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Possible ecological risks of transgenic organism release when transgenes affect mating success: sexual selection and the Trojan gene hypothesis.

Widespread interest in producing transgenic organisms is balanced by concern over ecological hazards, such as species extinction if such organisms were to be released into nature. An ecological risk associated with the introduction of a transgenic organism is that the transgene, though rare, can spread in a natural population. An increase in transgene frequency is often assumed to be unlikely because transgenic organisms typically have some viability disadvantage. Reduced viability is assumed to be common because transgenic individuals are best viewed as macromutants that lack any history of selection that could reduce negative fitness effects. However, these arguments ignore the potential advantageous effects of transgenes on some aspect of fitness such as mating success. Here, we examine the risk to a natural population after release of a few transgenic individuals when the transgene trait simultaneously increases transgenic male mating success and lowers the viability of transgenic offspring. We obtained relevant life history data by using the small cyprinodont fish, Japanese medaka (Oryzias latipes) as a model. Our deterministic equations predict that a transgene introduced into a natural population by a small number of transgenic fish will spread as a result of enhanced mating advantage, but the reduced viability of offspring will cause eventual local extinction of both populations. Such risks should be evaluated with each new transgenic animal before release.

Animals↗

Transgene and host growth hormone gene expression in pituitary and nonpituitary tissues of normal and growth hormone transgenic salmon.

Growth hormone (GH) gene expression has been examined in control and transgenic coho salmon containing a transgene comprised of the sockeye salmon GH1 gene under the control of the MT-B promoter from the same species. This transgene dramatically enhances the growth of salmonids, and raises serum GH levels some forty-fold. Transcript levels from this transgene were detected by RT-PCR using construct-specific GH primers in all tissues examined (liver, kidney, skin, intestine, stomach, muscle, spleen, pyloric caeca), and ranged from 0.1 - 9.4 pg/50 microg total RNA in different tissues as estimated by dot blot analysis. Interestingly, GH gene expression was also observed in intestine of control coho salmon by RT-PCR capable of detecting host and transgene transcripts using general primers. Sequence analysis of the intestinal GH mRNA from controls indicated it was derived from the coho GH2 gene. GH mRNA abundance analyzed by northern analysis indicates lower levels are found in large (400-500 g) than small transgenic salmon (20-21 g). No molecular evidence for transgene expression was obtained in tissues from transgenic fry, despite an obvious increase in size relative to control siblings, suggesting very low levels of transgene expression early in development. GH mRNA levels (per microg RNA) were also examined in the pituitary gland, and were found to be significantly lower (P < 0.01) in transgenic coho compared to nontransgenic animals of the same size. Pituitary glands of transgenic animals were also smaller than control animals of the same size, and pituitary size, expressed as a proportion of body weight, decreased with body size in transgenic but not control animals. These results imply that pituitary GH expression is regulated by negative feed-back controls as occurs in other vertebrate systems. GH mRNA was examined in pituitary glands by whole-mount in situ hybridization, and, whereas overall levels appeared reduced in transgenic animals, the site of hybridization did not differ between transgenic and control glands.

Animals↗

Loss of critical palindromic transgene promoter sequence in chemically induced Tg.AC mouse skin papillomas expressing transgene-derived mRNA.

The Tg.AC transgenic mouse carries a v-Ha-ras transgene. Skin papillomas develop in Tg.AC mice upon repeated dermal application of tumor promoters and carcinogens. The transgene is inserted at a single site on chromosome 11 in a multiple-copy array. Although most of the >or= 40 copies are arranged in a direct-repeat orientation, two copies of the transgene are inserted in a palindromic, inverted-repeat orientation. Deletion of the palindromic transgene promoter sequence is associated strongly with and diagnostic of loss of phenotypic responsiveness to Tg.AC papillomagens, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Unexpectedly, a loss of palindromic transgene sequence, in the absence of an observable reduction in copy number of the direct-repeat-oriented transgene sequence, is seen in DNA from papillomas when compared to genomic DNA from tail clips or skin samples away from the application site. Transgene-derived transcripts were detectable in all Tg.AC papillomas sampled. The transgene locus was hypomethylated in papillomas but not in samples from tail clips from the same animal or from skin samples away from the application site in responder Tg.AC mice, as shown by loss of resistance to digestion by HpaII. A cell line derived from a Tg.AC squamous cell carcinoma showed complete loss of the palindromic transgene sequence, hypomethylation of the transgene locus, and strong expression of v-Ha-ras mRNA. These data indicate that the palindromic transgene sequence, which appears to be necessary for initial responsiveness to tumorigens, may be susceptible to deletion during rapid cellular proliferation and is not required for transgene expression in later phases of papilloma growth.

