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PubMed1992

Hypothalamic preprosomatostatin messenger ribonucleic acid expression in mice transgenic for excess or deficient endogenous growth hormone.

The influence of altered endogenous GH status on somatostatin (somatotropin release-inhibiting hormone; SRIF) gene expression was studied in two transgenic mouse models. Transgenic dwarf mice carried the rat GH gene promoter fused to the diphtheria toxin A-chain gene, placing toxin expression under GH promoter control. As a result, the toxic product of the transgene ablated all GH-expressing cells, resulting in undetectable circulating GH, reduced weight (10.6 +/- 1.0 g for transgenic dwarfs vs. 29.5 +/- 1.7 g for controls; P less than 0.001), and no detectable somatotrophs. Transgenic giant mice contained a construction combining a widely expressed metallothionein promoter and the human GH-releasing hormone (hGHRF) structural gene. Transgene expression of hGHRF resulted in overproduction of endogenous mouse GH in the anterior pituitary and weight increases (42.7 +/- 2.7 g for giants vs. 29.5 +/- 1.7 g for controls; P less than 0.005). Using in situ hybridization, control mice, transgenic dwarfs, and transgenic giants were compared for levels of prepro-SRIF mRNA. Hybridization signal intensities for prepro-SRIF mRNA were similar in transgenic dwarfs to those in littermate nontransgenic mice in non-GH-regulating regions of the brain, such as cortex (control, 31 +/- 2 U; dwarf, 27 +/- 2) and reticulothalamic nucleus (control, 41 +/- 2 U; dwarfs, 39 +/- 3). Transgenic giant mice had hybridization intensity of SRIF mRNA similar to that of normals in cortex (controls, 31 +/- 2 U; giant, 27 +/- 1) and reticulothalamic nucleus (controls, 41 +/- 2 U; giant, 40 +/- 4). In the GH-regulating neurons of the anterior periventricular hypothalamus (PeN), prepro-SRIF mRNA signal in transgenic dwarf mice decreased to 60% of that in controls (88 +/- 13 U for dwarfs vs. 147 +/- 17 U for controls; P less than 0.01), although the numbers of mRNA-expressing cells in the PeN were not different between the transgenic dwarfs and controls (dwarfs, 69 +/- 6 cells; controls, 72 +/- 4 cells). The transgenic giant mice had 230% higher prepro-SRIF mRNA signal than control mice in the PeN (343 +/- 30 U in giants vs. 147 +/- 17 U in controls; P less than 0.001). Again, the numbers of mRNA-expressing cells were not different in giants (57 +/- 9) and normals (72 +/- 4). These results suggest that while the lack of endogenous GH is accompanied by a slight decrease in transcriptional expression of SRIF in the PeN, the overproduction of endogenous GH greatly stimulates hypothalamic SRIF steady state mRNA levels.

Animals↗
PubMed1993

Effects of age and endogenously secreted human growth hormone on the regulation of gonadotropin secretion in female and male transgenic mice expressing the human growth hormone gene.

