 of the 23 transgenic lines and resulted in distorted segregation of transgenic phenotypes in 10 lines. Silencing and inheritance distortions were irregular and unpredictable. They were often reversible in a subsequent generation of self-pollinated progeny and abnormally segregating progenies were as likely to trace back to parents that exhibited normal segregation in a previous generation as to parents showing segregation distortions. Possible causes of the irregular patterns of transgene silencing are discussed.

Acetyltransferases↗
PubMed2004

Human apoB contributes to increased serum total apo(a) level in LPA transgenic mice.

BACKGROUND: The Lp(a) lipoprotein (Lp(a)) consists of the polymorphic glycoprotein apolipoprotein(a) (apo(a)), which is attached by a disulfide bond to apolipoprotein B (apoB). Apo(a), which has high homology with plasminogen, is present only in primates and hedgehogs. However, transgenic mice and rabbits with high serum apo(a) levels exist. Liver is the main site for apo(a) synthesis, but the site of removal is uncertain. To examine differences between transgenic mice expressing the LPA gene and mice capable of forming Lp(a) particles, LPA-YAC transgenic mice and hAPOB transgenic mice were crossed and their offspring examined. RESULTS: Comparison of LPA-YAC with LPA-YAC/hAPOB transgenic mice showed that LPA-YAC/hAPOB transgenic mice have higher serum total apo(a) and total cholesterol level than mice lacking the hAPOB gene. However, hepatic apo(a) mRNA level was higher in LPA-YAC transgenic mice than in LPA-YAC/hAPOB transgenic mice. Feeding of a high-cholesterol/high-fat diet to male LPA-YAC transgenic mice with or without the hAPOB gene resulted in reduced serum total apo(a) and hepatic apo(a) mRNA level. CONCLUSION: In conclusion, the higher serum total apo(a) level in LPA-YAC/hAPOB transgenic mice than in LPA-YAC transgenic mice is not caused by increased apo(a) synthesis. Lower hepatic apo(a) mRNA level in LPA-YAC/hAPOB than in LPA-YAC transgenic mice may suggest that the increase in total apo(a) level is a result of apo(a) accumulation in serum. Furthermore, observed higher serum total cholesterol level in LPA-YAC/hAPOB transgenic mice than either in wild type or LPA-YAC transgenic mice may further suggest that human APOB transgenicity is a factor that contributes to increased serum total apo(a) and cholesterol levels. Our results on reduced serum total apo(a) and hepatic apo(a) mRNA levels in HCHF fed male LPA-YAC transgenic mice confirm earlier findings in females, and show that there are no sex difference in mechanisms for lowering apo(a) level in response to HCHF feeding.

Journal Article↗
PubMed2002

Lactational performance of first-parity transgenic gilts expressing bovine alpha-lactalbumin in their milk.

The goal of this study was to determine whether the presence of the bovine alpha-lactalbumin transgene in first-lactation gilts enhances lactational performance and litter growth. Transgenic and sibling nontransgenic gilts were bred to nontransgenic boars. Litters were standardized to 10 piglets within 24 h of farrowing. Milk production was measured by the weigh-suckle-weigh method on d 3, 6, 9, and 12 of lactation. Bovine alpha-lactalbumin was present in the colostrum and milk of transgenic gilts throughout lactation. The expression of the transgene was associated with alterations in composition of mammary secretions, especially in early lactation. Lactose concentrations were greater (P < 0.05) in mammary secretions of transgenic gilts during the first 12 h postpartum compared with controls. In contrast, total solids concentration in mammary secretions from transgenic gilts were lower (P < 0.05) relative to controls during the first 6 h postpartum. Transgenic gilts produced more milk than controls on d 3, 6, and 9 of lactation (P < 0.01). By d 12, differences in milk production between transgenic and control sows were no longer different. Lactose intake by transgenic-reared litters was greater than lactose intake by control-reared litters on d 6 of lactation (P < 0.05). Total solids intake was significantly greater (P < 0.05) by transgenic-reared litters on d 3 and 6 compared to control-reared litters. The day x genotype interaction on litter weight gain after birth was highly significant (P = 0.011), with transgenic-reared litters gaining weight at a greater rate than control-reared piglets. Expression of the transgene was associated with increased milk production in lactating gilts and increased growth of transgenic-reared piglets. Increased lactose synthesis in response to the presence of the transgene may result in increased milk production in early lactation, leading to increased milk component intake by transgenic litters, and ultimately to increased growth of litters reared by first-parity transgenic gilts.

