Transgene transmission to progeny by oMt1a-oGH transgenic mice

Most studies utilizing transgenic technology focus on the impact to traits of interest, rather than propagation of the transgene to offspring. In animals containing growth hormone constructs, transgene transmission to progeny follows a Mendelian pattern of inheritance in the first few generations following generation of a founder animal, but decreases in subsequent generations. In the present study, the ovine metallothionein 1a-ovine growth hormone (oMt1a-oGH) transgenic mouse was used to determine whether transgene transmission rate to progeny was affected by overexpression of ovine growth hormone in the transgenic parent. The oMt1a-oGH mouse is a useful model for assessing transgene transmission, as the construct is easily regulatable and transgene inactivation results in a return of plasma GH to basal levels. Male and female hemizygous oMt1a-oGH mice were assigned to 1 of 3 treatment groups: (1) mice never actively expressing the transgene, (2) mice actively expressing the transgene from 3 weeks of age, and (3) mice actively expressing the transgene from 3 to 11 (males) or 3 to 8 (females) weeks of age. Transgenic mice were mated to wild type animals and the resulting progeny were genotyped. Males never actively expressing the transgene passed on the transgene to progeny in a Mendelian fashion, while males actively expressing the transgene transmitted the transgene to a smaller than expected number of progeny. However, following inactivation of the oMt1a-oGH construct in transgenic males, subsequent offspring demonstrated Mendelian inheritance of the transgene. In contrast, females expressing the transgene from 3 to 8 weeks of age were able to pass on the oMt1a-oGH construct in a Mendelian fashion, but females from other treatment groups were not. In oMt1a-oGH males, reduced transgene transmission appears to be due to selection against transgenic gametes. In females, however, selection against the transgenic genotype likely occurs at the embryonic level.     

Expression of an ovine growth hormone transgene in mice increases archidonic acid in cellular membranes

The degree of unsaturation of membrane lipids has been implicated in a number of physiological disorders, yet its regulation remains poorly understood, especially the regulation of the synthesis and distribution of arachidonic acid levels, the most abundant long chain polyunsaturated fatty acid in membranes. Transgenic mice expressing the ovine metallothionein 1a — ovine growth hormone (oMt1a-oGH) fusion gene exhibited significantly elevated levels of a number of long chain polyusaturated fatty acids in serum, including arachidonic acid. In oMt1a-oGH transgenic mice the products of all three desaturation pathways are affected by the expression of the ovine growth hormone trangene. The essential precursors of membrane long chain polyunsturated fatty acids, 18:2n-6 and 18:3n-3, were reduced in transgenic relative to controls, and their desaturation and elongation products, arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3), were elevated. As rare intermediate long chain polyunsaturated fatty acids such as eicosatrienoic acid (20:3n-9) were also signficantly elevated, we conclude that these observations reflect increased activity of the Δ-5 and Δ-6 desaturase enzymes. In contranst, the products of the stearoyl CoA or Δ-9 desaturase, were significantly reduced in oMt1a-oGH expressing transgenics relative to their levels in control mice.     

Development of obesity following inactivation of a growth hormone transgene in mice

