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PubMed1996

Evolution of neoplastic development in the liver of transgenic mice co-expressing c-myc and transforming growth factor-alpha.

We have previously shown that co-expression of c-myc and transforming growth factor (TGF)-alpha as transgenes in mouse liver results in major enhancement of neoplastic development in this organ as compared with expression of either of these transgenes alone. In this report we describe in detail the progression from liver cell dysplasia to hepatocellular carcinomas (HCCs) occurring in the liver of c-myc/TGF-alpha and c-myc transgenic mice. Despite morphological similarities in the sequence of events between the two transgenic lines, the dramatic acceleration, extent, and severity of hepatic lesions in c-myc/TGF-alpha mice clearly demonstrated the synergistic effects of this transgenic combination. Although c-myc/TGF-alpha and c-myc females displayed longer latency and lower tumor incidence, the pathological changes were the same as those seen in the male mice, including the formation of HCCs, which are absent in TGF-alpha single-transgenic females. Tumors in single- and double-transgenic mice showed induction of the endogenous c-myc and TGF-alpha and, most frequently, unchanged or decreased epidermal growth factor receptor, further indicating the collaborative role of c-myc and TGF-alpha in providing a selective growth advantage to tumor cells independently of the epidermal growth factor receptor levels. To identify possible tumor precursors, we focused particularly on the dysplastic changes preceding and accompanying the appearance of preneoplastic and neoplastic lesions in the double-transgenic mice. Early on, these changes were characterized by the appearance of large dysplastic hepatocytes, mostly pericentrally, expressing high levels of TGF-alpha and uPA, as well as TGF-beta 1, particularly in apoptotic cells. After a short period of replication and expansion into the liver parenchyma, as well as penetration into the central veins, these cells underwent apoptotic cell death while preneoplastic and neoplastic lesions were forming. The peritumorous tissues also contained small dysplastic hepatocytes and oval-like cells, similar to those found in the tumors. Transplantation of the transgenic liver tissues harboring only dysplasia with or without vascular lesions onto nude mice was able to yield HCCs composed of small diploid cells, suggesting that initiated cells are generated during the early dysplastic phase and can progress to HCC. It is therefore likely that large dysplastic hepatocytes undergo apoptosis, which may be closely associated with the up-regulation of TGF-beta 1 and uPA, whereas other cells evolve into the precursor population for HCC. Due to the simultaneous presence of c-myc, TGF-alpha, and dysplasia in premalignant human liver diseases, our transgenic mouse system appears to be an appropriate model for studying human hepatocarcinogenesis.

Animals↗
PubMed1995

Overexpression of cyclin A in the mammary glands of transgenic mice results in the induction of nuclear abnormalities and increased apoptosis.

Aberrant expression of several cyclin genes has been demonstrated to be associated with many types of tumors. To determine the capacity of cyclin A to function as an oncogene in vivo, wild-type and mutant cyclin A proteins were specifically overexpressed in the mammary glands of transgenic mice using regulatory sequences from the ovine beta-lactoglobulin gene. Several lines of transgenic mice were generated that expressed human cyclin A or a nondegradable mutant version of human cyclin A, in which the amino-terminal 89 amino acids encompassing the cyclin destruction box were removed. The cyclin A transgene products were localized in the nuclei of mammary epithelial cells, and the transgenic mammary glands had an increase in cyclin A- and cdk2-associated H1 kinase activity. Many mammary epithelial cells in the transgenic glands exhibited nuclear abnormalities, including multinucleation and karyomegaly, which were suggestive of preneoplastic alterations. The abnormalities were more severe in mammary glands of the mutant cyclin A transgenics, which expressed a stabilized cyclin A protein. In situ analysis of mid-lactation mammary gland sections revealed increased numbers of apoptotic cells in the transgenic glands. Double transgenic animals were generated that expressed both the mutant human cyclin A and human cdk2 transgenes, and a more pronounced phenotype resulted. The bigenic mammary glands exhibited focal areas of hyperplasia, as well as a greater incidence of apoptosis than observed in the single transgenic glands, demonstrating in vivo cooperation between these genes in transformation and apoptotic signaling pathways.

Amino Acid Sequence↗
PubMed1999

Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection.

Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.