Animals↗
PubMed2000

DNA preparation method can influence outcome of transgenic animal experiments.

In our continuing quest to improve the efficiency of producing transgenic animals, we have compared the influence of two transgene purification techniques on the efficiency of creating transgenic sheep and mice. Three hundred eighty-seven sheep zygotes and 2,737 mouse zygotes were microinjected with one of four transgenes. Transgenes were isolated from plasmid sequences either by agarose gel electrophoresis followed by gel extraction or by a single step sodium chloride gradient fractionation technique. Four transgenic sheep and 61 transgenic mice were produced. Both sheep and mice embryos responded similarly to transgene preparation methods. Overall, pregnancy rate was higher for recipients that received embryos injected with NaCl purified DNA (mean +/- SEM: 64 +/- 7% vs. 38 +/- 7%). Furthermore, offspring per zygote transferred (NaCl, 22 +/- 3% vs. Gel, 12 +/- 3%) and transgenics born per zygote transferred (NaCl, 3.9 +/- 0.6% vs. Gel, 1.5 +/- 0.6%) were higher when the NaCl purified DNA was used. However, the proportion of offspring born that were identified as transgenic did not differ between transgene purification methods. Transgenes responded differently to methods of preparation. One of the four genes yielded a significantly higher proportion of transgenics when the transgene was prepared by NaCl purification. These data suggest that on average the NaCl gradient purification technique results in a higher embryo survival rate to term for both sheep and mice, but the technique has no influence on rate of transgene integration.

Animals↗
PubMed1999

Calcium regulatory proteins and their alteration by transgenic approaches.

Abnormalities in calcium flux have been linked to abnormal contractile behavior of the heart in patients with congestive heart failure as well as in animal models. Decreased activity or levels of the calcium adenosine triphosphatase of the sarco(endo)plasmic reticulum (SERCA2) particularly have been known to cause a delay in calcium transients. The SERCA2 protein pumps 2 moles of calcium per mole of adenosine triphosphate (ATP) split from the cytoplasm into the sarcoplasmic reticulum, thus lowering the free cytoplasmic calcium concentration. It therefore is of interest to identify mechanisms by which SERCA activity could be increased in the heart. To determine influences of increased expression of the SERCA2 gene on calcium transient and contractile behavior, we constructed transgenic mice and rats expressing a SERCA2 transgene in their heart. In these animals, a 20% increase in SERCA levels occurs due to additional expression of the SERCA transgene. This leads to a corresponding increase in contractile activity as determined by the increase in left ventricular pressure measured as dP/dt(max) and decrease in diastolic ventricular pressure determined as dP/dt(min). Similarly, isolated cardiac myocytes obtained from the heart of transgenic mice showed an accelerated calcium transient and increased speed of shortening and relengthening as determined by edge detection. To determine if SERCA2 transgene expression could have a compensatory effect on the contractile behavior of the heart in transgenic mice expressing SERCA2, these mice were made hypothyroid, and papillary muscle function was determined. Contractile behavior of the papillary muscle of wild-type hypothyroid mice showed a significant increase in muscle relaxation time (RT50). In contrast, SERCA2 transgenic hypothyroid mice showed normal contractile behavior of papillary muscle. A compensatory effect of SERCA transgene expression was therefore demonstrated. In addition, we constructed transgenic rats expressing a SERCA2 transgene in which constriction of the ascending aorta induced cardiac hypertrophy and a delayed contraction of papillary muscle. In preliminary results, we found that SERCA2 transgenic rats submitted to ascending aortic constriction did not show the delayed relaxation of papillary muscle as was found in wild-type rats submitted to aortic constriction. In addition, adenoviral vectors expressing transgenes for calcium-handling proteins can be used to improve cardiac myocyte contraction. Adenoviruses expressing a SERCA transgene or a mutant phospholamban transgene exhibiting dominant negative action were used to infect isolated myocytes treated with a phorbol ester (phorbol 12-myristate 13-acetate), which delays the calcium transients. The calcium transients and contractile behavior of the isolated myocytes indicated that increased SERCA expression or increased expression of mutant phospholamban transgene led to increased SERCA2 activity, resulting in an increased contractile phenotype. Recent findings by other investigators also indicate that decreased SERCA2 activity can be increased under in vivo conditions using adenoviral vector-based SERCA2 expression. A gene therapy type of approach delivering increased amounts of SERCA or phospholamban mutants leading to increased SERCA activity should therefore be considered in the future.

