Mitochondrial DNA sequence and phylogenetic evaluation of geographically disparate Sus scrofa breeds.

Next generation sequencing of mitochondrial DNA (mtDNA) facilitates studies into the metabolic characteristics of production animals and their relation to production traits. Sequence analysis of mtDNA from pure-bred swine with highly disparate production characteristics (Mangalica Blonde, Mangalica Swallow-bellied, Meishan, Turopolje, and Yorkshire) was initiated to evaluate the influence of mtDNA polymorphisms on mitochondrial function. Herein, we report the complete mtDNA sequences of five Sus scrofa breeds and evaluate their position within the phylogeny of domestic swine. Phenotypic traits of Yorkshire, Mangalica Blonde, and Swallow-belly swine are presented to demonstrate their metabolic characteristics. Our data support the division of European and Asian breeds noted previously and confirm European ancestry of Mangalica and Turopolje breeds. Furthermore, mtDNA differences between breeds suggest function-altering changes in proteins involved in oxidative phosphorylation such as ATP synthase 6 (MT-ATP6), cytochrome oxidase I (MT-CO1), cytochrome oxidase III (MT-CO3), and cytochrome b (MT-CYB), supporting the hypothesis that mtDNA polymorphisms contribute to differences in metabolic traits between swine breeds. Our sequence data form the basis for future research into the roles of mtDNA in determining production traits in domestic animals. Additionally, such studies should provide insight into how mtDNA haplotype influences the extreme adiposity observed in Mangalica breeds.

Simian virus 40 (SV40)-transgenic mice that develop tumors are specifically tolerant to SV40 T antigen.

The ability to mount an immune response to simian virus 40 (SV40) T antigen was evaluated using mice from two distinct SV40 transgenic lines derived from injection of the same gene construct. Our studies demonstrate functional immune tolerance to SV40 T antigen in a SV40 transgenic line that consistently develops tumors of the choroid plexus by 7 mo of age. Antibodies to SV40 T antigen are undetectable in the serum of these animals; furthermore, mice from this line are unable to generate SV40-specific CTL after primary or secondary immunization with the virus, although they mount a normal CTL response to vaccinia virus when appropriately immunized. In contrast, we find that mice from a second transgenic line of low tumor incidence can mount a humoral response to SV40 T antigen, and upon immunization they generally respond with a vigorous cytotoxic T cell response to SV40 T antigen. These data suggest that specific immune tolerance to the product of an integrated viral oncogene may be induced, and is likely a reflection of the time in development at which the gene product first appears. Immune tolerance or responsiveness to the endogenous oncogene product may in turn play a role in the tumorigenic potential of such genes.

The E mu-myc transgenic mouse. A model for high-incidence spontaneous lymphoma and leukemia of early B cells.

Mice transgenic for a c-myc gene driven by the IgH enhancer (E mu-myc) were shown to almost invariably develop lymphomas, 90% succumbing in the first 5 mo of life. The tumors typically presented as rapidly progressive lymphadenopathy with thymic involvement and were highly malignant by transplantation assay. Morphologically, they were lymphoblastic lymphomas, usually accompanied by lymphoid leukemia and granulocytosis, and were distinct from the tumors that arose much later in 37% of nontransgenic mice of the same (C57BL/6 x SJL)F2 genetic background. Cell-surface markers on 31 E mu-myc tumors identified 52% as pre-B lymphomas, 29% as mixed pre-B and B lymphomas, and 19% as B lymphomas. The tumors appeared to arise at random from a population of pre-B cells expanded by constitutive expression of the myc transgene. A majority of the animals initiated malignancy at the rate of 17% per week. The rate at which the cycling, benign pre-B cells spontaneously convert to malignancy was estimated to about 10(-10) per cell per generation. A transient leukocytosis identified in young E mu-myc mice was developed into a rapid assay for inheritance of the transgene.

A transgenic mouse model of the chronic hepatitis B surface antigen carrier state.