In aging rats and humans, GH secretion is reduced. In transgenic mice bearing the human (h) GH gene, hGH is secreted during the entire lifespan. To evaluate the effects of endogenously secreted hGH on age-related changes in hypothalamic-pituitary function, the following two experiments were conducted in young (2.5-4 months of age) and old (11-14 months of age) female and male transgenic mice expressing the hGH gene and their normal siblings. In Exp I, young and old female transgenic mice and their normal siblings were ovariectomized. On days 8 and 9 after ovariectomy, mice were injected (sc) with oil or primed with 0.5 micrograms estradiol benzoate (EB) in oil, 24 h later treated with 10 micrograms EB/100 g BW, and a day later bled for the determination of FSH, LH, PRL, and hGH levels by RIAs. In Exp II, young and old male transgenic mice and their normal littermates were castrated and injected with either peanut oil or testosterone propionate (TP; 1 microgram/g BW) in oil. Blood samples were obtained 18-20 h after oil or TP injection. Plasma FSH, LH, PRL, and hGH levels were measured by RIAs. hGH was present in the circulation of young and old transgenic mice, but not in normal siblings. Circulating FSH and LH levels were significantly lower in ovariectomized young and old transgenic mice than in similarly treated young and old normal siblings. The suppressive effect of EB on LH secretion was reduced in ovary-ablated young and old transgenic mice. The PRL response to EB treatment was increased (P < 0.005) in old ovariectomized transgenic mice. In the male, the castration-induced increase in plasma LH levels was higher (P < 0.05) in young transgenic mice. Administration of TP failed to suppress the absolute plasma LH levels in castrated young and old transgenic mice. However, the percent decrease in circulating LH levels was lower in young and old transgenic mice. The castration-induced increase in FSH secretion was reduced (P < 0.005) in aged transgenic mice. The effects of TP on plasma FSH levels were similar to its effects on LH secretion. Gonad-ablated young and old transgenic mice of both sexes are hypoprolactinemic. These observations demonstrate that endogenously secreted hGH modulates gonadotropin and PRL secretion in young and old mice bearing the hGH gene and indicate that the changes in the hypothalamic-pituitary system of mice expressing the hGH gene are similar to those observed in aging human subjects.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1999

Targeted overexpression of galanin in lactotrophs of transgenic mice induces hyperprolactinemia and pituitary hyperplasia.

We generated transgenic mice that carry 4.6 kb of the mouse galanin gene fused to 2.5 kb of the rat PRL promoter. In all transgenic lines that carried and transmitted the transgene, there were significant increases in galanin messenger RNA and peptide levels in the anterior pituitary in both male and female transgenic mice, and the elevation of galanin was restricted to the anterior lobe. Furthermore, galanin release from pituitary cells in vitro of both male and female transgenic mice was dramatically increased compared with that in control mice. At 2-4 months of age, pituitary PRL contents in female transgenic mice were increased compared with those in normal controls. Moreover, PRL messenger RNA levels were increased in female transgenic mice. However, plasma levels of PRL in female transgenic mice were not significantly higher until 6 months of age. By 11 months of age, cell numbers in the anterior pituitary were increased in female, but not male, transgenic mice. The percentage of lactotrophs in female transgenic mice as well as PRL gene expression per cell were significantly higher. No differences were detected in PRL content, gene expression, or release between normal and transgenic male mice. Six weeks of estrogen treatment significantly increased anterior pituitary weights and PRL secretion in male transgenic mice compared with that in normal male mice. In addition, anterior pituitary weights and PRL secretion were decreased in female transgenic mice compared with controls 6 weeks after ovariectomy. We conclude that overexpression of galanin in lactotrophs stimulates PRL synthesis and secretion and acts as a growth factor resulting in the formation of pituitary hyperplasia and hyperprolactinemia. Furthermore, estrogen appears critical for these galanin-mediated events.

Aging↗
PubMed2000

Pituitary hormone gene expression and secretion in human growth hormone-releasing hormone transgenic mice: focus on lactotroph function.

The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA) levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per microg total RNA) were unchanged. In contrast, total PRL contents were unchanged, whereas the concentrations of PRL (micrograms of PRL per mg total protein) were decreased 3-fold. Hypothalamic tyrosine hydroxylase steady state mRNA levels were not altered in the hGHRH transgenic mice, but hypothalamic tyrosine hydroxylase activity was increased 2-fold in transgenic mice. Dopamine D2 receptor mRNA concentrations in the anterior pituitary were increased 2.5-fold in hGHRH transgenic mice, and total pituitary D2 receptor mRNA levels were increased nearly 10-fold. Furthermore, the basal secretory capacity of lactotrophs from transgenic mice was increased significantly at the level of the single cell, and dopamine inhibited the secretion of PRL to a greater extent in hGHRH transgenic mice. Thus, although the total number of lactotrophs is increased 2-fold in hGHRH transgenic mice, the present data are consistent with the hypothesis that increased hypothalamic dopamine synthesis and release coupled with an increase in D2 dopamine receptor gene expression and functional sensitivity in the pituitary result in normal plasma levels of PRL.