Animals↗
PubMed2001

Zebrafish tiggy-winkle hedgehog promoter directs notochord and floor plate green fluorescence protein expression in transgenic zebrafish embryos.

Zebrafish tiggy-winkle hedgehog (twhh) is a member of the hedgehog gene family that plays an important role in patterning brain, neural tube, somites, and eyes. To better understand the regulation of its tissue-specific expression, the activity of the twhh promoter was determined in zebrafish embryos by transient and transgenic expression analysis. Transient expression studies revealed that the 5.2-kb twhh promoter drove green fluorescence protein (GFP) expression in the notochord, floor plate, and branchial arches. Deletion analysis showed that distinct regions of the twhh promoter regulated the respective notochord or floor plate specific expression. To confirm the tissue specificity of the twhh promoter, transgenic zebrafish containing the twhh-GFP transgene were generated. GFP expression was analyzed in the F1, F2, and F3 generations of the transgenic embryos. The results confirmed the tissue-specific expression of the transgene in the notochord, floor plate, and branchial arches. In addition, GFP expression was also found in the pectoral fin buds, retina, and epithelial lining cells of the Kupffer's vesicle in the transgenic fish embryos. The expression pattern of the twhh-GFP transgene mimicked the expression of the endogenous twhh mRNAs in the floor plate, fin buds, branchial arches, retina, and epithelial lining cells of the Kupffer's vesicle. The expression in the notochord, however, did not mimic the pattern of the endogenous twhh expression. To determine whether no tail (ntl) or floating head (flh) mutants that have developmental defect in the notochord or the Kupffer's vesicle may affect the GFP expression in these regions, GFP expression was analyzed in ntl or flh transgenic embryos. No GFP expression could be detected in the midline region of the ntl transgenic embryos. However, in flh transgenic embryos, although GFP expression was affected in the midline region, its expression in the Kupffer's vesicle appeared normal. Together, these data indicated that the 5.2-kb twhh promoter contains regulatory elements for tissue-specific expression of twhh in the floor plate, pectoral fin bud, branchial arches, retina, and Kupffer's vesicle.

Animals↗
PubMed1996

Isolation of recombinant viruses between cauliflower mosaic virus and a viral gene in transgenic plants under conditions of moderate selection pressure.

We demonstrate that recombinant viruses formed between a wild-type virus and a viral transgene can be isolated from transgenic plants under conditions of moderate to weak selection pressure. We inoculated cauliflower mosaic virus (CaMV) strain W260 to transgenic Nicotiana bigelovii plants that expressed a copy of CaMV gene VI derived from CaMV strain D4, a gene that determines systemic infection of solanaceous species, including N. bigelovii. Because W260 infects nontransformed N. bigelovii systemically, a recombinant virus formed between W260 and the D4 transgene would be expected to have little selective advantage over the wild-type W260 virus W260 was inoculated to approximately 100 plants each of nontransformed and transgenic N. bigelovii and it systemically infected nearly all of the plants. An analysis of viral DNA recovered from 23 transgenic plants infected with W260 revealed that 20 infections resulted from the systemic movement of the wild-type W260 virus, while a recombinant between W260 and the D4 transgene was detected in three of the infections. To determine the percentage of recovery of recombinant viruses under strong selection pressure, we inoculated approximately 100 nontransformed and 100 D4 gene VI transgenic plants with CaMV strain CM1841, a virus that is unable to infect nontransformed N. bigelovii. CM1841 infected 36% of the transgenic plants systemically, but none of the nontransformed controls. An analysis of 24 infected plants showed that a recombination event occurred in every plant, demonstrating that under strong selection conditions, the recovery of CaMV recombinants from transgenic plants can be very high.

Brassica↗
PubMed1996

High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos.

For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.

Actins↗
PubMed1995

Molecular and genetic analysis of nitrite reductase co-suppression in transgenic tobacco plants.