Animals↗

Insect resistance to Nilaparvata lugens and Cnaphalocrocis medinalis in transgenic indica rice and the inheritance of gna+sbti transgenes.

Molecular genetic analysis and insect bioassay of transgenic indica rice 'Zhuxian B' plants carrying snowdrop lectin gene (gna) and soybean trypsin inhibitor gene (sbti) were investigated in detail. PCR, 'dot' blot and PCR-Southern blot analysis showed that both transgenes had been incorporated into the rice genome and transmitted up to R3 progeny in most lines tested. Some transgenic lines exhibited Mendelian segregation, but the other showed either 1:1 (positive: negative for the transgenes) or other aberrant segregation patterns. The segregation patterns of gna gene crossed between R2 and R3 progeny. In half of transgenic R3 lines, gna and sbti transgenes co-segregated. Two independent homozygous lines expressing double transgenes were identified in R3 progeny. Southern blot analysis demonstrated that the copy numbers of integrated gna and sbti transgenes varied from one to ten in different lines. Insect bioassay data showed that most transgenic plants had better resistance to both Nilaparvata lugens (Stahl) and Cnaphalocrocis medinalis (Guenee) than wild-type plants. The insect resistance of transgenic lines increased with the increase in transgene positive ratio in most of the transgenic lines. In all, we obtained nine lines of R3 transgenic plants, including one pure line, which had better resistance to both N lugens and C medinalis than wild-type plants.

Animals↗

Transgene expression and repression in transgenic rats bearing the phosphoenolpyruvate carboxykinase-simian virus 40 T antigen or the phosphoenolpyruvate carboxykinase-transforming growth factor-alpha constructs.

Transgenic Sprague-Dawley rats expressing either human transforming growth factor-alpha (TGFalpha) or simian virus 40 large and small T antigen (TAg), each under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, were developed as an approach to the study of the promotion of hepatocarcinogenesis in the presence of a transgene regulatable by diet and/or hormones. Five lines of PEPCK-TGFalpha transgenic rats were established, each genetic line containing from one to several copies of the transgene per haploid genome. Two PEPCK-TAg transgenic founder rats were obtained, each with multiple copies of the transgene. Expression of the transgene was undetectable in the TGFalpha transgenic rats and could not be induced when the animals were placed on a high-protein, low-carbohydrate diet. The transgene was found to be highly methylated in all of these lines. No pathological alterations in the liver and intestine were observed at any time (up to 2 years) during the lives of these rats. One line of transgenic rats expressing the PEPCK-TAg transgene developed pancreatic islet cell hyperplasias and carcinomas, with few normal islets evident in the pancreas. This transgene is integrated as a hypomethylated tandem array of 10 to 12 copies on chromosome 8q11. Expression of large T antigen is highest in pancreatic neoplasms, but is also detectable in the normal brain, kidney, and liver. Mortality is most rapid in males, starting at 5 months of age and reaching 100% by 8 months. Morphologically, islet cell differentiation in the tumors ranges from poor to well differentiated, with regions of necrosis and fibrosis. Spontaneous metastasis of TAg-positive tumor cells to regional lymph nodes was observed. These studies indicate the importance of DNA methylation in the repression of specific transgenes in the rat. However, the expression of the PEPCK-TAg induces neoplastic transformation in islet cells, probably late in neuroendocrine cell differentiation. T antigen expression during neoplastic development may result in a pervasive change in the islet cell growth properties with selection of a transformed phenotype as a possible requirement for cell viability.

Animals↗

Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants.