In an attempt to establish a model of the healthy carrier state in hepatitis B virus (HBV) infections, transgenic mice expressing HBV genes were produced. Fertilized one-cell eggs were microinjected with subgenomic fragments of HBV DNA containing the coding regions for the HBV surface antigen (HBsAg) and pre-S and X antigens. Either the normal (HBV) or metallothionein promoters were used to obtain expression of the HBV genes. There was no evidence of viral replication or tissue pathology. The integrated HBV DNA sequences were inherited in a normal Mendelian fashion. Three of 16 transgenic mice expressed HBV-encoded gene products to which they were immunologically tolerant. Expression was not tissue specific and may be influenced by the genomic integration site and cellular factors. Both HBsAg and pre-S antigen were detectable within the cytoplasm of hepatocytes and renal tubular epithelial cells. High serum concentrations of HBsAg were detectable and the secreted product appeared authentic as judged by mean density, morphology, mean particle diameter, polypeptide composition, and antigenicity. The absence of tissue pathology in these immunologically tolerant animals supports the hypothesis that cellular injury under these conditions is not a direct consequence of expression of the pre-S or HBs regions of the HBV genome.

Genetic engineering of livestock.

Genetic engineering of livestock is expected to have a major effect on the agricultural industry. However, accurate assessment of the consequences of transgene expression is impossible without multigenerational studies. A systematic study of the beneficial and adverse consequences of long-term elevations in the plasma levels of bovine growth hormone (bGH) was conducted on two lines of transgenic pigs. Two successive generations of pigs expressing the bGH gene showed significant improvements in both daily weight gain and feed efficiency and exhibited changes in carcass composition that included a marked reduction in subcutaneous fat. However, long-term elevation of bGH was generally detrimental to health: the pigs had a high incidence of gastric ulcers, arthritis, cardiomegaly, dermatitis, and renal disease. The ability to produce pigs exhibiting only the beneficial, growth-promoting effects of growth hormone by a transgenic approach may require better control of transgene expression, a different genetic background, or a modified husbandry regimen.

Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice.

The role of the immune response to hepatitis B virus (HBV)-encoded antigens in the pathogenesis of liver cell injury has not been defined because of the absence of appropriate experimental models. HBV envelope transgenic mice were used to show that HBV-encoded antigens are expressed at the hepatocyte surface in a form recognizable by major histocompatibility complex (MHC) class I-restricted, CD8+ cytotoxic T lymphocytes specific for a dominant T cell epitope within the major envelope polypeptide and by envelope-specific antibodies. Both interactions led to the death of the hepatocyte in vivo, providing direct evidence that hepatocellular injury in human HBV infection may also be immunologically mediated.

Identification of transgenic mice by PCR analysis of saliva.

As an alternative to surgically obtaining samples (e.g., tail or tissue biopsy, toe dock, or blood sampling) from weanling mice to screen for transgene integration or other genetic monitoring procedures, we offer a simpler, nonsurgical method. A small amount of saliva, obtained from weanling mice by oral wash using a plastic pipet tip, contains enough oral epithelial cells and lymphocytes to yield sufficient DNA for nested primer polymerase chain reaction (PCR) analysis. The procedure can be repeated many times with minimal stress to the animal, in contrast to tissue biopsy procedures such as tail cutting. Sample analysis is rapid and straightforward; saliva is applied to sample collection paper and then purified using a solid phase DNA purification system. The paper, containing purified DNA, is added directly to PCR cocktail for the first round of amplification. For weanling mice, in the second round of amplification, a small amount of product from the first round is removed and added to PCR cocktail containing the second set of primers. With adult mice, an adequate volume of saliva may be obtained (dependent upon the sensitivity of the particular reaction) to eliminate the need for second-round amplification with nested primers. This technique is reliable, does not require organic solvents, and is more humane than protocols currently in use. Furthermore, this technique could replace hundreds of thousands of surgical biopsies on rodents annually, which are performed for both transgene determination and genetic monitoring procedures.

Oncogene-induced liver neoplasia in transgenic mice.

