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.

Xenomitochondrial mice: investigation into mitochondrial compensatory mechanisms.

Xenomitochondrial mice, harboring evolutionarily divergent Mus terricolor mitochondrial DNA (mtDNA) on a Mus musculus domesticus nuclear background (B6NTac(129S6)-mt(M. terricolor)/Capt; line D7), were subjected to molecular and phenotypic analyses. No overt in vivo phenotype was identified in contrast to in vitro xenomitochondrial cybrid studies. Microarray analyses revealed differentially expressed genes in xenomitochondrial mice, though none were directly involved in mitochondrial function. qRT-PCR revealed upregulation of mt-Co2 in xenomitochondrial mice. These results illustrate that cellular compensatory mechanisms for mild mitochondrial dysfunction alter mtDNA gene expression at a proteomic and/or translational level. Understanding these mechanisms will facilitate the development of therapeutics for mitochondrial disorders.

Development and initial characterization of xenomitochondrial mice.

Xenomitochondrial mice harboring trans-species mitochondria on a Mus musculus domesticus (MD) nuclear background were produced. We created xenomitochondrial ES cell cybrids by fusing Mus spretus (MS), Mus caroli (MC), Mus dunni (Mdu), or Mus pahari (MP) mitochondrial donor cytoplasts and rhodamine 6-G treated CC9.3.1 or PC4 ES cells. The selected donor backgrounds reflected increasing evolutionary divergence from MD mice and the resultant mitochondrial-nuclear mismatch targeted a graded respiratory chain defect. Homoplasmic (MS, MC, Mdu, and MP) and heteroplasmic (MC) cell lines were injected into MD ova, and liveborn chimeric mice were obtained (MS/MD 18 of 87, MC/MD 6 of 46, Mdu/MD 31 of 140, and MP/MD l of 9 founder chimeras, respectively). Seven MS/MD, 1 MC/MD, and 11 Mdu/MD chimeric founder females were mated with wild-type MD males, and 18 of 19 (95%) were fertile. Of fertile females, only one chimeric MS/MD (1% coat color chimerism) and four chimeric Mdu/MD females (80-90% coat color chimerism) produced homoplasmic offspring with low efficiency (7 of 135; 5%). Four male and three female offspring were homoplasmic for the introduced mitochondrial backgrounds. Three male and one female offspring proved viable. Generation of mouse lines using additional female ES cell lineages is underway. We hypothesize that these mice, when crossbred with neurodegenerative-disease mouse models, will show accelerated age-related neuronal loss, because of their suboptimal capacity for oxidative phosphorylation and putatively increased oxidative stress.

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.

Gestational, pathologic and biochemical differences between very long-chain acyl-CoA dehydrogenase deficiency and long-chain acyl-CoA dehydrogenase deficiency in the mouse.

Although many patients have been found to have very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, none have been documented with long-chain acyl-CoA dehydrogenase (LCAD) deficiency. In order to understand the metabolic pathogenesis of long-chain fatty acid oxidation disorders, we generated mice with VLCAD deficiency (VLCAD(-/-)) and compared their pathologic and biochemical phenotypes of mice with LCAD deficiency (LCAD(-/-)) and wild-type mice. VLCAD(-/-) mice had milder fatty change in liver and heart. Dehydrogenation of various acyl-CoA substrates by liver, heart and skeletal muscle mitochondria differed among the three genotypes. The results for liver were most informative as VLCAD(-/-) mice had a reduction in activity toward palmitoyl-CoA and oleoyl-CoA (58 and 64% of wild-type, respectively), whereas LCAD(-/-) mice showed a more profoundly reduced activity toward these substrates (35 and 32% of wild-type, respectively), with a significant reduction of activity toward the branched chain substrate 2,6-dimethylheptanoyl-CoA. C(16) and C(18) acylcarnitines were elevated in bile, blood and serum of fasted VLCAD(-/-) mice, whereas abnormally elevated C(12) and C(14) acylcarnitines were prominent in LCAD(-/-) mice. Progeny with the combined LCAD(+/+)//VLCAD(+/-) genotype were over-represented in offspring from sires and dams heterozygous for both LCAD and VLCAD mutations. In contrast, no live mice with a compound LCAD(-/-)//VLCAD(-/-) genotype were detected.

Membrane cofactor protein (MCP; CD46) expression in transgenic mice.

Human membrane cofactor protein (MCP; CD46) is a widely distributed complement regulator. In the mouse, expression of MCP is largely restricted to the testis while a related, widely expressed protein (Crry) appears to perform MCP's (CD46) regulatory activity. We have developed two mouse strains transgenic for human MCP (CD46) utilizing an approximately 400 kb YAC clone carrying the complete gene. A third mouse strain was generated using an overlapping YAC clone isolated from a second library. The expression of human MCP (CD46) in these mouse strains was characterized by immunohistochemistry, FACS, Western blotting and RT-PCR. No differences were detected in the isoform pattern or distribution among the three strains, although the expression level varied according to how many copies of the gene were integrated. The expression profile closely mimicked that observed in humans, including the same pattern of isoform expression as the donor. In addition, tissue-specific isoform expression in the kidney, salivary gland and brain paralleled that observed in man. The transgenic mice expressed low levels of MCP (CD46) on their E, in contrast to humans but in line with most other primates. These mice should be a useful tool to analyse tissue-specific expression, to establish animal models of infections and to characterize the role of MCP (CD46) in reproduction.