Silencing of Nia host genes and transgenes (encoding nitrate reductase) was previously achieved by introducing into tobacco plants the tobacco Nia2 cDNA cloned downstream of the cauliflower mosaic virus (CaMV) 35S promoter. To check whether Nii host genes and transgenes (encoding nitrite reductase, the second enzyme of the nitrate assimilation pathway) were also susceptible to silencing, a transgene consisting of the tobacco Nii1 gene with two copies of the enhancer of the 35S promoter cloned 1 kb upstream of the Nii promoter region was introduced into tobacco plants. Among nine independent transformants analysed, two showed silencing of Nii host genes and transgenes in some descendants after selfing, but never after back-crossing with wild-type plants, suggesting that silencing depends on the number of transgene loci and/or on certain allelic or ectopic combinations of transgene loci. In one transformant carrying a single transgene locus in a homozygous state, silencing was triggered in all progeny plants of each generation, 20 to 50 days after germination. Field trial analysis confirmed that silencing was not triggered when the transgene locus of this latter line was present in a hemizygous state. In addition, it was revealed that silencing can be triggered, albeit at low frequency and later during the development, when this transgene locus is brought into the presence of a non-allelic transgene locus by crossing, suggesting that a homozygous state is not absolutely required.

Cloning, Molecular↗
PubMed1999

Safety Evaluation of Transgenic Tilapia with Accelerated Growth.

Recent advances in modern marine biotechnology have permitted the generation of new strains of economically important fish species through the transfer of growth hormone genes. These transgenic fish strains show improved growth performance and therefore constitute a better alternative for aquaculture programs. Recently, we have obtained a transgenic tilapia line with accelerated growth. However, before introducing this line into Cuban aquaculture, environmental and food safety assessment was required by national authorities. Experiments were performed to evaluate the behavior of transgenic tilapia in comparison to wild tilapia as a way to assess the environmental impact of introducing transgenic tilapia into Cuban aquaculture. Studies were also conducted to evaluate, according to the principle of substantial equivalence, the safety of consuming transgenic tilapia as food. Behavior studies showed that transgenic tilapia had a lower feeding motivation and dominance status than controls. Food safety assessment indicated that tilapia growth hormone has no biological activity when administered to nonhuman primates. Furthermore, no effects were detected in human healthy volunteers after the consumption of transgenic tilapia. These results showed, at least under the conditions found in Cuba, no environmental implications for the introduction of this transgenic tilapia line and the safety in the consumption of tiGH-transgenic tilapia as an alternative feeding source for humans. These results support the culture and consumption of these transgenic tilapia.

Journal Article↗
PubMed2003

Peripheral insulin resistance develops in transgenic rats overexpressing phosphoenolpyruvate carboxykinase in the kidney.

AIMS/HYPOTHESIS: To study the secondary consequences of impaired suppression of endogenous glucose production (EGP) we have created a transgenic rat overexpressing the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in the kidney. The aim of this study was to determine whether peripheral insulin resistance develops in these transgenic rats. METHODS: Whole body rate of glucose disappearance (R(d)) and endogenous glucose production were measured basally and during a euglycaemic/hyperinsulinaemic clamp in phosphoenolpyruvate carboxykinase transgenic and control rats using [6-(3)H]-glucose. Glucose uptake into individual tissues was measured in vivo using 2-[1-(14)C]-deoxyglucose. RESULTS: Phosphoenolpyruvate carboxykinase transgenic rats were heavier and had increased gonadal and infrarenal fat pad weights. Under basal conditions, endogenous glucose production was similar in phosphoenolpyruvate carboxykinase transgenic and control rats (37.4+/-1.1 vs 34.6+/-2.6 micromol/kg/min). Moderate hyperinsulinaemia (810 pmol/l) completely suppressed EGP in control rats (-0.6+/-5.5 micromol/kg/min, p<0.05) while there was no suppression in phosphoenolpyruvate carboxykinase rats (45.2+/-7.9 micromol/kg/min). Basal R(d) was comparable between PEPCK transgenic and control rats (37.4+/-1.1 vs 34.6+/-2.6 micromol/kg/min) but under insulin-stimulated conditions the increase in R(d) was greater in control compared to phosphoenolpyruvate carboxykinase transgenic rats indicative of insulin resistance (73.4+/-11.2 vs 112.0+/-8.0 micromol/kg/min, p<0.05). Basal glucose uptake was reduced in white and brown adipose tissue, heart and soleus while insulin-stimulated transport was reduced in white and brown adipose tissue, white quadriceps, white gastrocnemius and soleus in phosphoenolpyruvate carboxykinase transgenic compared to control rats. The impairment in both white and brown adipose tissue glucose uptake in phosphoenolpyruvate carboxykinase transgenic rats was associated with a decrease in GLUT4 protein content. In contrast, muscle GLUT4 protein, triglyceride and long-chain acylCoA levels were comparable between PEPCK transgenic and control rats. CONCLUSIONS/INTERPRETATION: A primary defect in suppression of EGP caused adipose tissue and muscle insulin resistance.