Models of hepatocarcinogenesis were generated by directing the expression of SV40 T-antigens, an oncogenic mutant of c-H-ras, or c-myc to the liver of transgenic mice using the albumin enhancer/promoter. The majority of mice carrying the ras transgene (group A) were born with enlarged livers and atypical hepatic architecture, and these all died within several days of birth. The remaining ras transgenic mice (group B) had lower levels of hepatic ras expression, exhibited mild hepatic dysplasia but no liver enlargement, and all ultimately died from development of lung tumors. In contrast, the livers of mice expressing T-antigens were relatively normal at birth, by one month displayed marked dysplasia, and by three to seven months developed multiple nodular adenomas and carcinomas. Myc expression caused mild to severe hepatic dysplasia in young mice, and focal hepatic adenomas in some mice over fifteen months of age. Lines of mice expressing ras (group B), T-antigen, or myc were established and crossed with each other to generate dual transgenic mice expressing oncogene pairs. Each combination resulted in accelerated tumor development, suggesting that these oncoproteins can cooperate with one another during multistep hepatic transformation.

Histopathology associated with elevated levels of growth hormone and insulin-like growth factor I in transgenic mice.

Serum levels of GH and insulin-like growth factor I (IGF-I) were genetically increased to investigate the physiological activities of these proteins. Lines of mice expressing chimeric genes composed of bovine GH, human GRF, or human IGF-I coding sequences fused to the mouse metallothionein I promoter were examined for consequences of chronic exposure to high levels of these peptides. Animals with elevated serum levels of GH (either bovine GH or mouse GH) have selective splanchnomegaly coupled with glomerular sclerosis and hepatocellularmegaly. Serum levels of insulin and cholesterol are increased. In contrast (with the exception of selective enlargement of organs), the chronic expression of IGF-I results in a different pattern of abnormalities. These findings suggest that the pathogenesis of GH-related disorders is not mediated solely by IGF-I.

Expression of human or bovine growth hormone gene with a mouse metallothionein-1 promoter in transgenic swine alters the secretion of porcine growth hormone and insulin-like growth factor-I.

Endocrine profiles were examined in swine that had integrated and expressed a fusion gene consisting of mouse metallothionein-1 (MT) promoter fused to either a human (h) or bovine (b) GH structural gene. Eleven of 18 pigs that had integrated MT-hGH and eight of nine pigs that had integrated MT-bGH expressed the genes. The level of expression varied widely among pigs (14-4551 micrograms/l for MT-hGH and 23-1578 micrograms/l for MT-bGH). The level of expression varied over time within each pig with no general pattern. Concentrations of porcine GH (pGH) were lower in MT-hGH pigs that expressed the gene than in non-expressors or in littermate controls. Insulin-like growth factor-I (IGF-I) concentrations increased with age in all pigs and were raised threefold in pigs expressing either the MT-hGH or MT-bGH genes. Measurement of the foreign GH in samples taken at 15-min intervals failed to reveal any short-term fluctuations in concentration. Administration of hGH releasing factor (GRF) to pigs expressing MT-bGH resulted in attenuated release of pGH compared with that of contemporary controls. Concentrations of bGH did not change after GRF injection. Human and bovine GH expressed in transgenic pigs appear to be biologically active in that they induce IGF-I and suppress endogenous pGH secretion. The failure to find short-term fluctuations and the lack of response to GRF injections are consistent with a non-pituitary and non-GRF regulatable site of production.

Developmental fate of mitochondria microinjected into murine zygotes.

A greater understanding of the fate of mitochondria injected into early preimplantation embryos would provide insights into mitochondrial biology and dynamics associated with development and disease. The ability to introduce foreign mitochondria into mouse embryos provides a means of tracking or following mitochondrial populations in vivo. Previously, injection of foreign mitochondria into the cytoplasm of the zygote was used to produce heteroplasmic mice. However, populations of introduced mitochondria decreased rapidly during development beyond the blastocyst stage. Therefore, the fate of exogenous mitochondria introduced into mouse ova was examined to determine viability and localization in comparison to endogenous mitochondria. Microinjection of murine mitochondria labeled with mitochondria-specific MitoTracker fluorophores allowed evaluation of subsequent viability and functionality of exogenous mitochondria populations in vivo. Characterization of mitochondrial survival and migration following microinjection illustrated toxic effects of MitoTracker Red upon exposure to laser confocal examination. In contrast, mitochondrial-specific fluorophores effectively detected foreign mitochondrial migration post-microinjection. The subsequent viability of the introduced mitochondria was observed through the blastocyst stage. Through the use of mitochondria-specific fluorophores, newly introduced mitochondria were further characterized and tracked post-transfer.

