Paul E. Mozdziak

Development of transgenic chickens expressing bacterial β‐galactosidase

Replication‐defective retroviral vectors are efficient vehicles for the delivery of exogenous genes, and they may be used in the generation of transgenic animals. The replication‐defective retroviral SNTZ vector carrying the lacZ gene with a nuclear localized signal was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the lacZ gene by using the polymerase chain reaction. Eight of 15 male chickens that survived to sexual maturity contained the lacZ gene in their semen. Subsequently, these males were mated with wild‐type female chickens. From one of the eight lacZ‐positive G0 males, two lacZ‐positive male chickens were produced from a total of 224 G1 progeny for a germline transmission rate of 0.89%. Both G1 male chickens carrying the lacZ gene were mated with wild‐type female chickens and 46.5% of the G2 progeny contained the lacZ gene, which is consistent with the expected Mendelian 50% ratio for a heterozygous dominant allele. The product of the lacZ gene, nuclear localized β‐galactosidase, was expressed in primary myoblast cultures derived from G2 chickens, and it was also expressed in whole G2 chicken embryos. Developmental Dynamics, 2003.

Status of Transgenic Chicken Models for Developmental Biology

The chick embryo is a classic model that has been used to gain insight into developmental processes and cell fate within the embryo for over a century. For the most part, investigators have implanted quail cells into a chicken embryo. A more powerful tool for developmental biology research than the quail:chick chimera system would be to have lines of transgenic chickens expressing reporter genes that are readily available to the research community. However, avian transgenic technology has been fraught with technical difficulties, and transgenic chickens expressing reporter genes have only recently been developed. The goal of this review is to report the technologies that have been used to generate transgenic chickens and to discuss the challenges in generating avian transgenics for developmental biology research. Developmental Dynamics 229:414–421, 2004.

Hyperammonemia in cirrhosis induces transcriptional regulation of myostatin by an NF-κB–mediated mechanism

Significance Loss of skeletal muscle mass, or sarcopenia, is nearly universal in cirrhosis and adversely affects the outcome of these patients. There are no established therapies to prevent or reverse sarcopenia because the mechanisms are not known. We show that the expression of myostatin, a negative regulator of skeletal muscle mass, is increased in the cirrhotic muscle and is mediated by increased ammonia concentration. Skeletal muscle ammonia concentrations are significantly increased in cirrhosis, resulting in activation of the transcription factor NF-κB, which in turn increases the expression of myostatin. Given the high prevalence of cirrhosis, these studies are of broad general interest because ammonia-lowering strategies, NF-κB antagonists, and myostatin blocker are potential therapies to reverse sarcopenia of cirrhosis. Loss of muscle mass, or sarcopenia, is nearly universal in cirrhosis and adversely affects patient outcome. The underlying cross-talk between the liver and skeletal muscle mediating sarcopenia is not well understood. Hyperammonemia is a consistent abnormality in cirrhosis due to impaired hepatic detoxification to urea. We observed elevated levels of ammonia in both plasma samples and skeletal muscle biopsies from cirrhotic patients compared with healthy controls. Furthermore, skeletal muscle from cirrhotics had increased expression of myostatin, a known inhibitor of skeletal muscle accretion and growth. In vivo studies in mice showed that hyperammonemia reduced muscle mass and strength and increased myostatin expression in wild-type compared with postdevelopmental myostatin knockout mice. We postulated that hyperammonemia is an underlying link between hepatic dysfunction in cirrhosis and skeletal muscle loss. Therefore, murine C2C12 myotubes were treated with ammonium acetate resulting in intracellular concentrations similar to those in cirrhotic muscle. In this system, we demonstrate that hyperammonemia stimulated myostatin expression in a NF-κB–dependent manner. This finding was also observed in primary murine muscle cell cultures. Hyperammonemia triggered activation of IκB kinase, NF-κB nuclear translocation, binding of the NF-κB p65 subunit to specific sites within the myostatin promoter, and stimulation of myostatin gene transcription. Pharmacologic inhibition or gene silencing of NF-κB abolished myostatin up-regulation under conditions of hyperammonemia. Our work provides unique insights into hyperammonemia-induced myostatin expression and suggests a mechanism by which sarcopenia develops in cirrhotic patients.