Animals↗
PubMed2004

Overexpression of lipoprotein lipase improves insulin resistance induced by a high-fat diet in transgenic rabbits.

AIMS/HYPOTHESIS: Dysfunctions of lipoprotein lipase (LPL) have been found to be associated with dyslipidaemias, atherosclerosis, obesity and insulin resistance. There are two conflicting hypotheses regarding the roles of LPL in glucose metabolism and insulin resistance. Whether systemically increased LPL activity would be beneficial or detrimental to insulin sensitivity is yet to be resolved. To address this issue, we studied transgenic rabbits overexpressing human LPL transgene. METHODS: LPL transgenic and control rabbits were fed a 10% high-fat diet (HFD) for 16 weeks. To evaluate glucose metabolism, we compared plasma levels of glucose and insulin in transgenic rabbits with control rabbits and performed an intravenous glucose tolerance test. In addition, we measured adipose tissue accumulation in HFD-fed rabbits. RESULTS: Increased LPL activity in transgenic rabbits resulted in a significant reduction of plasma triglycerides and non-esterified fatty acids, but not in basal levels of glucose and insulin. HFD feeding induced an elevation of plasma glucose levels accompanied by hyperinsulinaemia in control rabbits, but was significantly inhibited in transgenic rabbits. The intravenous glucose tolerance test showed that transgenic rabbits had faster glucose clearance associated with lower levels of insulin secretion than control rabbits. In addition, there was a significant reduction of body adipose tissue in transgenic rabbits compared with in control rabbits fed an HFD. Scanning electron microscopic examination revealed that adipocytes in transgenic rabbits were predominately small cells. CONCLUSIONS/INTERPRETATION: Our results showed that systemically increased LPL activity improves insulin resistance and reduces adipose accumulation in transgenic rabbits, indicating that systemic elevation of LPL may have potential benefits for the treatment of insulin resistance and obesity.

Adipocytes↗
PubMed2001

Combination of Trichoderma harzianum endochitinase and a membrane-affecting fungicide on control of Alternaria leaf spot in transgenic broccoli plants.

Progeny from transgenic broccoli (cv. Green Comet) expressing a Trichoderma harzianum endochitinase gene were used to assess the interaction between endochitinase and the fungicide Bayleton in the control of Alternaria brassicicola. In vitro assays have shown synergistic effects of endochitinase and fungicides on fungal pathogens. Our study examined the in planta effects of endochitinase and Bayleton, individually and in combination. Two month old transgenic and non-transgenic plants were sprayed with ED50 levels of Bayleton and/or inoculated with an A. brassicicola spore suspension. Disease levels in non-sprayed transgenic plants were not statistically different from sprayed transgenic plants nor from sprayed non-transgenic controls. Thus endochitinase-transgenic plants alone provided a significant reduction of disease severity, comparable to the protection by fungicide on non-transgenic plants. Comparison of the expected additive and observed effects revealed no synergism between endochitinase and Bayleton (at ED50 level), and usually less than an additive effect. Some transgenic lines sprayed with fungicide at doses higher than ED50 showed resistance similar to the non-sprayed transgenic lines, again suggesting no synergistic effect. Lack of synergism may be due to incomplete digestion of the cell wall by endochitinase, so that the effect of Bayleton at the cell membrane is not enhanced.

Alternaria↗
PubMed2006

Production of transgenic-clone pigs by the combination of ICSI-mediated gene transfer with somatic cell nuclear transfer.

The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.

Animals↗
PubMed1993

A new method for manipulating transgenes: engineering heat tolerance in a complex, multicellular organism.