Animals↗
PubMed2001

Transgenic mice with overexpression of human scavenger receptor A on endothelial cells.

OBJECTIVES: To establish a new transgenic mouse model for determining the function and role of human scavenger receptor A (SR-A) in atherosclerosis in vivo. METHODS: Human scavenger receptor minigene-driven mouse tie-1 promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and Southern blot were used to screen the positive transgenic mice. RT-PCR and immunohistochemical analysis were used to detect the level and location of human SR-AI expression in transgenic mice. The activity of human SR-AI was determined by morphologic observation of aortic endothelial cells of transgenic mice under transmission electron microscopy. RESULTS: The electrophoresis assay showed the expected 4 fragments of 0.9 kb, 1.1 kb, 1.2 kb and 4.2 kb in the Sma I digest and 2 fragments of 0.8 kb and 6.7 kb in Bgl II digest of plasmids pTie-1/hSR-A. The fragment sequence of tie-1 promoter and human SR-A cDNA in plasmids pTie-1/hSR-A was correct and no ATG before the translation initiation sites of human SR-A was found by sequence analysis. 561 injected and surviving embryos with the purified human SR-A minigene were implanted into the oviducts of 19 ICR pseudopregnant mice. Among the 54 surviving pups from 13 foster mothers, 7 were identified by PCR and Southern blot analysis. The results of RT-PCR and immunohistochemical analysis showed human SR-A was specifically expressed on vascular endothelial cells of the aorta and renal artery, as well as hepatic sinusoidal endothelial cells in transgenic mice. Transmission electron microscope (TEM) of aorta of transgenic mice showed that a large number of vesicles, multivesicle bodies and swollen mitochondria filled the plasma of endothelial cells. CONCLUSIONS: A transgenic mouse model with overexpression of human SR-A in endothelial cells was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Tie-1 promoter controlled the transgene to express in endothelial cells in mice. Pinocytic activity of aortic endothelial cells in transgenic mice was higher than that of C57BL/6J mice. Our studies will provide a new transgenic model for investigation of atherosclerosis and functions of human SR-A.

Animals↗
PubMed2003

[Escape of transgenes and its ecological risks].

The rapid development of biotechnology, particularly the transgenic technology, has brought us with tremendous opportunities to solve the world's starvation problems that have been caused by the continued expanding of the global population. However, the application of transgenic biotechnology and the environmental release of transgenic organisms have evoked a series of extraordinary debates on biosafety issues related to the prosperity and the future of transgenic technology. The public and scientific communities are desperately interested in knowing whether the transgenic products would pose negative influences on plants and animals, human life and health, as well as on genetic resources and environment. These concerns have become universal hot topics over the last decade. Among the most debated biosafety issues caused potentially by transgenic products, transgene escape to the environment and its consequent ecological risks become one of the appealing focal points. In this review, a series of biosafety issues concerned by public, including the possibility of transgene escape and its various paths, as well as the potential ecological risks caused by such escape were discussed, and various approaches for controlling for transgene escape and the factors to consider when designing safety isolation distance between transgenic varieties and other concerned plants were also examined. The objective of this review is to allow readers to understand the potential biosafety problems caused by environmental release of transgenic crops and by the escape of foreign transgenes in particular, and to use the effective tools to control and avoid transgene escape.

Animals↗
PubMed1990

A peripheral mechanism preserves self-tolerance to a secreted protein in transgenic mice.