Transgenic studies of fatty acid oxidation gene expression in nonobese diabetic mice.

Type 1 diabetes mellitus is a devastating disorder affecting both glucose and lipid metabolism. Using the nonobese diabetic (NOD) mouse model, we found that diabetic mice had a liver-specific increase in steady state mRNA levels for enzymes involved in oxidation of fatty acids. Increased mRNA abundance was observed in very long-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase (LCAD), medium-chain acyl-CoA dehydrogenase (MCAD), carnitine palmitoyltransferase I (CPT-1a), and the gluconeogenic enzyme phosphoenolpyruvate carboxykinase, whereas short-chain acyl-CoA dehydrogenase mRNA remained unchanged. In contrast, minimal elevations in LCAD and CPT-1a mRNA were observed in hearts of diabetic mice with no significant differences found for the other enzymes. We developed NOD mice with transgenes containing regulatory elements of human MCAD gene controlling a reporter gene to determine if the increase in MCAD gene expression occurred via the well-characterized nuclear receptor response element (NRRE-1). These results demonstrated that the transgene containing the NRRE-1 and adjacent 5' sequences had elevated liver expression in diabetic mice compared with prediabetic or normal control mice. Surprisingly, the transgene that contains NRRE-1 with adjacent 3' sequences and the transgene with the NRRE-1 deleted showed minimal response to the fulminant diabetic condition.Collectively, these results indicate that in type 1 diabetes there exists an excessive and liver-specific activation of fatty acid oxidation gene expression. Using human MCAD as a prototype gene, we have shown that this increased expression is mediated at the transcriptional level but does not occur via the well-characterized NRRE-1 site responsible for baseline expression in normal mice.

The C(H)1 and transmembrane domains of mu in the context of a gamma2b transgene do not suffice to promote B cell maturation.

Mice carrying a gamma2b transgene have been shown previously to be deficient in B cell development. In particular, a developmental block exists at the pre-B cell stage. The few B cells that develop all express endogenous micro heavy chains. The phenotype suggests that gamma2b exerts a strong feedback inhibition on endogenous Ig gene rearrangement, but, unlike micro, cannot support further B cell development. In this study we have created hybrid transgenes between gamma2b and micro. Transgenic mice with a C(H)1 domain of micro, or both a C(H)1 and transmembrane/cytoplasmic domain of micro replacing the respective domains of a gamma2b transgene, have the same B cell defect as gamma2b transgenic mice. Interestingly, the severity of the defect is correlated with the level of expression of the transgene, suggesting that the degree of feedback inhibition of Ig gene rearrangement depends on the level and timing of Ig production. Crossing the gamma2b/micro transgenes into a Bcl-x(L) transgenic line allows immature gamma2b B cells to survive, but not to develop to maturity. Therefore, the missing function of micro is not simply an anti-apoptotic effect.

Isolation and microinjection of somatic cell-derived mitochondria and germline heteroplasmy in transmitochondrial mice.

At present, there are no means for creation of relevant animal models of human mitochondrial DNA (mtDNA)-based diseases in a directed fashion. As an initial step towards this end, we have developed a microinjection technique for transfer of isolated, viable mitochondria between two mouse species. Previously, we reported detection, by nested PCR with species-specific primer sets, of Mus spretus mtDNA in Mus musculus domesticus blastocyts following zygote microinjection and culture. We now report the production of transmitochondrial founder mice, and germline transmission of the heteroplasmic state in a maternal lineage. Heteroplasmic mice produced by this technique will be useful in the study of mitochondrial dynamics and may hasten the creation of animal models of human mtDNA-based diseases.

Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation.

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/- mice yielded an abnormally low number of LCAD +/- and -/- offspring, indicating frequent gestational loss. LCAD -/- mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD -/- males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD -/- mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.

Functional correction of short-chain acyl-CoA dehydrogenase deficiency in transgenic mice: implications for gene therapy of human mitochondrial enzyme deficiencies.

We report the therapeutic effects of liver-specific expression of a short-chain acyl-CoA dehydrogenase (SCAD) transgene in the SCAD-deficient mouse model. Transgenic mice were produced with a rat albumin promoter/enhancer driving a mouse SCAD minigene (ALB-SCAD) on both the SCAD normal genetic background and a SCAD-deficient background. In three transgenic lines produced on the SCAD-deficient background, recombinant SCAD activity and antigen in liver mitochondria were found up to 7-fold of normal control values. All three lines showed a markedly reduced organic aciduria and fatty liver, which are sensitive indicators of the metabolic abnormality seen in this disease found in children. We found no detrimental effects of high liver SCAD expression in transgenic mice on either background. These studies provide important basic and practical therapeutic information for the potential gene therapy of nuclear-encoded mitochondrial enzyme deficiencies, as well as insights into the mechanisms of the disease.