RNA-mediated virus resistance has recently been shown to be the result of post-transcriptional transgene silencing in transgenic plants. This study was undertaken to characterize the effect of transgene length and nontarget DNA sequences on RNA-mediated tospovirus resistance in transgenic plants. Transgenic Nicotiana benthamiana plants were generated to express different regions of the nucleocapsid (N) protein of tomato spotted wilt (TSWV) tospovirus. Transgenic plants expressing half-gene segments (387-453 bp) of the N gene displayed resistance through post-transcriptional gene silencing. Although smaller N gene segments (92-235 bp) were ineffective in conferring resistance when expressed alone in transgenic plants, these segments conferred resistance when fused to the nontarget green fluorescent protein gene DNA. These results demonstrate that (i) a critical length of N transgene (236-387 bp) is required for a high level of transgene expression and consequent gene silencing, and (ii) the post-transcriptional gene silencing mechanism can trans-inactivate the incoming tospovirus genome with homologous transgene segments that are as short as 110 bp. Therefore, the activation of post-transcriptional transgene silencing requires a significantly larger transgene than is required for the trans-inactivation of the incoming viral genome. These results raise the possibility of developing a simple new strategy for engineering multiple virus resistance in transgenic plants.

Plant Diseases↗

Chemically induced forestomach papillomas in transgenic mice carry mutant human c-Ha-ras transgenes.

Forestomach papillomas and skin papillomas were induced very efficiently by a single dose administration of the chemical carcinogen methylnitrosourea (MNU) in transgenic mice (rasH2 line) carrying human hybrid c-Ha-ras genes, which encode the prototype p21 gene product. The incidence of forestomach papillomas was dose dependent; when 50 mg/kg of MNU were administered i.p., all of the transgenic mice (56 of 56) developed forestomach papillomas within 12 weeks after administration, whereas 5 and 0.5 mg/kg of MNU induced papillomas in 2 of 19 and 1 of 19 mice, respectively. Nine of 56 transgenic mice (16%) also developed skin papillomas at sites wounded by bites or scratches. Only 1 of 77 nontransgenic littermates developed forestomach papillomas after administration of 50 mg/kg of MNU, and no skin papillomas appeared within 12 weeks after MNU administration. The transgenes (integrated copy number, 5-6) in the tumors developed in 55 of 56 affected transgenic mice (98%) contained at least 1 copy of the transgene that was activated by somatic point mutation at the 12th codon, from GGC (Gly) to GAC (Asp). Because somatic point mutations at the 12th or 61st codon of transgenes have never been detected in normal tissues of transgenic mice thus far examined, these mutational activations of transgenes are tumor-specific events. RNA expression of these activated transgenes was also detected. From these results, it is suggested that somatic mutational activation of the human c-Ha-ras transgene plays a causative role in the occurrence of forestomach and skin papillomas induced by MNU administration in these transgenic mice. This transgenic mouse provides a unique screening system for chemicals that induce or suppress papillomagenesis.

Animals↗

Testes-specific transgene expression in insulin-like growth factor-I transgenic mice.

Insulin-like growth factor-I (IGF-I) is a low molecular weight peptide that mediates the cell proliferating actions of growth hormone. Evidence exists indicating that IGF-I is produced by various cell types and this growth factor has been implicated in a variety of reproductive processes. To investigate the effect of IGF-I over-expression on reproductive systems, we generated three independent lines of transgenic mice harbouring a human IGF-I cDNA (hIGF-I) under the control of a Cytomegalovirus immediate early (CMV) promoter. The CMV promoter was used in an attempt to direct expression of IGF-I into a variety of tissues both reproductive and non-reproductive. Yet expression of the foreign hIGF-I gene, determined by Northern blot, was found to occur only in the testicular tissues of the male mice, apparently due to methylation of the transgene in all the tissues tested except the testes, which demonstrate transgene hypomethylation. Evaluation of the transgene expression during testicular development revealed that expression begins between 10 and 15 days of development, coinciding with the appearance of the zygotene and pachytene primary spermatocytes during early spermatogenesis, therefore indicating germ line expression of the transgene. Extensive study of the CMV-hIGF-I transgenic lines of mice has revealed that the effects of the transgene expression do not extend beyond the testicular tissues. No significant differences (P > 0.05) in the IGF-I serum levels, growth rates, or testicular histology have been observed between transgenic and non-transgenic male siblings. The ability of transgenic males to produce offspring also appears unaffected. Evaluation of the IGF binding protein (IGFBP) levels in the testicular tissues of CMV-hIGF-I transgenic mice by Western ligand blot revealed an increase in the concentration of testicular proteins with molecular weights corresponding to IGFBP-2 and IGFBP-3. These results suggest that the testicular over-expression of IGF-I induces increased IGFBP localization in this tissue. Inhibition of IGF activity by the IGFBPs would explain the lack of a dramatic physiological effect in the CMV-hIGF-I transgenic mice, despite the presence of elevated testicular IGF-I. The observation that testis specific IGF-I overexpression induces localization of IGFBPs in this tissue confirms the existence of a well regulated testicular IGF system and supports the convention that this growth factor plays an important role in testicular function.