Species variations in cDNA sequence and exon splicing patterns in the extensible I-band region of cardiac titin: relation to passive tension

473 Titin is believed to playa major role in passive tension development in cardiac muscle . The eDNA sequence of cardiac titin in the I-band sarcomeric region was determined for several mammalian species. Contiguous sequences of 3749, 12,230, 6602, and 11,850 base pairs have been obtained for the rat N2B, rat N2BA, dog N2B, and dog N2BA isoforms respectively. The length of the PEVK region of the N2B isoform did not correlate with rest tension properties since the only species showing an altered length was the dog that expressed a shorter form . No differences were found between the N2B PEVK lengths in ventricular and atrial muscle. New N2BA splicing pathways in the first tandem Ig region were found in human and dog cardiac muscle . Most of the rat and dog sequences were 8595% identical with the reported human sequence. However, the N2B unique amino acid sequences of rat and dog were only 51 and 67% identical to human. The rat N2B unique sequence was 526 amino acids in length compared to 572 in human. The difference in length was due to deletion of amino acid segments from six different regions of the N2B unique domain. Patterns of PEVK exon expression were also much different in the dog, human, and rat. Six separate dog N2BA PEVK clones were sequenced, and all had different exon splice combinations yielding PEVK lengths ranging from 703 to 900 amino acids. In contrast a rat N2BA clone had a PEVK length of 525 amino acids , while a human clone had an 908 amino acid PEVK segment. Thus, in addition to the higher proportion of the shorter N2B isoform found in rat compared with dog cardiac muscle observed previously, shorter N2B unique and N2BA PEVK segments may also contribute to the greater passive tension in cardiac muscle from rats .

Satellite cells express distinct patterns of myogenic proteins in immature skeletal muscle

Satellite cells are the myogenic cells lying between the myofiber sarcolemma and basal lamina. The objective of this study was to determine the expression patterns of MyoD, myogenin, and Pax7 within the satellite cell population in the growing rat soleus and extensor digitorum longus (EDL) muscles. Secondly, the expression of the myogenic markers was also studied within the interstitial cell compartment and myonuclei. It was discovered that the soleus contained a higher number of Pax7, MyoD, or myogenin‐positive nuclei compared with the EDL. Similarly, myogenin was expressed at a lower level in the myonuclei of the soleus compared with the EDL, and myogenin was expressed at a higher level in the interstitial compartment of the soleus compared with the EDL. When interstitial nuclei, myonuclei, and double‐labeled nuclei were used in the estimate of the satellite cell population, it was discovered that approximately of 13% of the myofibers in a transverse section of the soleus muscle and 4.1% of EDL myofibers exhibit a labeled satellite cell nucleus. Overall, results from this study suggest that expression patterns of these markers vary predictably among muscles with different growth dynamics and phenotypic characteristics. Developmental Dynamics 235:3230–3239, 2006.

The effect of in vivo and in vitro irradiation (25 Gy) on the subsequent in vitro growth of satellite cells

Abstract.The effect of in vivo and in vitro irradiation on subsequent satellite cell growth, in vitro, was investigated to ascertain the ability of a 25 Gy dose to inhibit satellite cell proliferation. Satellite cells were isolated from the left (irradiated) and right (non-irradiated) Pectoralis thoracicus of two-week-old tom turkeys 16 h (n=3) and seven weeks (n=2) after the left Pectoralis thoracicus had been irradiated (25 Gy). Satellite cells isolated from the irradiated and non-irradiated muscles exhibited similar (P>0.10) in vitro proliferation indicating that a population of satellite cells survived an in vivo dose of 25 Gy. In additional experiments, satellite cell cultures derived from tom turkey Pectoralis thoracicus were irradiated (25 Gy) in vitro. The number of satellite cells did not (P>0.05) increase in irradiated cultures for 134 h following irradiation, while satellite cells in non-irradiated cultures proliferated (P<0.05) over this time. At later time periods, satellite cell number increased (P<0.05) in irradiated cultures indicating that a population of satellite cells survived irradiation. The results of these in vitro experiments suggest that a 25 Gy dose of irradiation does not abolish satellite cell divisions in the turkey Pectoralis thoracicus.

