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At least 937 records · Page 52Linked to original sources
PubMed1999

A transgenic rabbit model for human hypertrophic cardiomyopathy.

Certain mutations in genes for sarcomeric proteins cause hypertrophic cardiomyopathy (HCM). We have developed a transgenic rabbit model for HCM caused by a common point mutation in the beta-myosin heavy chain (MyHC) gene, R400Q. Wild-type and mutant human beta-MyHC cDNAs were cloned 3' to a 7-kb murine beta-MyHC promoter. We injected purified transgenes into fertilized zygotes to generate two lines each of the wild-type and mutant transgenic rabbits. Expression of transgene mRNA and protein were confirmed by Northern blotting and 2-dimensional gel electrophoresis followed by immunoblotting, respectively. Animals carrying the mutant transgene showed substantial myocyte disarray and a 3-fold increase in interstitial collagen expression in their myocardia. Mean septal thicknesses were comparable between rabbits carrying the wild type transgene and their nontransgenic littermates (NLMs) but were significantly increased in the mutant transgenic animals. Posterior wall thickness and left ventricular mass were also increased, but dimensions and systolic function were normal. Premature death was more common in mutant than in wild-type transgenic rabbits or in NLMs. Thus, cardiac expression of beta-MyHC-Q(403) in transgenic rabbits induced hypertrophy, myocyte and myofibrillar disarray, interstitial fibrosis, and premature death, phenotypes observed in humans patients with HCM due to beta-MyHC-Q(403).

Animals↗
PubMed2004

Suppression of apoptosis by bcl-2 overexpression in lymphoid cells of transgenic zebrafish.

The zebrafish is an attractive vertebrate model for genetic studies of development, apoptosis, and cancer. Here we describe a transgenic zebrafish line in which T- and B-lymphoid cells express a fusion transgene that encodes the zebrafish bcl-2 protein fused to the enhanced green fluorescence protein (EGFP). Targeting EGFP-bcl-2 to the developing thymocytes of transgenic fish resulted in a 2.5-fold increase in thymocyte numbers and a 1.8-fold increase in GFP-labeled B cells in the kidney marrow. Fluorescent microscopic analysis of living rag2-EGFP-bcl-2 transgenic fish showed that their thymocytes were resistant to irradiation- and dexamethasone-induced apoptosis, when compared with control rag2-GFP transgenic zebrafish. To test the ability of bcl-2 to block irradiation-induced apoptosis in malignant cells, we compared the responsiveness of Myc-induced leukemias with and without EGFP-bcl-2 expression in living transgenic zebrafish. T-cell leukemias induced by the rag2-EGFP-Myc transgene were ablated by irradiation, whereas leukemias in double transgenic fish expressing both Myc and EGFP-bcl-2 were resistant to irradiation-induced apoptotic cell death. The forward genetic capacity of the zebrafish model system and the ability to monitor GFP-positive thymocytes in vivo make this an ideal transgenic line for modifier screens designed to identify genetic mutations or small molecules that modify bcl-2-mediated antiapoptotic pathways.

Amino Acid Sequence↗
PubMed2004

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle.

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 microg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.

Animals↗
PubMed2005

Comparative development and impact of transgenic papayas in Hawaii, Jamaica, and Venezuela.

We present data concerning the creation of transgenic papayas resistant to Papaya ringspot virus (PRSV) and their adoption by three different countries: the United States (e.g., Hawaii), Jamaica, and Venezuela. Although the three sets of transgenic papayas showed effective resistance to PRSV, the adoption rate in each country has varied from full utilization in Hawaii to aggressive testing but delay in deregulating of the product in Jamaica to rejection at an early stage in Venezuela. Factors that contributed to the rapid adoption in Hawaii include a timely development of the transgenic product, PRSV causing severe damage to the papaya industry, close collaboration between researchers and the industry, and the existence of procedures for deregulating a transgenic product. In Jamaica, the technology for developing the initial field-testing of the product progressed rather rapidly, but the process of deregulation has been slowed down owing to the lack of sustained governmental efforts to complete the regulatory procedures for transgenic crops. In Venezuela, the technology to develop and greenhouse test the transgenic papaya has moved abreast with the Jamaica project, but the field testing of the transgenic papaya within the country was stopped very early on by actions by people opposed to transgenic products. The three cases are discussed in an effort to provide information on factors, other than technology, that can influence the adoption of a transgenic product.