Mice with a temporally regulatable ovine metallothionein 1a—ovine growth hormone transgene (oMT1a-oGH) were utilized to study the effects of withdrawal of elevated circulating levels of growth hormone (GH) on growth and body composition. The transgene was activated from 21–42 days of age by provision of zinc sulfate in the drinking water. At 42 days, mice were allocated to either activated transgenic (remain on zinc sulfate) or inactivated transgenic (removal of zinc sulfate) groups, and to receive eitherad libitum or restricted (80–90% ofad libitum) access to feed. Non-transgenic control mice were treated similarly. Body weights and intakes were recorded weekly. Mice were killed at 70 d and epididymal and subcutaneous fat pads, trimmed hind carcass and various organs were weighed. The main findings of this study are: (1) food-restricted mice possessing an activated oMT1a-oGH transgene fail to demonstrate increased growth, but exhibit significantly reduced levels of fat (P<0.05) relative to all other genotype x feed level combinations; and (2) inactivation of the oMT1a-oGH transgene, following a period of elevated GH levels, leads to development of obesity as evidenced by two to three fold increases in epididymal and subcutaneous fat pad weights (P<0.01) relative to both activated transgenic and non-transgenic control mice. These large increases in fat deposition also occurred when intake was restricted to 80–90% ofad libitum levels, indicating that metabolic changes independent of intake occur in these inactivated transgenic mice. It is possible that highly elevated production of GH in activated oMT1a-oGH transgenic mice leads to (1) enhanced promotion of preadipocyte differentiation, leading to increased numbers of adipocytes that, upon cessation of oGH production, are available for lipid deposition resulting in obesity, or (2) alterations in production of or responsiveness to insulin, leading to increased fat deposition upon removal of the chronic anti-lipogenic actions of GH. The oMT1a-oGH transgenic mouse line should provide a new genetic model with which to investigate the mechanisms by which growth hormone affects obesity.     

Effect of genetic background on growth of mice hemizygous for wild-type or dwarf mutated bovine growth hormone transgenes

The effects of a high-growth genetic background on the growth of mice hemizygous for one of two growth hormone transgenes were examined. Male mice hemizygous for wild-type (W) and dwarf mutant (M) bovine growth hormone (bGH) transgenes were crossed with females of a high-growth selected (S) and control (C) line as follows: W x S, W x C, M x S and M x C. Body weights of progeny were recorded weekly from 2 to 10 weeks of age. F₁ progeny were classified as carriers (P) or non-carriers (N) of the transgene by assaying tail DNA for bGH using the polymerase chain reaction and agarose gel electrophoresis. A deficiency in the number of f₁ progeny carrying the W (P<0.05) and M (P<0.01) bGH transgene was most likely due to differential prenatal and early postnatal mortality. Bodyweight means of wild-type transgenic mice were larger (P < 0.05) than those of non-transgenic littermates by 3 weeks of age in a C background in contrast to 5 weeks in S. The wild-type bGH transgene increased adult body weights more in the C (155%) than in the S (136%) background, indicating transgene expression by selection background interaction (P < 0.05). However, the growth response to the wild-type transgene in the S background was still large. The dwarf mutant transgene had a greater effect on growth reduction in the S (70%) than in the C (84%) background, thus causing transgene expression by selection background interaction (P < 0.05). Gender by wild-type transgene effect interactions (P < 0.001) for adult body weight were caused by the transgene reducing the gender difference for body weight in C and eliminating it in S. The dwarf mutant caused a larger negative effect on growth in males than in females, resulting in a gender by dwarf mutant transgene interaction (P < 0.001) for adult body weights. Results indicate that the effect of a GH transgene on growth can be affected both by a high-growth genetic background and the gender of progeny.     

Evaluating the fitness of human lysozyme transgenic dairy goats: growth and reproductive traits

While there are many reports in the literature describing the attributes of specific applications of transgenic animals for agriculture, there are relatively few studies focusing on the fitness of the transgenic animals themselves. This work was designed to gather information on genetically modified food animals to determine if the presence of a transgene can impact general animal production traits. More specifically, we used a line of transgenic dairy goats expressing human lysozyme in their mammary gland to evaluate the reproductive fitness and growth and development of these animals compared to their non-transgenic counterparts and the impact of consuming a transgenic food product, lysozyme-containing milk. In males, none of the parameters of semen quality, including semen volume and concentration, total sperm per ejaculate, sperm morphology, viability and motility, were significantly different between transgenic bucks and non-transgenic full-sib controls. Likewise, transgenic females of this line did not significantly differ in the reproductive traits of gestation length and litter size compared to their non-transgenic counterparts. To evaluate growth, transgenic and non-transgenic kid goats received colostrum and milk from either transgenic or non-transgenic does from birth until weaning. Neither the presence of the transgene nor the consumption of milk from transgenic animals significantly affected birth weight, weaning weight, overall gain and post-wean gain. These results indicate that the analyzed reproductive and growth traits were not regularly or substantially impacted by the presence or expression of the transgene. The evaluation of these general parameters is an important aspect of defining the safety of applying transgenic technology to animal agriculture.     