We have examined mechanisms of tolerance to circulating self-proteins in mice that are transgenic for human insulin. Normal, nontransgenic mice develop serum antibody responses when injected with human insulin in CFA; syngeneic transgenic mice do not. B cell responsiveness was assessed by immunizing with human insulin coupled to a T-independent Ag, Brucella abortus. No differences were found in the numbers of insulin-specific splenic plaque-forming cells between transgenic and nontransgenic mice suggesting that insulin-specific B cells are not tolerant in transgenic mice. Similarly, APC from transgenic and nontransgenic mice display no differences in their ability to process and present human insulin to human insulin-specific T cells in vitro. However, marked differences were detected between transgenic and nontransgenic T cells. Lymph node T cells from transgenic mice primed with human insulin provided no detectable helper activity for secondary antibody responses to human insulin whereas, lymph node T cells from nontransgenic mice did. Nevertheless, lymph node T cells from transgenic mice developed significant proliferative responses to human insulin. Lymph node T cells obtained from transgenic and nontransgenic mice were fused to BW5147 and human insulin-specific T cell hybridomas were generated. The fact that human insulin-specific T cell hybridomas were obtained from the transgenic mice suggests that these T cells were not clonally deleted. In addition, APC from transgenic mice did not stimulate human insulin-specific hybridomas from normal mice in the absence of exogenous insulin. We suggest that T cells specific for human insulin are not deleted in the thymus of transgenic mice because APC in the thymus do not bear the requisite levels of endogenous human insulin/Ia complexes. Therefore, we conclude that tolerance in the transgenic mice is preserved by peripheral mechanisms.

Animals↗
PubMed1999

Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.

Animals↗
PubMed2002

Production of transgenic silver sea bream (Sparus sarba) by different gene transfer methods.

We have been interested in developing convenient mass gene transfer methods for producing strains of silver sea bream (Sparus sarba) with superior genetic traits for aquaculture. A transgene construct carrying rainbow trout growth hormone (rtGH) complementary DNA driven by a common carp b-actin promoter was introduced into silver sea bream by electroporating the sperm with the rtGH transgene and using the treated sperm to fertilize eggs stripped from mature females. The presence of the GH transgene in presumptive transgenic individuals was detected by polymerase chain reaction (PCR) analysis. Between 56% and 70% of the animals carried the GH transgene. We refer to this method as sperm-mediated gene transfer (SMGT). Since the handling stress of stripping gametes from female sliver sea bream brood fish could cause severe mortality, an alternative gene transfer method would be highly desirable. We developed a liposome-based method to transfer the GH transgene into the fish. This method, referred as testis-mediated gene transfer (TMGT), involves injecting the liposome-transgene mixture into the gonads of male sea bream at least 48 hours before spawning. The males were mated to reproductively active females, and fertilized eggs were collected for further incubation. Between 59% and 76% of the hatched fry were found by PCR analysis to carry the rtGH transgene. The efficiency of gene transfer was improved more than 80% by injecting multiple doses of the liposome-transgene mixture into the gonads of treated males. Results of Southern blot analysis of DNA isolated from PCR-positive animals showed that the transgene was integrated into the host genome and could be transmitted to its offspring. The rtGH transgene was expressed in many of the rtGH-transgenic fish. Several P1 GH-transgenic silver sea bream exhibited significant growth enhancement compared with nontransgenic controls. Our studies showed that faster-growing silver sea bream could be produced by a variety of mass gene transfer technologies. These gene transfer technologies would be of great value to aquaculture.

Journal Article↗
PubMed2002

Decreased susceptibility to NMU-induced mammary carcinogenesis in transgenic rats carrying multiple copies of a rat ras gene driven by the rat Harvey ras promoter.