Elevated PC responsive B cells and anti-PC antibody production in transgenic mice harboring anti-PC immunoglobulin genes.

The rearrangement of heavy and light chain immunoglobulin genes is necessary for the production of functional antibody molecules. The myeloma MOPC 167 produces specific antibodies to the antigen phosphorylcholine (PC), which is present on bacterial surfaces, fungi and other environmental contaminants. Rearranged heavy and light chain immunoglobulin genes cloned from MOPC 167 were microinjected into mouse eggs. Within the resulting transgenic mice, expression of the transgenes were limited to lymphoid tissues. Transgenic mice produced elevated levels of anti-PC antibodies constitutively, at 16 days of age, when normal non-transgenic mice were not fully immunocompetent. A triggering antigenic stimulus was not necessary to evoke anti-PC immunoglobulin production. Additionally, the frequency of PC-responsive B cells in these transgenic mice was further increased upon specific immunization.

Progress on gene transfer in farm animals.

Transgenic pigs and sheep have been produced by the microinjection of single-cell zygotes and two-cell ova with linear molecules of mouse metallothionein I (MT) promoter/regulator fused to either the human growth hormone (hGH) or bovine growth hormone (bGH) structural genes. The foreign genes integrated into the chromosomes of 3 of 111 lambs or fetuses and 31 of 341 pigs or fetuses examined. Immunoreactive hGH or bGH was present in the plasma of two transgenic lambs and 19 transgenic pigs. The hGH concentration in plasma varied greatly among pigs and was unrelated to the number of gene copies that had integrated. Rate of growth was not enhanced in any of the transgenic pigs in comparison to their littermate controls. However, bGH and hGH exerted definite biological effects in transgenic pigs as evidenced by significantly depressed backfat measurements, elevated levels of insulin-like growth factor (IGF-I), stimulation of mammary development (by hGH) and reduction in porcine growth hormone (pGH) to nondetectable levels in plasma. Five of six founder transgenic pigs transmitted the MT-hGH gene construct to one or more progeny. Three progeny of a boar that expressed hGH also expressed the foreign gene.

Characterization of transgenic livestock production.

The objective of transgenic livestock improvement projects is to develop and bring to market superior breeding stock, as well as germplasm for the artificial insemination and embryo transfer industries. Livestock animal biotechnology programs hold the promise of achieving, in a single generation, improvements in commercially important livestock species previously possible only through long-term traditional selective breeding practices or by chance mutation. Transgenic farm animals harboring growth hormone or metabolically related structural genes have been created. Studies of these animals demonstrate the effects of inadequate regulation of transgene expression. Research continues to explore the intricacies of developmental regulation of such genes and phenotypic consequences of mammalian gene transfer. Ultimately, genetically engineered livestock will provide producers with the benefit of increased production efficiencies while the consumer will have healthier animal food products. Conceivably, products will be produced with lower levels of fat, cholesterol, feed additives and pharmaceutical residues from animals with altered carcass composition that will result in greater nutritional benefit for the consumer.

Optimization of embryo transfer protocols for mice.

The efficiency of ova transfer and subsequent survivability were explored in this study. The goals of the experiment were to 1) determine the minimum number of ova necessary for pregnancy maintenance, 2) ascertain if the number of zygotes used in ova transfer approaches or exceeds uterine capacity, and 3) establish if location of deposition of ova influences embryo survival. A total of 1647 pronuclear zygotes were transferred in groups of 1, 2, 4, 6, 15 or 25 on Day 1 of gestation either via the oviducal ampulla or ostium to 156 nulliparous ICR pseudopregnant female mice. Pregnancy status was determined on Day 12 or Day 19 of gestation. Results indicated that pregnancy rates were not significantly increased by transferring larger numbers of zygotes (P < 0.1504) and that beyond transfer of 15 zygotes, the progressive increase in fetal numbers per litter declined. However, on Day 19 of gestation, no definitive evidence of limitation of uterine capacity was obtained with the numbers of zygotes transferred (P < 0.0531), and the estimates of numbers of viable and resorbed fetuses differed when determinations were made on Day 12 versus Day 19 of gestation. Mean numbers of developed fetuses per recipient declined (P < 0.0001), whereas the number of resorptions (partially resorbed fetuses or resorption sites) increased (P < 0.0001) over this period, reflecting fetal loss in mid- to late-gestation and possibly the transient nature of resorptions prior to Day 12. Additionally, there was no difference in pregnancy outcome when transferring ova into the oviducal ostium or isthmus (P < 0.5256). Finally, these results illustrated that when large numbers of zygotes were transferred into the oviducal ampulla, equivalent numbers of ova eventually implanted in the uterus; however, proportionally more of them began resorption.