Effect of transgenesis on reproductive traits of rabbit males

The influence of foreign transgene integration on the reproductive capabilities of rabbit males is not known. Therefore, we analyzed their ejaculate characteristics, reproductive capabilities, occurrence of pathological spermatozoa and histological structure of the testis. We have generated transgenic rabbits by microinjection of WAP-hFVIII gene into pronucleus of fertilized egg. We observed that the libido, volume and pH value of the ejaculate did not differ significantly between transgenic and non-transgenic male lines. The motility, concentration, osmolarity, thermoresistant test of spermatozoa (at 1 or 6 h) and the percentage of alive spermatozoa were significantly different (p < 0.001) among transgenic and non-transgenic males. No significant differences were found between transgenic and non-transgenic male lines in the occurrence of pathological spermatozoa and histology of the testis. The ability of spermatozoa from transgenic and non-transgenic males to fertilize eggs was ranged within 96 and 100%; while the yield of transgenic embryos ranged from 43 to 57%. Our results show that mammary gland specific over-expression mWAP-hFVIII gene construct does not affect reproductive traits of transgenic rabbit males.     

Competitive performance of transgenic wheat resistant to powdery mildew

Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes.     

Mitigation of establishment of Brassica napus transgenes in volunteers using a tandem construct containing a selectively unfit gene

Transgenic oilseed rape ( Brassica napus ) plants may remain as 'volunteer' weeds in following crops, complicating cultivation and contaminating crop yield. Volunteers can become feral as well as act as a genetic bridge for the transfer of transgenes to weedy relatives. Transgenic mitigation using genes that are positive or neutral to the crop, but deleterious to weeds, should prevent volunteer establishment, as previously intimated using a tobacco ( Nicotiana tabacum ) model. A transgenically mitigated (TM), dwarf, herbicide-resistant construct using a gibberellic acid-insensitive (Δ gai ) gene in the B. napus crop was effective in offsetting the risks of transgene establishment in volunteer populations of B. napus . This may be useful in the absence of herbicide, e.g. when wheat is rotated with oilseed rape. The TM dwarf B. napus plants grown alone had a much higher yield than the non-transgenics, but were exceedingly unfit in competition with non-transgenic tall cohorts. The reproductive fitness of TM B. napus was 0% at 2.5-cm and 4% at 5-cm spacing between glasshouse-grown plants relative to non-transgenic B. napus . Under screen-house conditions, the reproductive fitness of TM B. napus relative to non-transgenic B. napus was less than 12%, and the harvest index of the TM plants was less than 40% of that of the non-transgenic competitors. The data clearly indicate that the Δ gai gene greatly enhances the yield in a weed-free transgenic crop, but the dwarf plants can be eliminated when competing with non-transgenic cohorts (and presumably other species) when the selective herbicide is not used.     

Fitness of transgenic Anopheles stephensi mosquitoes expressing the SM1 peptide under the control of a vitellogenin promoter

Three transgenic Anopheles stephensi lines were established that strongly inhibit transmission of the mouse malaria parasite Plasmodium berghei. Fitness of the transgenic mosquitoes was assessed based on life table analysis and competition experiments between transgenic and wild-type mosquitoes. Life table analysis indicated low fitness load for the 2 single-insertion transgenic mosquito lines VD35 and VD26 and no load for the double-insertion transgenic mosquito line VD9. However, in cage experiments, where each of the 3 homozygous transgenic mosquitoes was mixed with nontransgenic mosquitoes, transgene frequency of all 3 lines decreased with time. Further experiments suggested that reduction of transgene frequency is a consequence of reduced mating success, reduced reproductive capacity, and/or insertional mutagenesis, rather than expression of the transgene itself. Thus, for transgenic mosquitoes released in the field to be effective in reducing malaria transmission, a driving mechanism will be required.     