Animals↗

Transgenic complementation of leptin receptor deficiency. II. Increased leptin receptor transgene dose effects on obesity/diabetes and fertility/lactation in lepr-db/db mice.

We have generated mice that are homozygous for a leptin receptor transgene that is expressed exclusively in neurons (NSE-LEPR-B). We had previously shown that this transgene in the hemizygous state is effective in ameliorating almost all aspects of leptin receptor deficiency. Now, we show that the transgene, in the homozygous state, almost fully corrects the excess adiposity of LEPR-deficient (db/db) mice. Body composition analyses indicate that the transgene is able to restrain the massive increase in adiposity observed in LEPR-deficient mice. Examination of hypothalamic agouti gene-related peptide and proopiomelanocortin mRNA shows normalization of these leptin-regulated transcripts. Interestingly, despite normalization of circulating leptin concentrations by the transgene in the fed state, transgenic db3J/db mice did not show fasting-induced reductions of circulating leptin. Increased adiposity of the transgenic db/db mice at 4 wk of age, immediately postweaning, suggests that the transgene is less effective in correcting the preferential fat deposition caused by LEPR deficiency. We noted that the morphology of brown adipose tissue is nearly normal, concordant with the cold tolerance conferred by the transgene. Aspects of the diabetes phenotype are also corrected: glucose and insulin concentrations are nearly normal, and islet hyperplasia is greatly diminished. The transgene also corrects the infertility of db/db females and confers the ability to lactate sufficiently to nurse normal-sized litters. Finally, the slightly increased adiposity and mild insulin resistance of transgenic db/db dams were not a contributory factor to the increased fat content of transgenic db/db male progeny.

Adaptation, Physiological↗

Expression of genomic and cDNA transgenes after co-integration in transgenic mice.

In general, genomic transgenes are expressed efficiently in mice, while their cDNA-based transgenes are frequently silent. Clark et al. (1992) have shown that silent cDNA transgenes under the control of the sheep beta-lactoglobulin promoter can be activated after co-injecting them with a genomic sheep beta-lactoglobulin transgene. We have tested the general utility of this concept using mouse whey acidic protein (WAP) transgenes. Here we show that WAP cDNA transgenes are virtually silent in transgenic mice. In contrast, WAP transgenes containing all introns are expressed in approximately 50% of the lines at levels ranging from 1% to more than 100% of the endogenous RNA (McKnight et al., 1992). When a WAP-genomic transgene was co-injected with a WAP-cDNA, basal activation of the cDNA was possible. However, the activity of the WAP-cDNA transgene did not exceed 1% of the WAP-genomic transgene. This suggests that a permissive integration site capable of supporting basal level transcription can be established, but further events are required for full activation of the transgene.

Animals↗

Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley.

In order to evaluate the long-term stability of transgene expression driven by the B(1)- and D-hordein promoters in transgenic barley ( Hordeum vulgare L., 2 n=2 x=14), we analyzed plants from 15 independent transgenic barley lines [6 for uidA and 9 for sgfp(S65T)] produced via microprojectile bombardment of immature embryos; 4 were diploid and 11 were tetraploid. The expression and inheritance of transgenes were determined by analysis of functional transgene expression, polymerase chain reaction and fluorescence in situ hybridization (FISH). Ability to express transgenes driven by either B(1)- or D-hordein promoter was inherited in T(4) and later generations: T(4) (2 lines), T(5) (8 lines), T(6) (3 lines), T(8) (1 line) and T(9) (1 line). Homozygous transgenic plants were obtained from 12 lines [5 for uidA and 7 for sgfp(S65T)]; the remaining lines are currently being analyzed. The application of the FISH technique for physical mapping of chromosomes was useful for early screening of homozygous plants by examining for presence of the transgene. For example, one line expressing uidA, and shown to have doublet fluorescence signals on a pair of homologous chromosomes was confirmed as a homozygous line by its segregation ratio; additionally this line showed stable inheritance of the transgene to T(9) progeny. The expression of transgenes in most lines (14 out of 15 lines) driven by hordein promoters was stably transmitted to T(4) or later generations, although there was a skewed segregation pattern (1:1) from the T(1) generation onward in the remaining line. In contrast, transgene silencing or transgene loss under the control of the maize ubiquitin promoter was observed in progeny of only 6 out of 15 lines.