Quantitation of Satellite Cell Proliferation in Vivo Using Image Analysis

A nonisotopic, double fluorescence technique was developed to study myogenic satellite cell proliferation in posthatch turkey skeletal muscle. Labeled satellite cell nuclei were identified on enzymatically isolated myofiber segments using a mouse monoclonal antibody (anti-BrdU) followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibody. Myofiber nuclei (myonuclei+satellite cell nuclei) were counterstained with propidium iodide (PI). The myofiber segment length, myofiber segment diameter, and the number of PI and FITC labeled nuclei contained in each segment was determined using a Nikon fluorescence microscope, a SIT video camera and Image-1 software. Data collected by three different operators of the image analysis system revealed 5.0 +/- 1.4 satellite cell nuclei per 1000 myofiber nuclei and 5284 +/- 462 microns3 of cytoplasm surrounding each myofiber nucleus in the pectoralis thoracicus of 9-week-old tom turkeys. BrdU immunohistochemistry coupled with the new approach of PI staining of whole myofiber mounts is an effective combination to allow the use of an efficient semi-automated image analysis protocol.

Superior preservation of the staphylococcal glycocalyx with aldehyde-ruthenium red and select lysine salts using extended fixation times.

The utility of lysine‐based aldehyde‐ruthenium red fixatives for the preservation and/or staining of the fibrous staphylococcal glycocalyx was improved by substitution of alternative forms of lysine for the free amino form. Paraformaldehyde‐glutaraldehyde fixatives containing alternative lysines, with or without ruthenium red, were compared at short 20‐minute prefixation times and at extended overnight fixation times. Although inclusion of paraformaldehyde made longer fixation times possible, the length of time for “safe” fixation varied per sample and could not be predicted. All alternative lysine forms permitted fixation of at least 24 hours without sample loss. The L‐lysine monohydrochloride or L‐lysine acetate forms permitted longer fixation times than the L‐lysine free amino form, and they had comparable or better preservation of the staphylococcal glycocalyx. Thus, the usefulness of aldehyde‐lysine‐based fixatives with minor changes has been enhanced. Microsc. Res. Tech. 41:291–297, 1998.

The incredible, edible, and therapeutic egg

The domestic fowl has a long and unique history, serving multiple purposes in society and science. A cursory review of the Nobel Prize awards in physiology or medicine since 1901 points to the significance of birds, and the chicken in particular, as the premier nonmammalian vertebrate animal model (see www.fbresearch.org/education/nobels.htm and http://nobelprize.org/nobel_prizes/medicine/laureates). The domestic fowl aided in the discovery of essential vitamins and gave the first clue to differences between T and B cells. In fact, B cell nomenclature is based on the origin of B cells from the avian bursa of Fabricius. In addition, the chicken model provided the foundation for understanding the chemical processes for vision, insights into animal behavior, and our first introduction to tumor viruses [e.g., Rous sarcoma virus (RSV)] and the cellular origin of retroviral oncogenes. Even today, avian oncogenic viruses provide valuable models for human disease. Furthermore, for many developmental biologists, the avian embryo remains the premier animal model (1). On the practical side, the general public is protected from yearly influenza outbreaks through vaccine production in chicken eggs. In addition to its scientific and biomedical importance, poultry as an agricultural commodity has grown over the last 60 years into a global industry providing billions of people with inexpensive high-quality animal protein in the form of meat and eggs. Much of the success of the poultry industry is directly related to the application of population genetics for the selection of commercial lines for efficient protein production (2). Today, estimates of the cost of egg production in the U.S. hover around 5 cents per egg. Given that the albumin from a single egg contains ≈3.6 g of protein, the domestic laying hen is a very efficient protein bioreactor. Now, with the report of Lillico et al. (3) in this issue of …

PARAFORMALDEHYDE EFFECT ON RUTHENIUM RED AND LYSINE PRESERVATION AND STAINING OF THE STAPHYLOCOCCAL GLYCOCALYX

The utility of lysine in glutaraldehyde—ruthenium red fixatives for the preservation and/or staining of the fibrous staphylococci glycocalyx was improved by inclusion of paraformaldehyde. Short, 20 min prefixation times for paraformaldehyde‐glutaraldehyde fixatives containing lysine, with or without ruthenium red, were compared to an extended overnight fixation. Samples were often lost in fixatives that did not contain paraformaldehyde at extended fixation times hampering the effective use of these fixatives for clinical or environmental applications. Inclusion of paraformaldehyde in the fixation with lysine permitted longer fixation times as well as stabilized the staphylococcal glycocalyx. Thus, the technical usefulness of fixatives employing lysine was significantly improved. Microsc. Res. Tech. 36:422–427, 1997.