Amino Acid Sequence↗
PubMed2005

Making better transgenic models: conditional, temporal, and spatial approaches.

Over the last decade transgenic mouse models have become a common experimental tool for unraveling gene function. During this time there has been a growing expectation that transgenes resemble the in vivo state as much as possible. To this end, a preference away from heterologous promoters has emerged, and transgene constructs often utilize the endogenous promoter and gene sequences in BAC, PAC and YAC form without the addition of selectable markers, or at least their subsequent removal. There has been a trend toward controlled integration by homologous recombination, either at a characterized chromosomal localization or in some cases within the allele of interest. Markers such as green fluorescent protein (GFP), beta-galactosidase (LacZ), and alkaline phosphatase (AP) continue to be useful to trace transgenic cells, or transgene expression. The development of technologies such as RNA interference (RNAi), are introducing new ways of using transgenic models. Future developments in RNAi technology may revolutionize tissue specific inactivation of gene function, without the requirement of generating conditionally targeted mice and tissue specific recombinase mice. Transgenic models are biological tools that aid discovery. Overall, the main consideration in the generation of transgenic models is that they are bona fide biological models that best impart the disease model or biological function of the gene that they represent. The main consideration is to make the best model for the biological question at heart and this review aims to simplify that task somewhat. Here we take a historical perspective on the development of transgenic models, with many of the important considerations to be made in design and development along the way.

Animals↗
PubMed1999

Establishment of SV40-tsA58 transgenic rats as a source of conditionally immortalized cell lines.

To isolate a variety of rat cell lines with differentiated functions, we established transgenic rat lines expressing the temperature-sensitive large T-antigen of simian virus 40 (SV40) tsA58 mutant under the control of the SV40 large T-antigen itself. We microinjected the DNA into 564 eggs of Wistar rat and 23 independent transgenic candidates were obtained. Ten pups died before weaning and eight transgenic rats could not transmit the transgene to the progeny. Finally, five lines of the transgenic rat were established. Although one line (#1511-6) had low reproductivity, the other four lines reproduced normally. Three out of the four lines (#1507-2, #1509-7, #1519-8) appeared normal but the other line had tumors in the brain and subcutaneous tissue at 3 weeks of age (#1511-6), and in the kidneys and subcutaneous tissue at 18 to 19-weeks of age (#1507-5). Fibroblast cells prepared from transgenic fetuses of lines #1507-5 and #1519-8 expressed the transgene and exhibited temperature-dependent growth. Both of the lines (#1507-5 and #1519-8) were successfully generated to be homozygous by sibling mating of transgenic offspring. These transgenic rat lines have bred through many generations and have been established to be a ready source of novel conditionally immortalized cell lines.

Animals↗
PubMed2005

Effect of Cry3Bb1-expressing transgenic corn on plant-to-plant movement by western corn rootworm larvae (Coleoptera: Chrysomelidae).

Dispersal of larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, in specific combinations of transgenic corn expressing the Cry3Bb1 protein and nontransgenic, isoline corn was evaluated in a 2-yr field study. In total, 1,500 viable western corn rootworm eggs were infested in each subplot. Each year, plant damage and larval recovery were evaluated among four pedigree combinations (straight transgenic; straight nontransgenic corn; nontransgenic corn with a transgenic central, infested plant; and transgenic corn with a nontransgenic central, infested plant) on six sample dates between egg hatch and pupation. For each subplot, the infested plant, three successive plants down the row (P1, P2, and P3), the closest plant in the adjacent row of the plot, and a control plant were sampled. The number of western corn rootworm larvae recovered from transgenic rootworm-resistant plants adjacent to infested nontransgenic plants was low and not statistically significant in either 2001 or 2002. In 2001, significantly fewer larvae were recovered from transgenic rootworm-resistant plants than from nontransgenic plants when both were adjacent to infested, nontransgenic plants. In 2002, significantly more neonate western corn rootworm larvae were recovered from nontransgenic plants adjacent to infested, transgenic rootworm-resistant plants than nontransgenic plants adjacent to infested, nontransgenic plants on the second sample date. Together, these data imply that both neonate and later instar western corn rootworm larvae prefer nontransgenic roots to transgenic rootworm-resistant roots when a choice is possible. However, when damage to the infested, nontransgenic plant was high, western corn rootworm larvae apparently moved to neighboring transgenic rootworm-resistant plants and caused statistically significant, although only marginally economic, damage on the last sample date in 2001. Implications of these data toward resistance management plan are discussed.