Chromosome Mapping↗

Matrix attachment region from the chicken lysozyme locus reduces variability in transgene expression and confers copy number-dependence in transgenic rice plants.

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. In this study, BP-MAR, a 1.3-kb upstream fragment of the 5'MAR flanking the chicken lysozyme locus, was tested for its effects on integration and expression of transgenes in transgenic rice plants. Using the Agrobacterium-mediated method, we transformed rice with nine different constructs containing seven and six different promoters and coding sequences, respectively. Genomic Southern blot analyses of 357 independent transgenic lines revealed that in the presence of BP-MAR, 57% of the lines contained a single copy of the transgene, whereas in its absence, only 20% of the lines contained a single copy of the transgene. RNA gel-blot and immunoblot experiments demonstrated that in the presence of BP-MAR, transgene expression levels were similar among different lines. These data were in direct contrast to those derived from transgenes expressed in the absence of BP-MAR, which varied markedly with the chromosomal integration site . Thus, it can be concluded that BP-MAR significantly reduces the variability in transgene expression between independent transformants. Moreover, the presence of BP-MAR appears to confer a copy number-dependent increase in transgene expression, although it does not increase expression levels of individual transgenes. These data contrast with results previously obtained with various MARs that increased expression levels of transgene significantly. Therefore, we conclude that the incorporation of BP-MAR sequences into the design of transformation vectors can minimize position effects and regulate transgene expression in a copy number-dependent way.

5' Flanking Region↗

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.).

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp b-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

Animals↗

Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement.

To more fully characterize the internal structure of transgene loci and to gain further understanding of mechanisms of transgene locus formation, we sequenced more than 160 kb of complex transgene loci in two unrelated transgenic oat (Avena sativa L.) lines transformed using microprojectile bombardment. The transgene locus sequences from both lines exhibited extreme scrambling of non-contiguous transgene and genomic fragments recombined via illegitimate recombination. A perfect direct repeat of the delivered DNA, and inverted and imperfect direct repeats were detected in the same transgene locus indicating that homologous recombination and synthesis-dependent mechanism(s), respectively, were also involved in transgene locus rearrangement. The most unexpected result was the small size of the fragments of delivered and genomic DNA incorporated into the transgene loci via illegitimate recombination; 50 of the 82 delivered DNA fragments were shorter than 200 bp. Eleven transgene and genomic fragments were shorter than the DNA lengths required for Ku-mediated non-homologous end joining. Detection of these small fragments provided evidence that illegitimate recombination was most likely mediated by a synthesis-dependent strand-annealing mechanism that resulted in transgene scrambling. Taken together, these results indicate that transgene locus formation involves the concerted action of several DNA break-repair mechanisms.

Avena↗

Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats.

The production of valuable pharmaceutical proteins using transgenic animals as bioreactors has become one of the goals of biotechnology. However, the efficiency of producing transgenic animals by means of pronuclear microinjection is low. This may be attributed in part to the low integration rate of foreign DNA. Therefore, a large number of recipients are required to produce transgenic animals. We recently developed a transgenic procedure that combined the techniques of goat oocyte in vitro maturation (IVM), in vitro fertilization (IVF), microinjection, preimplantation selection of the transgenic embryos with nested PCR and transferring the transgenic embryos into the recipient goat uterus to produce transgenic goats. Thirty-seven transgenic embryos determined by nested PCR were transferred to thirty-two recipient goats. In the end, four live-born kids were produced. As predicted, all the live kids were transgenic as identified by PCR as well as Southern blot hybridization, The integration rate was 100% (4/4) which was completely in accordance with the results of embryo preimplantation detection. The results showed a significant decrease in the number of recipients required as only 8 recipients (32/4) were needed to obtain one live transgenic goat. We suggest that the transgenic system described herein may provide an improved way to efficiently produce transgenic goats on a large scale.

Animals↗

Relative fitness of transgenic vs. non-transgenic maize x teosinte hybrids: a field evaluation.