Ras protein over-expression has been observed in human breast cancers although the significance of Ras over-expression in the etiology of breast cancer is unknown and its contribution to breast cancer prognosis is still debated. In this study, the over-expression of both wild-type Harvey and Kirsten Ras proteins as contributors to rat mammary carcinogenesis were examined using a transgenic rat model. Three rat transgenic lines (designated HrHr transgenics) carrying three to six copies of wild-type rat Harvey ras driven by the wild-type rat Harvey ras promoter were produced. In addition, transgenic lines carrying either three or seven copies of the Kirsten ras gene under the same promoter (HrKr) were produced. No pathological changes in the mammary gland were observed in any of the HrHr or HrKr transgenic rat line heterozygotes. Two of the Ras transgenic lines, HrHr (R8) and HrKr (4334), had a significant reduction in NMU-induced rat mammary cancer when compared to their non-transgenic littermates. All five Ras transgenic lines developed fewer carcinomas than their non-transgenic littermates following NMU exposure. The percentage of NMU-induced G35 to A35 activating mutations in the endogenous Harvey ras gene in mammary carcinomas from the HrHr, HrKr transgenic rats and their non-transgenic littermates was similar ( approximately 50%). In contrast, less than 1% of the NMU-induced carcinomas in these Ras transgenic rats had an activating ras mutation in their transgenes. These findings highlight the potential of Ras to function as a modifier gene in repressing mammary carcinogenesis.

Animals↗
PubMedDate not supplied

Hybridization and backcrossing between transgenic oilseed rape and two related weed species under field conditions.

Determining the frequency of crop-wild transgene flow under field conditions is a necessity for the development of regulatory strategies to manage transgenic hybrids. Gene flow of green fluorescent protein (GFP) and Bacillus thuringiensis (Bt) transgenes was quantified in three field experiments using eleven independent transformed Brassica napus L. lines and the wild relatives, B. rapa L. and Raphanus raphanistrum L. Under a high crop to wild relative ratio (600:1), hybridization frequency with B. rapa differed among the individual transformed B. napus lines (ranging from ca. 4% to 22%), however, this difference could be caused by the insertion events or other factors, e.g., differences in the hybridization frequencies among the B. rapa plants. The average hybridization frequency over all transformed lines was close to 10%. No hybridization with R. raphanistrum was detected. Under a lower crop to wild relative ratio (180:1), hybridization frequency with B. rapa was consistent among the transformed B. napus lines at ca. 2%. Interspecific hybridization was higher when B. rapa occurred within the B. napus plot (ca. 37.2%) compared with plot margins (ca. 5.2%). No significant differences were detected among marginal plants grown at 1, 2, and 3 m from the field plot. Transgene backcrossing frequency between B. rapa and transgenic hybrids was determined in two field experiments in which the wild relative to transgenic hybrid ratio was 5-15 plants of B. rapa to 1 transgenic hybrid. As expected, ca. 50% of the seeds produced were transgenic backcrosses when the transgenic hybrid plants served as the maternal parent. When B. rapa plants served as the maternal parent, transgene backcrossing frequencies were 0.088% and 0.060%. Results show that transgene flow from many independent transformed lines of B. napus to B. rapa can occur under a range of field conditions, and that transgenic hybrids have a high potential to produce transgenic seeds in backcrosses.

Agriculture↗
PubMed2004

Growth and tissue accretion rates of swine expressing an insulin-like growth factor I transgene.