Animals↗
PubMed2002

Measuring Gene Flow in the Cultivation of Transgenic Barley.

Genetic engineering is becoming a useful tool in the improvement of plants and plant-based raw materials. Varieties with value-added traits are developed for nonfood use in industrial and medical production, and different production lines must be kept separate. For good management practices, knowledge of relevant gene flow parameters is required. In the present study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. A transgenic barley (Hordeum vulgare L.) line carrying a marker gene coding for neomycin phosphotransferase II (nptII) was used as a pollen donor. For maximum resolution, a cytoplasmically male-sterile barley line was utilized as recipient and the flow of nptII transgene was monitored at distances of 1, 2, 3, 6, 12, 25, 50, and 100 m from the donor plots of 225 and 2000 m(2). Male-fertile plots at a distance of 1 m were included to measure the transgene flow in normal barley. The number of seeds obtained from male-sterile heads diminished rapidly with distance and only a few seeds were found at distances of 50 and 100 m. Molecular genetic analysis (polymerase chain reaction-PCR) revealed that all seeds obtained from male-sterile heads at a distance of 1 m were transgenic, as anticipated. However, only 3% of the distant seeds (50 m) actually carried the transgene, whereas most of them resulted from fertilization with nontransgenic background pollen. This background pollen was mainly due to pollen leakage in some male-sterile heads. In normal male-fertile barley, the cross-fertilization frequency with transgenic pollen varied from 0 to 7% at a distance of 1 m, depending on weather conditions on the heading day. We conclude that, because of competing self-produced and nontransgenic background pollen, the possibility of cross-pollination is very low between a transgenic barley field and an adjacent field cultivated with normal barley. However, adequate isolation distances and best management practices are needed for cultivation of transgenic barley.

Journal Article↗
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↗
PubMed2003

Oral immunization of animals with transgenic cherry tomatillo expressing HBsAg.

AIM: To investigate the expression of recombinant HBsAg (rHBsAg) in transgenic cherry tomatillo in order to explore the feasibility of producing HBV oral vaccine with cherry tomatillo by animal immune tests. METHODS: The recombinant plant expression vector containing HBsAg gene was constructed. Mediated with Agrobacterium tumefaciens, HBsAg gene was transferred into cotyledons of cherry tomatillo. Transformed cherry tomatillos were obtained through hygromycin delay-selection. Integrated DNA in transgenic cherry tomatillo was confirmed by hygromycin resistance selection, Gus detection, polymerase chain reaction (PCR) and dot blotting analysis. Antigenicity of rHBsAg was examined by ELISA and the immunogenicity of rHBsAg derived from transgenic cherry tomatillo tissues was confirmed by oral feed of transformed tissues to BALB/c mice primed with commercial HBV vaccines. Specific antibody titers in mice's serum were examined by ELISA every week. RESULTS: By far, 10 positive lines of transgenic cherry tomatillos containing HBsAg gene were obtained. Among different organs of the same transgenic cherry tomatillo, level of rHBsAg expressed in leaves was the highest with the yield up to 300ng/g fresh weight. And the rHBsAg expression level in fruits was about 10 ng/g fresh weight. In animal immune tests, oral delivery with transgenic tissues to mice primed with commercial vaccine instead of naive mice resulted in significant immune response. CONCLUSION: The result of this animal immune test indicated the rHBsAg derived from transgenic cherry tomatillo possessed normal immunogenicity. This work demonstrated the feasibility to generate oral immunogenic rHBsAg in transgenic cherry tomatillo, and would provide some experimental approach for the production of low-cost oral vaccines using transgenic cherry tomatillo in large scale.

Administration, Oral↗
PubMed1998

Cloning of 1-aminocyclopropane-1-carboxylate (ACC) synthetase cDNA and the inhibition of fruit ripening by its antisense RNA in transgenic tomato plants.