In vitro fertilization in mice: Strain differences in response to superovulation protocols and effect of cumulus cell removal.

Strain differences have proven to be crucial components in mouse in vitro fertilization (IVF) and superovulatory protocols. To maximize the yield of IVF-derived mouse eggs, a series of experiments was conducted using different injection timing intervals for administration of pregnant mare serum gonadotropin (PMSG) and hCG to induce follicular development and ovulation. Strains were chosen that were representative of those commonly used in genetic engineering experimentation. These strains included ICR outbred, C57BL/6 inbred, and B6SJLF1 hybrid (C57BL/6J x SJL/J F1) mice. Females were superovulated using 4 PMSG/hCG/IVF timing regimens (group), with sperm obtained from males of the same strain. Group designations were based on the following PMSG/hCG and hCG/oocyte collection intervals, respectively: Group 1, 55 and 21.5 h; Group 2, 60 and 14.5 h; Group 3, 55 and 14.5 h; Group 4, 48 and 14.5 h. After overnight culture of ova, fertilization rates (development to the 2-cell stage) were assessed. A logistic regression was performed using indicator variables for both strain and group. There was a significant strain influence on ova fertilization rate, based on the coefficients of mouse strain (ICR, beta = -1.1067, P = 8E-17 and C57BL/6, beta = -0.5172, P = 8E-06). Additionally, group affected the proportion of fertilized ova obtained (coefficient of Group 1, beta = -1.3152, P = 0.00 and Group 3, beta = 0.9531, P = 3E-12). From the coefficients for the interaction terms, the effect of groups varies across mouse strain. Therefore, the treatment that produces the highest fertilization rate is related to and contingent upon the strain of mouse. In the second study, the Group 3 protocol was used to evaluate fertilization differences between cumulus-intact and cumulus-free oocytes. Again, there was a significant strain influence on ova fertilization rate based on the coefficients of mouse strain (ICR, beta = -2.6639, P = 0.00; C57BL/6, beta = -2.5114, P = 0.00). However, there was no difference between Cumulus and No Cumulus groups (cumulus coefficient, beta = 0.1640, P = 0.59872), indicating that there was no affect of cumulus presence on fertilization rate. In summary, responses to standardized mouse IVF protocols vary significantly. The efficiency of IVF procedures can be optimized between and within specific mouse strains by the timing of superovulatory regimens. However, absence of cumulus cells during the IVF procedure does not adversely affect fertilization rate.

Regression of induced corpora lutea by human chorionic gonadotropin in prepuberal gilts.

The effect of daily injections of human chorionic gonadotropin (HCG) on luteal maintenance in hysterectomized prepuberal gilts induced to ovulate and in hysterectomized mature gilts was studied. Twenty-four pre-puberal gilts, 120 to 130 d of age, were induced to ovulate with 1,000 IU pregnant mare serum gonadotropin followed 72 h later with 500 IU HCG. Nine of the 24 prepuberal gilts (bred controls) were artificially inseminated on d 0 (d 0 = d after HCG). Mature gilts that had displayed one or more estrous cycles of 17 to 22 d were used (d 0 = onset of estrus). All gilts, except the bred controls, were totally hysterectomized on d 6 to 9 and their corpora lutea (CL) marked with charcoal. From d 10 through 29, eight prepuberal and 10 mature hysterectomized gilts received daily injections of 500 IU HCG in saline while seven prepuberal and eight mature hysterectomized gilts received daily injections of saline vehicle. Jugular blood samples were quantitated by radioimmunoassay for estrogen and 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM), a metabolite of prostaglandin F2 alpha. One bred control gilt was pregnant on d 30, indicating that the prepuberal gilts used in the experiment were prepuberal. All mature gilts and six of seven prepuberal gilts that received saline had maintained CL to d 30. Eight of 10 mature gilts that received HCG had maintained CL to d 30, while only two of eight (P less than .05) prepuberal gilts that received HCG maintained CL to d 30. All gilts receiving HCG had numerous follicles and accessory luteal structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum concentrations of pituitary and adrenal hormones in female pigs exposed to two photoperiods.