The goal of this research was to determine whether directing expression of an insulin-like growth factor I (IGF-I) transgene specifically to striated muscle would alter the growth characteristics in swine. Transgenic pigs were produced with a fusion gene composed of avian skeletal alpha-actin regulatory sequences and a cDNA encoding human IGF-I. Six founder transgenic pigs were mated to nontransgenic pigs to produce 11 litters of G1 transgenic and sibling control progeny. Birth weight, weaning weight, and proportion of pig survival did not differ between transgenic and control pigs. The ADG of pigs as they grew incrementally from 20 to 60 kg, 60 to 90 kg, and 90 to 120 kg, respectively, did not significantly differ between transgenic and control pigs. Efficiency of feed utilization (gain:feed) was also similar for transgenic and control pigs. Plasma IGF-I and porcine growth hormone (pGH) concentrations were determined at 60, 90, and 120 kg body weight. Plasma IGF-I concentrations were 19% higher in transgenic gilts than control gilts and 11.1% higher in transgenic boars than control boars (P=0.0005). Plasma IGF-I concentrations for boars were also higher than for gilts (P=0.0001). At 60, 90, and 120 kg body weight each pig was scanned by dual energy X-ray absorptiometry (DXA) to derive comparative estimates of carcass fat, lean, bone content of the live animal. Control pigs had more fat and less lean tissue than transgenic pigs at each of the scanning periods and the difference became more pronounced as the pigs grew heavier (P<0.005 at each weight). Transgenic pigs also had a slightly lower percentage of bone than control pigs (P<0.05 at each weight). While daily rates of lean tissue accretion did not differ for transgenic and control pigs, daily rates of fat accretion were lower in transgenic pigs than in control pigs (P<0.05). Based on these results we conclude that expression of IGF-I in the skeletal muscles gradually altered body composition as pigs became older but did not have a major affect on growth performance.

Absorptiometry, Photon↗
PubMed2004

Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress.

Physiological responses to water stress in hybrid poplar (INRA 7171-B4, Populus tremula L. x P. alba L.) lines transformed to overexpress a pine cytosolic glutamine synthetase (GS1) gene were compared with those of non-transgenic plants. Before, during and after a drought treatment, net photosynthetic rates (Anet) were higher in transgenic than in non-transgenic plants. Stomatal conductance (gs) was higher in transgenic than in non-transgenic plants before, but not after exposure to drought. Before drought treatment, a sudden reduction in photosynthetic photon flux caused a greater burst of CO2 efflux in transgenic than non-transgenic plants, indicating greater photorespiratory activity. Drought caused greater reductions in photochemical quenching, photosystem II (PSII) antennae transfer efficiency (Fv'/Fm') and light-adapted PSII yield (PhiPSII) in non-transgenic than in transgenic plants, especially at low irradiances. Antennae-based thermal dissipation was higher in transgenic plants than in non-transgenic plants both during the imposition of drought and 1 or 3 days after the relief of drought. Under severe water stress and subsequently, transgenic plants maintained a higher expression of glutamine synthetase, glutamate synthase and Rubisco and higher concentrations of chlorophyll and glycine than non-transgenic plants. These findings indicate that overexpression of pine cytosolic GS1 enhanced sustained photosynthetic electron transport capacity during severe stomatal limitation. The data also suggest that ectopic expression of cytosolic GS increases photorespiratory activity, and that this serves as a protective sink for electrons from photosynthetic reaction centers.

Chlorophyll↗
PubMed1997

Viability of transgenic rat embryos after freezing and thawing.

In-vivo viability of frozen-thawed embryos derived from transgenic rats, as well as the transmission and the expression of transgenes in the resultant newborn rats, was investigated. Three strains of transgenic rats, carrying human growth hormone gene connected downstream to the promoter region of the bovine alpha-lactalbumin gene (alpha LA/hGH), bovine beta-casein gene (beta CN/hGH) or bovine alpha-S1 casein gene (alpha S1CN/hGH), were used. Two-cell stage embryos (non-transgenic Wistar female x heterozygous transgenic male) were placed in 10% (v/v) dimethylsulfoxide (DMSO) solution and cooled from -7 to -30 degrees C at -0.5 degree C/min before being plunged into liquid nitrogen. After 2 to 4 years storage, the embryos were thawed by rapid warming. The intact embryos were transferred into the oviducts of Day 1 pseudopregnant recipients. The postthaw survival rate of frozen embryos was high in all 3 transgenic strains (88 to 92%), which was similar to that of control (non-transgenic) frozen embryos (95%). Development to newborn rats following transfer of embryos derived from the 3 strains (64 to 68%) was also similar to that of control embryos (60%). These transgenes (alpha LA/hGH, beta CN/hGH and alpha S1CN/hGH) were detected in the DNA extracts from tail tissue of the newborn rats, but the transmission rates (41, 23 and 32%, respectively) were lower than 50% which is expected in the Mendelian fashion. In a transgenic line carrying alpha S1CN/hGH, hGH levels of secretion into the milk of transgenic newborn rats derived from frozen-thawed embryos and her transgenic offspring were the same mg/ml-level as that of their founder rat. Two-step freezing of embryos derived from transgenic rats was therefore an effective method for the long-term cryopreservation of transgene.