A 1.7 kb fragment of ACC synthetase cDNA, one member of the ACC synthetase multigene family, was amplified from total tomato cDNA through a polymerase chain reaction (PCR) and cloned in E. coli. Restriction mapping and sequencing analysis confirmed its fidelity and correctness. The cloned ACC synthetase gene was then inserted into a binary vector pBin437, in an inverted orientation between the CaMV 35S promoter with duplicated enhancers and the Nos 3' transcriptional termination sequence, to construct an expression vector pBACC. Transgenic tomato plants were obtained by A. tumefaciens-mediated transformation of cotyledons. PCR detection and Southern blot analysis confirmed the integration of the antisense ACC synthetase gene in the transformed tomato genome. The results from RT-PCR of RNAs isolated from transgenic tomato leaves confirmed that antisense ACC synthetase RNA was synthesized in these transgenic plants. The amount of ethylene released from transgenic tomato fruits was reduced significantly to about 30% of that released by non-transformed controls. The inhibition effect of antisense RNA on fruit ripening was observed in transgenic plants and their progeny (T1). The shelf life of transgenic tomato fruits was at least 60 days at room temperature without significant change in hardness and color. After 15-20 days of treatment of the transgenic fruits with ethylene, most of them reached the ripe stage. The antisense ACC synthetase gene was inherited as a single gene in the progenies of transgenic tomatoes determined by T1 progeny analysis, consistent with the results of Southern blot analysis. Transgenic homozygotes expressing antisense ACC synthetase RNA showed prolonged shelf life in the T2 progeny.

Cloning, Molecular↗
PubMed2002

[Studies of F1 of transgenic allotetraploid hybrids of Carassius auratus red var. (Female) x Cyprinus carpio (Male)].

The tetraploid fish has been developed by assortative breeding the hybrids of Carassius auratus red var. (Female) x cyprinus carpio (Male), which has the stable genetic characters and can reproduce themselves. An "all-fish" recombinant DNA construct (pCAgcGHc) containing common carp beta-actin gene promoter and cDNA for grass carp (Ctenopharyngodon idella) growth hormone gene was introduced into fertilized eggs of the allotetroploid fish through microinjection as soon as artificial insemination was done. Artificial insemination was carried out between the female and the male transgenic allotetraploid fish which contain the "all-fish" recombinant DNA construct (pCAgcGHc) and are the biggest in the size. Fifty F1 samples of transgenic allotetraploid fish of 150 days and 50 allotetraploid fish (regarded as the control) were chosen, and the weight and the body length of each were measured, the results showed that F1 of transgenic allotetraploid fish of 150 days had obvious growth dominance compared with the control. Genomic DNA of tail fin was extracted from 20 F1 of transgenic allotetraploid fish of 150 days and the control. Proper primers were introduced to check whether the sample had the transgene. Pa, the upstream primer, is located in beta-actin promoter, and Pg, the downstream primer, is located in growth hormone cDNA for grass carp (gcGHc). The transgene was detected in 90% F1 of transgenic allotetraploid fish in tail fin DNA by polymerase chain reaction (PCR) amplification. Sperm could be squeezed out from a few F1 of transgenic allotetraploid fish of 150 days, however, this phenomenon did not exist in the controls. The importance of forming the pure line of transgenic allotetraploid was elucidate in the paper.

Animals↗
PubMed2003

[Virus resistance in transgenic watermelon plants containing a WMV-2 coat protein gene].

Virus disease is a major cause that affects the quality and output of watermelon which is an important fruit in summer. So it is really urgent to develop disease resistance plants. But it takes a long time to breed such plants in conventional ways, and it is very difficult to get ideal result. With the development of plant genetic engineering, new ways have been found to breed plants with disease resistance. By using plant transgenic technique, much progress was been made in plant improvement. There are many successful cases of transgenic plants against corresponding virus disease through transferring coat protein gene. This paper reports the results of inheritance, segregation, expression of WMV-2 coat protein gene in inbred transgenic watermelon and its resistance to virus. Through PCR analysis of inbred plants, we found WMV-2 coat protein gene in the genome of progeny R1 separated with 3:1. After successive selection and identification of 4 generations, 8 transgenic pure lines with almost the same agronomic traits were obtained from 3 independent transformants of T7, T11 and T32. The result of Western blotting shows all 3 different transgenic lines of R4T7-1, R4T11-3 and R4T32-7 can produce coat protein. Disease resistance experiment on transgenic plants with WMV-2 shows that, compared with the control groups, transgenic plants can delay the disease infection and reduce the incidence and the symptoms of virus disease. And the transgenic line R4T32-7 expressed high resistance to infection by WMV-2, which lays a foundation for breeding of disease resistant varieties through plant transgenic technique.