Serum concentrations of pituitary and adrenal hormones were determined in lactating sows and ovariectomized (OVX) gilts exposed to 8 h (8L:16D) or 16 h of light (16L:8D). In addition serum prolactin (PRL) concentrations were determined after a thyrotropin releasing hormone (TRH) challenge. At 103 +/- 2 d of gestation or 3 wk after ovariectomy of nulliparous gilts on d 7 to 9 of the estrous cycle (d - 10), blood samples were collected from jugular vein cannulae at 30-min intervals for 8 h beginning at 0800 h. Immediately after the last sample, 13 sows and five OVX gilts were assigned to 8L:16D and 14 sows and five OVX gilts were assigned to 16L:8D/d and placed in two identical chambers in the farrowing house. Blood sampling was repeated on d 7, 14 and 21 of lactation in the sows and on d 7, 14, 21 and 28 in the OVX gilts. In Exp. 1, serum cortisol (C) concentrations were similar for sows exposed to 8L:16D (n = 7) and 16L:8D (n = 6) treatments, whereas in Exp. 2, serum C concentrations for sows exposed to 8L:16D (n = 6) were lower than those exposed to 16L:8D (n = 6) on d 7, 14 and 21. Photoperiod failed to influence serum concentrations of PRL, luteinizing hormone (LH) and growth hormone in the lactating sows or PRL in the OVX gilts. Photoperiod also failed to affect mean basal serum concentrations, peak height and peak frequency for PRL and LH in the lactating sows or for PRL in the OVX gilts.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum luteinizing hormone and estrogen profiles before puberty in the gilt.

The hypothesis that an increase in serum luteinizing hormone (LH) concentration occurs before the onset of puberty in the gilt was tested. Blood samples were collected from 14 gilts, 166 d of age and 72.6 +/- 1.8 kg body weight, every 15 min for 8 h 1 d after jugular cannulation. Gilts were checked daily for estrus with a boar. The sampling procedure was repeated 21 and 33 d later, by which time three gilts had attained puberty (first estrus with continued regular estrous cycles), and then every 7 d thereafter until an additional five gilts attained puberty. Serum LH and estrogen (E) concentrations were quantitated by radioimmunoassay. Bleeding periods were grouped into seven common physiological ages before puberty; age 1 greater than or equal to 57 d (n = 4), age 2 = 43 to 50 d (n = 5), age 3 = 31 to 38 d (n = 4), age 4 = 22 to 26 d (n = 5), age 5 = 17 to 19 d (n = 4), age 6 = 10 to 12 d (n = 5) and age 7 = 3 to 5 d (n = 5). Mean serum LH concentration (ng/ml, mean +/- SE) at age 7 (1.0 +/- .1) was greater (P less than .01) than at ages 2, 3, 4 and 5 (6 +/- .1) but not ages 1 and 6 (.8 +/- .1).(ABSTRACT TRUNCATED AT 250 WORDS)

Production of functional human hemoglobin in transgenic swine.

A construct containing the locus control region (LCR) from the human beta globin locus together with two copies of the human alpha 1 gene and a single copy of the human beta A gene was used to obtain three transgenic pigs. The transgenic pigs are healthy, not anemic, and grow at a rate comparable to non-transgenic littermates. All animals expressed the human genes. However, alpha globin was consistently expressed at higher levels than beta globin. Isolation of the human hemoglobin from both porcine hemoglobin and other non-hemoglobin proteins was accomplished by ion exchange chromatography. The purified porcine derived human hemoglobin exhibited an oxygen affinity similar to that of human derived human hemoglobin.