Animals↗
PubMed2004

[Nutrition assessment of transgenic rice].

OBJECTIVE: To observe the nutrition effects between transgenic rice and non-transgenic rice. METHODS: Wistar rats were divided into three groups according to their sex respectively: transgenic rice group, non-transgenic rice group and AIN93G normal control group. They were fed with corresponding food for 28 days. Indicator as follows: food intake, protein effective ratio, body weight, body length, blood routine test, blood biochemistry test, organ index and bone density. RESULTS: In male groups, expect that liver/body ratio was higher in transgenic rice group than that in non-transgenic rice group, all other indicators had no significant difference each other. In female groups, liver/body weight ratio, blood calcium and bone density were higher in transgenic group than those in non-transgenic group. All other indicators had no significant difference between transgenic rice group and non-transgenic rice group. Body weight, bone density and blood calcium were higher and liver/body weight ratio had no significant difference in transgenic rice group than those in AIN93G group. CONCLUSION: Transgenic rice had good nutrition effects on rat development and no toxicity and adverse effects were found in transgenic rice group.

Animals↗
PubMed2025

Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.

Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.

Populus↗
PubMed2002

Multiple effects of genetic background on variegated transgene expression in mice.

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.

Animals↗
PubMed2003

Expression of a muscle-specific, nitric oxide synthase transgene prevents muscle membrane injury and reduces muscle inflammation during modified muscle use in mice.

Nitric oxide (NO) can function as either a pro-inflammatory or anti-inflammatory molecule, depending upon its concentration and the microenvironment in which it is produced. We tested whether muscle-derived NO affects muscle inflammation and membrane lysis that occur in modified muscle use. Transgenic mice with muscle-specific over-expression of neuronal NO synthase (nNOS) were generated in which transgene expression was driven by the human skeletal muscle actin promoter. Transgenic mice and non-transgenic littermates were subjected to hindlimb muscle unloading followed by reloading, which causes muscle inflammation and membrane lysis. NOS expression decreased in transgenic and non-transgenic mice during muscle unloading. Muscle inflammation was assessed by immunohistochemistry after 24 h of muscle reloading following 10 days of unloading. Soleus muscles of non-transgenic mice showed significant increases in the concentrations of neutrophils (4.8-fold) and macrophages (11.3-fold) during reloading, compared to mice that experienced unloading only. Muscles of transgenic mice showed 51 % fewer neutrophils in reloaded muscles than those of non-transgenic mice, but macrophage concentrations did not differ from non-transgenic mice. Muscle membrane damage was determined by measuring influx of an extracellular marker dye. Significantly more membrane damage occurred in muscles of non-transgenic mice experiencing reloading than in ambulatory controls. However, membrane damage in the reloaded muscles of transgenic mice did not differ from that in ambulatory mice. In vitro cytotoxicity assays confirmed that mouse neutrophils lyse muscle cell membranes, and showed that inhibition of NOS in muscle and neutrophil co-cultures significantly increased neutrophil-mediated lysis of muscle cells. Together, these data show that muscle-derived NO can function as an anti-inflammatory molecule in muscle that experiences modified loading, and that NO can prevent neutrophil-mediated damage of muscle cell membranes in vivo and in vitro.

Animals↗