Mycorrhizal colonization of transgenic aspen in a field trial

Mycorrhizal colonization of genetically modified hybrid aspen (Populus tremula x P. tremuloides Michx.) was investigated over 15 months in a field experiment. The aspen carried the rolC gene from Agrobacterium rhizogenes under control of either the constitutive cauliflower mosaic virus 35S promoter or the light-inducible rbcS promoter. Arbuscular mycorrhizas (AMs) were rare in all root samples, while fully developed ectomycorrhizas (EMs) were found in all samples. No significant differences in the degree of mycorrhizal colonization between aspen lines were seen with either AMs or EMs. The EM community on the release area was dominated by four fungal species that formed more than 90% of all mycorrhizas, while eleven EM types were found occasionally. Mycorrhizal diversity did not differ between transgenic and non-transgenic trees. The structure of mycorrhizal communities was similar for most aspen lines. The sole significant difference was found in the abundance and development of one of the four common EM morphotypes, which was rare and poorly developed on roots from the transgenic aspen line Esch5:35S-rolC-#5 compared with non-transgenic controls. This effect is clone specific as the formation of this EM type was not affected by the transgene expression in the other transgenic line, Esch5:35S-rolC-#1. This is the first demonstration of a clonal effect influencing the ability of a transgenic plant to form a mycorrhizal symbiosis with a potential fungal partner.     

Decreased frequency of the rat growth hormone transgene in mouse populations with or without selection for increased adult body weight

Summary Frequencies of mice with the rat growth hormone (rGH) transgene were examined in lines derived from two genetic bases (P/W and P/C). The genetic bases were developed from males (P) with the rGH transgene, mated with non-transgenic females of different origin: a line previously selected for large body size (W) and a corresponding unselected control line (C). They were maintained for six generations under random mating with or without selection for increased 42-day body weight. The frequencies of P/W and P/C males with the rGH transgene wer 0.075 and 0.300, respectively at generation 0 of the genetic bases. They were significantly (P<0.05) lower than the expected frequency (about 0.5). At generation 6, the frequencies had decreased further both in selected and unselected lines (ranging from 0.025 to 0.125). Decreased frequencies of mice with the transgene were confirmed in a separate experiment testing segregation of the transgene. The reasons for these decreases are not clear. The results suggest that transgenes need to be monitored when transgenic animals are mated with animals of different origin.Animal Research Centre Contribution No. 1697     

Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica)

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic proteins found primarily in plants. The barley hva1 gene encodes a group 3 LEA protein and is induced by ABA and water deficit conditions. We report here the over expression of hva1 in mulberry under a constitutive promoter via Agrobacterium-mediated transformation. Molecular analysis of the transgenic plants revealed the stable integration and expression of the transgene in the transformants. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of hva1 in conferring tolerance. The transgenic plants showed better cellular membrane stability (CMS), photosynthetic yield, less photo-oxidative damage and better water use efficiency as compared to the non-transgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants show many fold increase in proline concentration than the non-transgenic plants and under water deficit conditions proline is accumulated only in the non-transgenic plants. Results also indicate that the production of HVA1 proteins helps in better performance of transgenic mulberry by protecting membrane stability of plasma membrane as well as chloroplastic membranes from injury under abiotic stress. Interestingly, it was observed that hva1 conferred different degrees of tolerance to the transgenic plants towards various stress conditions. Amongst the lines analysed for stress tolerance transgenic line ST8 was relatively more salt tolerant, ST30, ST31 more drought tolerant, and lines ST11 and ST6 responded well under both salinity and drought stress conditions as compared to the non-transgenic plants. Thus hva1 appears to confer a broad spectrum of tolerance under abiotic stress in mulberry.     

Direct genetic and postnatal maternal genetic effects on body composition in mice selected for body weight.