Concern has been often expressed regarding the impact and persistence of transgenes that enter wild populations via gene flow. The impact of a transgene and its persistence are largely determined by the relative fitness of transgenic hybrids and hybrid derivatives compared to non-transgenic plants. Nevertheless, few studies have addressed this question experimentally in the field. Despite the economic importance of maize, and the fact that it naturally hybridizes with the teosinte taxon Zea mays ssp. mexicana, sometimes known as "chalco teosinte," the question has received little experimental attention in this system. Using a glyphosate-tolerant maize cultivar and chalco teosinte as parental lines, we carried out a field experiment testing (1) the relative fitness of maize x teosinte hybrids, compared to their parental taxa, as well as (2) the relative fitness of transgenic hybrids compared to non-transgenic hybrids created from the same parental stock. In order to evaluate the influence of the transgenic construct in different genetic backgrounds, our study included transgenic and non-transgenic pure maize progeny from the cultivar as well. We measured both vegetative and reproductive parameters. Our results demonstrated that hybrids have greater vigor and produced more seeds than the wild parent. However, in the absence of selective pressure from glyphosate herbicide, we did not observe any direct positive or negative impact of the transgene on the fitness or vigor of either the hybrids or pure maize progeny. We discuss our results in terms of the potential for spontaneous transgene flow and introgression from transgenic maize into sympatric teosinte.

Germination↗

Assessment of the proportion of transgene-bearing sperm by fluorescence in situ hybridization: a novel approach for the detection of germline mosaicism in transgenic male founders.

Genetic mosaicism is frequent among transgenic animals produced by pronuclear microinjection. A successful method for the screening of founder animals for germline mosaicism prior to mating would greatly reduce the costs associated with the propagation of the transgenic lines, and improve the efficiency of transgenic livestock production. With this aim, we have devised a simple method to detect integrated transgenes in individual spermatozoa using fluorescence in situ hybridization (FISH). The experiments reported here were undertaken to investigate the efficiency of this FISH-based approach to accurately evaluate the proportion of transgene-bearing sperm and to be applied for the detection of potential germline mosaics. Sperm samples from mice homozygous and hemizygous for a beta-lactoglobulin transgene were analyzed in a first set of experiments. A high hybridization efficiency was achieved, and the proportions of transgene-positive sperm cells in both homozygous (94.8-98.2%) and hemizygous (49.8-51.9%) animals were close to the expected frequencies (100 and 50%, respectively). To evaluate the sensitivity of the assay more directly, simulated mosaic samples with 5, 10, 15, 20 and 40% of transgene-bearing spermatozoa were then prepared and analyzed by FISH. Significant differences in the frequency of transgene-positive sperm were observed between all mosaic samples, indicating that even small deviations (5%) from the expected 50% transgene transmission rate in a founder animal could be reliably detected with our assay. Therefore, the method proposed represents a novel approach for the identification of germline mosaic founder males in livestock transgenic projects and a much more economic and faster alternative to breeding.

Animals↗

A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission.

The Cre/loxP site-specific recombination system derived from bacteriophage P1 provides a convenient tool for directed modifications of genomes in various organisms. To exploit Cre-mediated manipulation of mouse genomic sequences at the zygote stage, we have developed a transgenic mouse line carrying the CAG-cre transgene in which the cre gene is under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid (CAG) promoter. The activity of the Cre recombinase at early stages of development was examined by crossing the CAG-cre transgenic mice to another transgenic mouse line carrying a reporter gene construct, CAG-CAT-Z, which directs expression of the E. coli lacZ gene upon Cre-mediated excision of the loxP-flanked chloramphenicol acetyltransferase (CAT) gene located between the CAG promoter and the lacZ gene. PCR-based analysis of F1 progeny from CAG-cre males x CAG-CAT-Z females showed that transmission of the CAG-cre transgene was accompanied by the complete deletion of the CAT gene of the CAG-CAT-Z transgene in all tissues, and that this deletion was never observed in the progeny without transmission of the CAG-cre gene. On the other hand, analysis of F1 mice from CAG-CAT-Z males x CAG-cre females showed that the CAG-CAT-Z transgene had undergone complete deletion of the CAT gene in all tissues irrespective of the cotransmission of the CAG-cre gene. This Cre-mediated recombination in F1 mice occurred before the two-cell stage of embryonic development, as shown by X-gal staining. The results suggest that the CAG-cre transgene is expressed in developing oocytes of CAG-cre transgenic mice, and Cre mRNA and/or protein are retained in mature oocytes irrespective of the transmission of the CAG-cre transgene, resulting in efficient Cre-mediated recombination of paternally derived target genes upon fertilization. The CAG-cre transgenic mouse should serve as a useful tool to introduce prescribed genetic modifications into the mouse embryo at the zygote stage.

Animals↗