Adenosine Triphosphate↗
PubMed2006

Inheritance analysis of herbicide-resistant transgenic soybean lines.

Four transgenic soybean lines generated via Agrobacterium-mediated transformation were used to analyze inheritance of the transgenes. Seed chip GUS assay and herbicide leaf painting and spraying assays were applied to test the gus reporter gene and the herbicide resistant bar selectable marker gene, respectively. Three of the four transgenic soybean lines were stably inherited in a Mendelian fashion with co-segregation of both transgenes in a 3:1 segregation ratio in the T(1) progeny, indicating that both transgenes were integrated into the same locus of the soybean genome. Homozygous transgenic progeny plants were obtained in the T(2) generation of these lines, and the transgenes were inherited in five successive generations. However, in one transgenic line, all the T(1) progeny plants showed GUS negative and herbicide sensitive. Southern blotting analysis confirmed that the transgenes were passed into the T(1) progeny, indicating that the transgenes were both silenced. To test if the transgene silencing was due to transcriptional or post-transcriptional level, Soybean mosaic virus (SMV) was inoculated on leaf tissues of the T(1) plants to test possible reverse effects on transgene silencing. Infection with SMV did not suppress transgene silencing, suggesting that transgene silencing in this transgenic line may not be due to post-transcriptional gene silencing.

Gene Silencing↗
PubMed2000

Modeling Alzheimer's disease in transgenic mice: effect of age and of presenilin1 on amyloid biochemistry and pathology in APP/London mice.

In transgenic mice that overexpress mutant Amyloid Precursor Protein [V717I], or APP/London (APP/Lo) (1999a. Early phenotypic changes in transgenic mice that overexpress different mutants of Amyloid Precursor Protein in brain. J. Biol. Chem. 274, 6483-6492; 1999b. Premature death in transgenic mice that overexpress mutant Amyloid precursor protein is preceded by severe neurodegeneration and apoptosis. Neuroscience 91, 819-830) the AD related phenotype of plaque and vascular amyloid pathology is late (12-15 months). This typical and diagnostic pathology is thereby dissociated in time from early symptoms (3-9 months) that include disturbed behavior, neophobia, aggression, glutamate excitotoxicity, defective cognition and decreased LTP. The APP/Lo transgenic mice are therefore a very interesting model to study early as well as late pathology, including the effect of age. In ageing APP*Lo mice, brain soluble and especially "insoluble" amyloid peptides dramatically increased, while normalized levels of secreted APPsalpha and APPsbeta, as well as cell-bound beta-C-stubs, remained remarkably constant, indicating normal alpha- and beta-secretase processing of APP. In double transgenic mice, i.e. APP/LoxPS1, clinical mutant PS1[A246E] but not wild-type human PS1 increased Abeta, and plaques and vascular amyloid developed at age 6-9 months. The PS1 mutant caused increasing Abeta42 production, while ageing did not. Amyloid deposits are thus formed, not by overproduction of Abeta, but by lack of clearance and/or degradation in the brain of ageing APP/Lo transgenic mice. The clearance pathways of the cerebral amyloid peptides are therefore valuable targets for fundamental research and for therapeutic potential. Although hyper-phosphorylated protein tau was evident in swollen neurites around the amyloid plaques, neurofibrillary pathology is not observed and the "tangle" aspect of AD pathology is therefore still missing from all current transgenic "amyloid" models. Also the "ApoE4" risk for late onset AD remains a problem for modeling in transgenic mice. We have generated transgenic mice that overexpress human ApoE4 (2000. Expression of Human Apolipoprotein E4 in neurons causes hyperphosphorylation of Protein tau in the brains of transgenic mice. Am. J. Pathol. 156 (3) 951-964) or human protein tau (1999. Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein. Am. J. Pathol. 155, 2153-2165) in their neurons. Both develop a similar although not identical axonopathy, with progressive degeneration of nerves and with muscle wasting resulting in motoric problems. Remarkably, ApoE4 transgenic mice are, like the tau transgenic mice, characterized by progressive hyper-phosphorylation of protein tau also in motor neurons which explains the motoric defects. Further crossing with the APP/Lo transgenic mice is ongoing to yield "multiple" transgenic mouse strains to study new aspects of amyloid and tau pathology.

Aging↗