Blotting, Western↗
PubMed2004

[Safety evaluation of food from transgenic fish and the molecular biological mechanism].

More progresses have been made in the studying of transgenic fish in China, but the studying work of safety evaluation of food from transgenic fish are started up just now. Compared to plants and animals on the land, it is more difficult to control the mobility of fish and fish can give birth to a large number of offsprings, so the ecological risk or hazard about transgenic fish is more critical than others. Another safety problem is the chimerism which is initiated by the gene transfer methods used in the transgenic fish. Getting sterile triploid transgenic fish and fixed point integration are efficient to solve the two problems above respectively. The solution of the two problems are also the basis of safety evaluation and detection of food from transgenic fish. Up to now, there are little reports on the safety evaluation of transgenic fish including nutritional evaluation and allergic reaction, and there are no basic research on the detection of transgenic fish for the aim of food safety. In brief, it is very urgent to start up the research on the safety evaluation and detection of transgenic fish for the control of food safety.

Animals↗
PubMed2006

[Transferring the Suaeda salsa glutathione S-transferase and catalase genes enhances low temperature stress resistance in transgenic rice seedlings].

The GST (glutathione S-transferase) and GST+CAT1 (catalase 1) of Suaeda salsa were introduced into a low temperature-sensitive rice cultivar (Oryza sativa cv. Zhonghua No.11) by Agrobacterium tumefaciens-mediated transformation under the control of cauliflower mosaic virus (CaMV) 35S promoter, and the transformed calli and plantlets were screened on Murashige and Skoog (1962) medium supplemented with hygromycin 25 microg/mL and cefotaxime 300 microg/mL. The putative primary transformants (T(0) generation) were acclimatized at 26 degrees C /22 degrees C in a greenhouse for 7 d, and then transplanted to the field, where they grew up to maturity under outdoor conditions. 25 and 14 independent transgenic lines of T(1) generation carrying the GST and GST+CAT1 genes, respectively, were identified by PCR amplification. Transgene expression was monitored by RNA-blot hybridization using total RNA samples from leaf tissues. To investigate whether expressing the Suaeda salsa GST and GST+CAT1 in transgenic rice increased low temperature stress tolerance, the T(4) 14-day-old transgenic and non-transgenic rice seedlings were transferred to a low temperature (day 7 degrees C/night 4 degrees C) growth chamber for 3-6 d. The experimental data showed that expressing the Suaeda salsa GST and GST+CAT1 enhanced low temperature stress resistance in transgenic rice seedlings. When treated with low temperature, both GST and CAT activity increased in the transformants with the time of temperature treatment. These transgenic rice plant seedlings exhibited a higher level of photosynthetic capacity than those of the non-transgenic control seedlings under low temperature treatment. Whereas, there were lower H(2)O(2) and MDA (malondialdehyde) content, and relative electrolyte leakage through the plasma membrane was also lower in transgenic rice seedlings than in the parent line under low temperature condition. The results also indicated that GST+CAT1 co-expression conferred greater level of low temperature stress tolerance to the transformed rice plants compared to the single GST transformed plants.

Acclimatization↗
PubMed1997

Increased inferior olivary neuron and cerebellar granule cell numbers in transgenic mice overexpressing the human Bcl-2 gene.

Neuron-target interactions during development are critical for determining the final numbers of neurons in the nervous system. To investigate the role of Purkinje cells and programmed cell death in the regulation of afferent neuron numbers, we have counted olivary neurons and granule cells in two lines of transgenic mice (NSE73a and NSE71) that overexpress a human gene for bcl-2 (Hu-bcl-2) in Purkinje cells and olivary neurons, but not in granule cells. Bcl-2 overexpression in vivo reduces naturally occurring neuronal cell death and cell death following axotomy, target removal, or ischemia. Olivary neuron numbers in NSE73a and NSE71 transgenic mice are significantly increased compared to controls by 28% and 27%, respectively, while granule cell numbers are only increased in NSE73a mice (29% above controls). We have previously shown that Purkinje cell number is increased by 43% in NSE73a transgenics and by 23% in NSE71 transgenics. The ratio of Purkinje cells to olivary neurons is not significantly different between the control and transgenic mice, while the ratio of granule cells to Purkinje cells is significantly decreased in the NSE71 transgenic mice compared to controls and NSE73a transgenics. The increased numbers of olivary neurons suggest that bcl-2 overexpression rescues these neurons from programmed cell death. The increase in granule cell number in only one transgenic line is discussed with respect to hypotheses that Purkinje cells regulate both granule cell progenitor proliferation and the survival of differentiated granule cells.