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.

Mitochondria transfer into mouse ova by microinjection.

A method for mitochondria isolation and interspecific transfer of mitochondria was developed in mice. Mitochondria were isolated from Mus spretus liver samples for microinjection into fertilized ova obtained from superovulated M. musculus domesticus females. Electron microscopic observations of mitochondria preparations used for microinjection demonstrated intact mitochondrial vesicles with little microsomal contamination. Species-specific nested PCR primers complementary to sequence differences in the mitochondrial DNA D-loop region revealed high rates of successful transfer of foreign mitochondria after isolation and injection into zygotes cultured through the blastocyst stage of embryonic development. Of 217 zygotes, 67 survived mitochondria injection and 23 out of 37 zygotes developed were at the blastocyst-stage of embryonic development after 4.5 days of in vitro culture. All 23 of these blastocysts contained detectable levels of foreign mitochondria. These results represent an initial step in developing a model system to study mitochondrial dynamics and development of therapeutic strategies for human metabolic diseases affected by aberrations in mitochondrial function or mutation.

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.

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.

Liver, renal and subcutaneous histopathology in PEPCK-bGH transgenic pigs.

Transgenic pigs were created that harboured a phosphoenolpyruvate carboxykinase-bovine growth hormone construct (PEPCK-bGH). Four founder animals and two transgenic offspring from one line were evaluated between 6 1/2 and 12 months of age. There was no evidence of severe hepatic or renal lesions in these pigs, which characterised transgenic PEPCK-bGH mice previously described. While glomerular and tubular lesions in kidney sections were not identified in the transgenic pigs, mesangial cell proliferation was observed in two transgenic offspring from a single line. Additionally, glomerular size was significantly increased in four of four puberal transgenic swine when compared to age- and sex-matched controls (28.30 +/- 4.1 vs. 14.2 +/- 2.7 x 10(5) microns 3; representing 3 transgenic lines, p < 0.05). Surprisingly, no mature adipocytes were observed in subcutaneous sections obtained in transgenic GH pigs. Histological evaluation of these transgenic pigs further illustrates the requirement for precise control of growth-related genes and their protein products.

Transgenic mice expressing a chimaeric anti-E. coli immunoglobulin alpha heavy chain gene.

To induce constitutive immunity against a pathogenic strain of Escherichia coli (K99), a rearranged immunoglobulin (Ig) heavy chain (HC) gene was constructed. Because the route of E. coli infection is enteric, an IgA transgene was desirable. A chimaeric gene construct was cloned that coded for a HC that recognized a specific E. coli pilus antigen. The construct comprised a kappa gene promoter, murine VDJ, and bovine alpha-HC constant region. Following microinjection of the HC construct into murine zygotes, of 50 liveborn mice, three were identified as transgenic. In all three transgenic founders, transgene-encoded mRNA expression was detected by northern blot. The transgenic founders were analysed for transgene-encoded RNA expression in splenic tissue before and after challenge with pathogenic E. coli. Founder 4-3 male expressed transgene-encoded RNA both before and after challenge; expression was detected in the other two founders only post-challenge. As no differences were found when sera were analysed for bovine IgA in control and transgenic mice, protein expression was assessed by challenge of HC founders with K99 E. coli by gavage. Control mice challenged with K99 E. coli were moribund within 24 h post-gavage, but there was no observable affect in the three transgenic founders. Unfortunately, after obtaining offspring from all founders, no transgenic offspring were identified (0/108). The low yield of transgenic founders, coupled with the apparent germ-line mosaicism may point to either mechanical or critical developmental anomalies.(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.

A strain-specific modifier on mouse chromosome 4 controls the methylation of independent transgene loci.

A transgene, pHRD, is highly methylated in 12 independent mouse lines when in a C57BL/6 strain background, but becomes progressively less methylated when bred into a DBA/2 background. Transgenes inherited from the mother are generally more methylated; however, this parental effect disappears following continued breeding into the nonmethylating strain. Mapping experiments using BXD recombinant inbred mice as well as other inbred strains indicate that a single strain-specific modifier (Ssm-1) linked to, but distinct from, Fv-1 is responsible for the strain effect. In addition to the methylated and unmethylated transgenic phenotypes, certain mice exhibit a partial methylation pattern that is a consequence of an unusual cellular mosaicism. The pHRD transgene, containing target sequences for the V(D)J recombinase, undergoes site-specific recombination only in lymphoid tissues. This V-J joining is restricted primarily to unmethylated transgene copies.