Line crossfostering techniques were used to study differences among selected and control lines of mice in direct genetic and postnatal maternal genetic influences on preweaning (day 12) body weight and composition. The lines were selected for high (H6) and low (L6) 6-week body weight and the control line (C2) was maintained by random selection. There were positive correlated responses to selection in both direct genetic and postnatal maternal genetic effects on body weight and weights of all body components (P less than 0.01) except for water and ash weight in H6. The correlated responses in postnatal maternal genetic effects were of the same order of magnitude as those in direct genetic effects. Correlated responses were greater in L6 than in H6. Correlated responses in direct genetic effects were positive (P less than 0.01) for water percent in H6 and ether extract percent in L6, and negative (P less than 0.01) for water percent and lean percent in L6. Correlated responses in postnatal maternal genetic effects were positive for ether extract percent and negative for water percent (P less than 0.01). Correlated responses were far greater in L6 than in H6 and were greater for postnatal maternal genetic effects than for direct genetic effects. Analyses of covariance results indicated line differences in the relative growth rates of the body components.     

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

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

Pathogenesis in Transgenic Mice Expressing Bovine Cellular Retinoic Acid-Binding Protein

Transgenic mice with ectopic expression of bovine CRABP under the control of the human metallotheionein IIA promoter have shown a variety of pathological consequences. Expression of the transgene has been detected in most of the tissues examined, including heart, lung, liver, spleen, kidney, intestine, testis, and ovary, except pancreas. Two independent lines have been able to produce normal non-transgenic F₁ animals of both sexes but only female transgenic progenies. All of these F₁ female transgenic animals derived from both lines are sterile, and the ovaries from these animals appear to be significantly smaller as compared to their non-transgenic littermates. Histopathological examinations have shown no maturing follicles in these transgenic ovaries in which abnormal cells have been observed. Another independent line has generated transgenic F₁ animals which have been growing retardly. These animals all have small spleen and liver and have become very sick at the age of 4 to 5 weeks. Histopathological examinations on these transgenic progenies have shown hepatocytes to be reduced in the cytoplasmic portion in which glycogen is highly depleted. The spleen is poorly developed as no well organized germinal centers can be observed in the spleen sections of these transgenic animals.     

Responses of MxPPO overexpressing transgenic tall fescue plants to two diphenyl-ether herbicides, oxyfluorfen and acifluorfen

We generated transgenic tall fescue (Festuca arundinacea Schreb. cv. Kentucky-31) plants harboring a synthetic Myxococcus xanthus protoporphyrinogen oxidase (MxPPO) gene through Agrobacterium-mediated gene transfer. Successful integration of the transgene into the genome of transgenic plants confirmed by polymerase chain reaction (PCR) and Southern blot analysis, and the functional expression of the MxPPO gene at the mRNA level in transgenic lines was validated by Northern blot analysis. Responses of transgenic and non-transgenic tall fescue plants to diphenyl-ether herbicides such as oxyfluorfen and acifluorfen have been evaluated in respect of various physiological and biochemical parameters. Differential responses were observed in chlorophyll content, in vivo H₂O₂ deposition and lipid peroxidation in both transgenic and non-transgenic plants exposed to oxyfluorfen or acifluorfen. Isozyme profiles of four antioxidant-enzymes, including peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), were also investigated in transgenic and non-transgenic plants using native PAGE analysis. Compared to the transgenic lines, higher staining activities of the examined antioxidant-enzymes observed in non-transgenic plants subjected to 100 μM of oxyfluorfen or acifluorfen suggests that non-transgenic plants are unable to prevent the photodynamic induced oxidative stress caused by herbicides. In addition, both transgenic and non-transgenic plants exposed to oxyfluorfen exhibited proportionally increased band-staining patterns in contrast to acifluorfen, which suggests that oxyfluorfen has relatively greater or more rapid effects on leaves than acifluorfen. Both Ki-Won Lee and Nagib Ahsan have contributed equally to this work.     

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

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