Animals↗
PubMed1998

Cyclin D3 and megakaryocyte development: exploration of a transgenic phenotype.

The roles of cell cycle regulatory proteins in megakaryocyte development are poorly understood. We have previously demonstrated that cyclin D3 is expressed in megakaryocytes and is induced upon treatment with Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the megakaryocytic lineage show features similar to in vivo Mpl ligand treatment, including increased megakaryocyte number and ploidy. Terminal maturation and platelet production are not enhanced, however, and transgenic megakaryocytes show a defect in demarcation membrane development. We have examined expression of the transcription factor nuclear factor (NF)-E2, known to be involved in cytoplasmic maturation and platelet fragmentation, in these transgenic mice and controls treated with Mpl ligand. Our findings demonstrate marked induction of NF-E2 mRNA in control megakaryocytes in response to Mpl ligand, but no NF-E2 increase in transgenic cells, potentially explaining the lack of platelet increase in these transgenic mice. Transgenic megakaryocytes treated with Mpl ligand display a limited increase in NF-E2. In response to literature reports of Mpl ligand-induced transient increases in p21Cip1/WAF1 mRNA in polyploidizing megakaryocytic cell lines, we have examined p21 transcript levels in both normal and transgenic megakaryocytes. In normal mouse spleen, only a small percentage of megakaryocytes express detectable levels of p21 mRNA, with the majority of these cells expressing at high intensity. p21 levels are not affected by treatment with Mpl ligand, while the frequency of expressing cells increases transiently. Transgenic megakaryocytes exposed to Mpl ligand also show an increased frequency of p21-positive cells, and stimulation with Mpl ligand resulted in a further increase in this frequency. The nature of this effect will require further investigation.

Animals↗
PubMed1996

The anti-Dextran response of scid mice transgenic for the heavy chain of a Dextran specific IgM antibody.

Mice homozygous for the scid mutation bear a severe defect in their ability to rearrange V(D)J gene segments to yield active genes for immunoglobulin and T cell receptor molecules. In older animals few clones of B and T cells can arise at random, a phenomenon called leakyness of the scid mutation. We established scid mice carrying as a transgene the rearranged heavy chain of the IgM/lambda1 antibody MOPC 104E with specificity for the alpha(1,3) glucosidic linkages in Dextran. Despite the scid defect one-third of these mice immunized with the thymus independent antigen Dextran at 2 weeks of age, and all of those immunized at 6 weeks responded with anti-Dextran antibodies bearing the lambda light chain. This indicates that despite the scid mutation these animals had at least once successfully rearranged their endogenous lambda1 light chain gene segments and harbor Dextran specific B cells. These mice thus provided for the first time the opportunity to study the immune response of B cells of a single specificity in an environment that should, as we shall argue, be devoid of regulatory B and T cells able to recognize the idiotype of the responding cells. One week after immunization the anti-Dextran response of 5- to 6-week-old mu-transgenic scid mice amounted to 30% of the response of mu-transgenic non-scid mice but in essence both responses followed the same kinetics, reaching antibody concentrations indistinguishable from each other 8 weeks after a single dose of Dextran. Furthermore, the ready response of young mu-transgenic scid mice to this antigen by employment of endogenously rearranged lambda1 light chains allowed experiments to be done to compare the frequency of lambda1 light chain rearrangements in mu-transgenic scid mice to that in mu-transgenic non-scid mice. This was done in limiting dilution assays counting B cell precursors responsive to mitogen and differentiating in vitro to produce antibodies toward Dextran. Specific precursors were reduced to about 1% in the spleen of mu-transgenic scid mice when compared to the spleen of mu-transgenic non-scid mice; those in the peritoneal cavity lymphocyte population were reduced to about 12%.

Animals↗