Coordinated patterns of gene expressions for adult muscle build-up in transgenic mice expressing myostatin propeptide.

BACKGROUND: Skeletal muscle growth and maintenance are essential for human health. One of the muscle regulatory genes, namely myostatin, a member of transforming growth factor-beta, plays a dominant role in the genetic control of muscle mass. Myostatin is synthesized as a precursor protein, which generates the N-terminal propeptide and the C-terminal mature myostatin peptide by a post-translational cleavage event. Previously, transgenic over-expression of myostatin propeptide in skeletal muscle results in significant muscle growth in early stages of development. The objectives of present study were to further characterize muscle growth in later stages of life and to identify genes and their expression patterns that are responsible for adult muscle build-up by myostatin propeptide. RESULTS: Immunohistochemical staining with an antibody to the N-terminus indicates a high level of myostatin propeptide present in the muscles of transgenic mice while there were no apparent differences in myostatin protein distribution in the muscle fibers between the transgenic and wild-type mice. Main individual muscles increased by 76-152% in the transgenic mice over their wild-type littermate mice at 12 months of age. A large number of nuclei were localized in the central and basal lamina of the myofibers in the transgenic mice as the number of nuclei per fiber and 100 microm(2) area was significantly higher in transgenic mice than wild-type mice. By systemic comparisons of global mRNA expression patterns between transgenic mice and wild-type littermates using microarray and qRT-PCR techniques, we have identified distinct gene expression patterns to support adult muscle build-up by myostatin propeptide, which are comprised of enhanced expressions of myogenic regulatory factors and extracelullar matrix components, and differentially down-regulated expressions of genes related to protein degradation and mitochondrial ATP synthesis. CONCLUSION: The results present a coordinated pattern of gene expressions for reduced energy utilization during muscle build-up in adult stage. Enhanced muscle buildup by myostatin propeptide is sustained by reduced ATP synthesis as a result of a decreased activity of protein degradation. Myostatin propeptide may have a therapeutic application to the treatment of clinical muscle wasting problems by depressing myostatin activity.

Engineering disease resistant cattle.

Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk. Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis causing organisms. Care will be taken to avoid altering milk's nutritional and manufacturing properties. Multi-cistronic constructs may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species.

Transgenic expression of myostatin propeptide prevents diet-induced obesity and insulin resistance.

Obesity and insulin resistance cause serious consequences to human health. To study effects of skeletal muscle growth on obesity prevention, we focused on a key gene of skeletal muscle named myostatin, which plays an inhibitory role in muscle growth and development. We generated transgenic mice through muscle-specific expression of the cDNA sequence (5'-region 886 nucleotides) encoding for the propeptide of myostatin. The transgene effectively depressed myostatin function. Transgenic mice showed dramatic growth and muscle mass by 9 weeks of age. Here we reported that individual major muscles of transgenic mice were 45-115% heavier than those of wild-type mice, maintained normal blood glucose, insulin sensitivity, and fat mass after a 2-month regimen with a high-fat diet (45% kcal fat). In contrast, high-fat diet induced wild-type mice with 170-214% more fat mass than transgenic mice and developed impaired glucose tolerance and insulin resistance. Insulin signaling, measured by Akt phosphorylation, was significantly elevated by 144% in transgenic mice over wild-type mice fed a high-fat diet. Interestingly, high-fat diet significantly increased adiponectin secretion while blood insulin, resistin, and leptin levels remained normal in the transgenic mice. The results suggest that disruption of myostatin function by its propeptide favours dietary fat utilization for muscle growth and maintenance. An increased secretion of adiponectin may promote energy partition toward skeletal muscles, suggesting that a beneficial interaction between muscle and adipose tissue play a role in preventing obesity and insulin resistance.

Comparison of gene expression in individual preimplantation bovine embryos produced by in vitro fertilisation or somatic cell nuclear transfer.

In vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) have been implicated in a variety of developmental abnormalities. Aberrant gene expression is likely to account for much of the diminished viability and developmental abnormalities observed. In the present study, the expression of multiple genes in IVF and SCNT bovine blastocyst-stage embryos were evaluated and compared with in vivo-produced embryos. Eleven genes expressed at and following maternal-zygotic transcription transition were evaluated in individual blastocysts by real-time polymerase chain reaction following RNA amplification. A subset of those genes was also evaluated in individual IVF and SCNT eight-cell embryos. A fibroblast-specific gene, expressed by nuclear donor cells, was also evaluated in IVF and SCNT embryos. The observed gene expression pattern at the eight-cell stage was not different between IVF and SCNT embryos (P > 0.05). In vitro fertilisation and SCNT blastocyst expression was lower (P < 0.01) for all genes compared with their in vivo-produced counterparts, except for lactate dehydrogenase isoenzyme A (P < 0.001). The patterns of gene expression of the IVF and SCNT blastocysts were indistinguishable. Neither SCNT eight-cell nor blastocyst-stage embryos expressed the gene used as a fibroblast marker (collagen VIalpha1). For the genes evaluated, the level of expression was influenced more by the environment than by the method used to produce the embryos. These results support the notion that if developmental differences observed in IVF- and SCNT-produced fetuses and neonates are the result of aberrant gene expression during the preimplantation stage, those differences in expression are subtle.

Genetically enhanced cows resist intramammary Staphylococcus aureus infection.

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

Expression of lysostaphin in milk of transgenic mice affects the growth of neonates.

As an initial step towards enhancing mastitis resistance in dairy animals, we generated BLG-Lys transgenic mice that secrete lysostaphin, a potent antistaphylococcal protein, in their milk. In the current study, we continue our assessment of lysostaphin as a suitable antimicrobial protein for mastitis resistance and have investigated mammary gland development and function in three lines of transgenic mice. As the lines were propagated, there was a tendency for fewer BLG-Lys litters to survive to weaning (51% as compared to 90% for nontransgenic lines, p = 0.080). Nontransgenic pups fostered on dams from these three lines exhibited diminished growth rates during the first week of lactation. Rates of gain became comparable to pups on nontransgenic dams at later time points. Initial slow growth also resulted in decreased weaning weights for pups nursed by transgenic dams (15.35 +/- 0.27 g) when compared to pups delivered and nursed by nontransgenic dams (18.61 +/- 0.61 g; p < 0.001), but the effect was temporary, as similar weights were attained by adulthood. Milk yield at peak lactation was not different between BLG-Lys (0.79 +/- 0.33 g) and nontransgenic (0.91 +/- 0.38 g; p = 0.166) dams. Histological examination of the transgenic mammary glands during gestation revealed no differences when compared to control glands; however, at early lactational stages, the BLG-Lys glands exhibited less alveolar area than control glands and a delay in lobulo-alveolar maturation. The results clearly demonstrate reduced growth of neonates on BLG-Lys dams; whether the poor pup performance can be attributed to delayed mammary development or the gland development merely reflects reduced suckling stimuli from the pups remains to be determined.

Molecular characterization of lysozyme type II gene in rainbow trout (Oncorhynchus mykiss): evidence of gene duplication.

Rainbow trout (Oncorhynchus mykiss) have two types of lysozyme. Type II lysozyme differs from type I by only one amino acid, but only type II lysozyme has significant bactericidal activity. Due to this novel antibacterial property, lysozyme type II appears to be a candidate gene for enhancing disease resistance in fish as well as livestock species. Using polymerase chain reaction the lysozyme type II gene was amplified from genomic DNA isolated from rainbow trout. Two amplified fragments of 2041 and 2589 bp were observed. Sequencing revealed both amplicons were lysozyme genes having nearly identical nucleotide sequences, except the longer fragment has 548 base pairs inserted in intron 2 at nucleotide position 513 and a few point mutations within intron 2. Both versions of trout lysozyme type II gene were comprised of four exons and three introns. We also demonstrated that trout lysozyme is most likely encoded by these two different genes.