BACKGROUND: Heat-shock proteins (hsps) are thought to protect cells against stresses, especially due to elevated temperatures. But while genetic manipulation of hsp gene expression can protect microorganisms and cultured metazoan cells against lethal stress, this has so far not been demonstrated in multicellular organisms. Testing whether expression of an hsp transgene contributes to increased stress tolerance is complicated by a general problem of transgene analysis: if the transgene cannot be targeted to a precise site in the genome, newly observed phenotypes may be due to either the action of the transgene or mutations caused by the transgene insertion. RESULTS: To study the relationship between heat tolerance and hsp expression in Drosophila melanogaster, we have developed a novel method for transgene analysis, based upon the site-specific FLP recombinase. The method employs site-specific sister chromatid exchange to create an allelic series of transgene insertions that share the same integration site, but differ in transgene copy number. Phenotypic differences between members of this series can be confidently attributed to the transgenes. Using such an allelic series and a novel thermotolerance assay for Drosophila embryos, we investigated the role of the 70 kD heat-shock protein, Hsp 70, in thermotolerance. At early embryonic stages, Hsp70 accumulation was rate-limiting for thermotolerance, and elevated Hsp70 expression increased survival at extreme temperatures. CONCLUSION: Our results provide an improved method for analyzing transgenes and demonstrate that, in Drosophila, Hsp70 is a critical thermotolerance factor. They show, moreover, that manipulating the expression of a single hsp can be sufficient to improve the stress tolerance of a complex multicellular organism.

Journal Article↗
PubMed2003

Transgenic rabbits as therapeutic protein bioreactors and human disease models.

Genetically modified laboratory animals provide a powerful approach for studying gene expression and regulation and allow one to directly examine structure-function and cause-and-effect relationships in pathophysiological processes. Today, transgenic mice are available as a research tool in almost every research institution. On the other hand, the development of a relatively large mammalian transgenic model, transgenic rabbits, has provided unprecedented opportunities for investigators to study the mechanisms of human diseases and has also provided an alternative way to produce therapeutic proteins to treat human diseases. Transgenic rabbits expressing human genes have been used as a model for cardiovascular disease, AIDS, and cancer research. The recombinant proteins can be produced from the milk of transgenic rabbits not only at lower cost but also on a relatively large scale. One of the most promising and attractive recombinant proteins derived from transgenic rabbit milk, human alpha-glucosidase, has been successfully used to treat the patients who are genetically deficient in this enzyme. Although the pronuclear microinjection is still the major and most popular method for the creation of transgenic rabbits, recent progress in gene targeting and animal cloning has opened new avenues that should make it possible to produce transgenic rabbits by somatic cell nuclear transfer in the future. Based on a computer-assisted search of the studies of transgenic rabbits published in the English literature here, we introduce to the reader the achievements made thus far with transgenic rabbits, with emphasis on the application of these rabbits as human disease models and live bioreactors for producing human therapeutic proteins and on the recent progress in cloned rabbits.

Animals↗
PubMed1998

Transgenic bioreactors.

Since the generation of the first transgenic mice in 1980, transgene technology has also been successfully applied to large farm animals. Although this technology can be employed to improve certain production traits of livestock, this approach has not been very successful so far owing to unwanted effects encountered in the production animals. However, by using tissue-specific targeting of the transgene expression, it is possible to produce heterologous proteins in the extracellular space of large transgenic farm animals. Even though some recombinant proteins, such as human hemoglobin, have been produced in the blood of transgenic pigs, in the majority of the cases mammary gland targeted expression of the transgene has been employed. Using production genes driven by regulatory sequences of milk protein genes a number of valuable therapeutic proteins have been produced in the milk of transgenic bioreactors, ranging from rabbits to dairy cattle. Unlike bacterial fermentors, the mammary gland of transgenic bioreactors appear to carry out proper postsynthetic modifications of human proteins required for full biological activity. In comparison with mammalian cell bioreactors, transgenic livestock with mammary gland targeted expression seems to be able to produce valuable human therapeutic proteins at very low cost. Although not one transgenically produced therapeutic protein is yet on the market, the first such proteins have recently entered or even completed clinical trials required for their approval.

Animals↗
PubMed1998

Expression of a novel piscine growth hormone gene results in growth enhancement in transgenic tilapia (Oreochromis niloticus).

Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced following egg injection with gene constructs carrying growth hormone coding sequences of fish origin. Using a construct in which an ocean pout antifreeze promoter drives a chinook salmon growth hormone gene, dramatic growth enhancement has been demonstrated, in which the mean weight of the 7 month old G2 transgenic fish is more than three fold that of their non transgenic siblings. Somewhat surprisingly G1 fish transgenic for a construct consisting of a sockeye salmon metallothionein promoter spliced to a sockeye salmon growth hormone gene exhibited no growth enhancement, although salmon transgenic for this construct do show greatly enhanced growth. The growth enhanced transgenic lines were also strongly positive in a radio-immuno assay for the specific hormone in their serum, whereas the non growth enhanced lines were negative. Attempts to induce expression from the metallothionein promoter by exposing fish to increased levels of zinc were also unsuccessful. Homozygous transgenic fish have been produced from the ocean pout antifreeze/chinook salmon GH construct and preliminary trials suggest that their growth performance is similar to that of the hemizygous transgenics. No abnormalities were apparent in the growth enhanced fish, although minor changes to skull shape and reduced fertility were noted in some fish. There is also preliminary evidence for improved food conversion ratios when growth enhanced transgenic tilapia are compared to their non-transgenic siblings. The long term objective of this study is to produce lines of tilapia which are both growth enhanced and sterile, so offering improved strains of this important food fish for aquaculture.

Animals↗
PubMed1998

Prediction of transgene integration by noninvasive bioluminescent screening of microinjected bovine embryos.

Transgenesis in domestic species, as a research tool and in biotechnological applications, has been limited by the expense of producing transgenic offspring by standard microinjection techniques. A major factor is the inefficiency of maintaining large numbers of recipient females, when a high percentage of these carry nontransgenic fetuses. There are two approaches to reduce this cost, the fusion of transfected fetal fibroblasts with enucleated oocytes, and the screening of microinjected embryos for transgene integration in blastocysts, prior to transfer. Here, we develop a luminescent screening system to select transgenic bovine embryos. A transgene with scaffold attachment regions flanking the murine HSP70.1 promoter linked to firefly luciferase cDNA, was microinjected into pronuclei of in vitro produced zygotes. At the blastocyst stage, the transgene was induced by heat shock (45 degrees C, 15 min) and 4-6 h later, luciferase expression was analyzed by photon counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transgene integration. When nonluminescent blastocysts were transferred, transgene integration was never observed. Of 13 fetuses derived from luminescent blastocysts, 3 contained integrated transgenes that were functional in all tissues examined. Image analysis of the signal emitted by positive blastocysts revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuses derived from blastocysts that emitted light over more than 70% of their surface were transgenic. Thus, by selecting luminescent blastocysts on the basis of both signal intensity and distribution, the number of recipient females required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of transgenesis by microinjection through studies in which vector design and integration conditions are examined at the blastocyst stage.

Animals↗
PubMed2001

Size matters: use of YACs, BACs and PACs in transgenic animals.

In 1993, several groups, working independently, reported the successful generation of transgenic mice with yeast artificial chromosomes (YACs) using standard techniques. The transfer of these large fragments of cloned genomic DNA correlated with optimal expression levels of the transgenes, irrespective of their location in the host genome. Thereafter, other groups confirmed the advantages of YAC transgenesis and position-independent and copy number-dependent transgene expression were demonstrated in most cases. The transfer of YACs to the germ line of mice has become popular in many transgenic facilities to guarantee faithful expression of transgenes. This technique was rapidly exported to livestock and soon transgenic rabbits, pigs and other mammals were produced with YACs. Transgenic animals were also produced with bacterial or P1-derived artificial chromosomes (BACs/PACs) with similar success. The use of YACs, BACs and PACs in transgenesis has allowed the discovery of new genes by complementation of mutations, the identification of key regulatory sequences within genomic loci that are crucial for the proper expression of genes and the design of improved animal models of human genetic diseases. Transgenesis with artificial chromosomes has proven useful in a variety of biological, medical and biotechnological applications and is considered a major breakthrough in the generation of transgenic animals. In this report, we will review the recent history of YAC/BAC/PAC-transgenic animals indicating their benefits and the potential problems associated with them. In this new era of genomics, the generation and analysis of transgenic animals carrying artificial chromosome-type transgenes will be fundamental to functionally identify and understand the role of new genes, included within large pieces of genomes, by direct complementation of mutations or by observation of their phenotypic consequences.

Animals↗