James N. Petitte

Imprint status of M6P/IGF2R and IGF2 in chickens.

Abstract. Genomic imprinting is a method of gene regulation whereby a gene is expressed in a parent-of-origin-dependent fashion; however, it is hypothesized that imprinting should not occur in oviparous taxa such as birds. Therefore, we examined the allelic expression of two genes in the chicken that are reciprocally imprinted in most mammals, mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and insulin-like growth factor 2 (IGF2). Single nucleotide polymorphisms were identified in these genes, and cDNA was prepared from several tissues of embryos heterozygous for these polymorphisms. Both alleles of M6P/IGF2R and IGF2 were expressed in all tissues examined by RT-PCR. Since the expression of these genes was independent of the parent from which they were inherited, we conclude that neither M6P/IGF2R nor IGF2 are imprinted in the chicken.

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.

Ovarian Adenocarcinomas in the Laying Hen and Women Share Similar Alterations in p53, ras, and HER-2/neu

We examined alterations in the p53 tumor suppressor gene and the ras and HER-2/neu oncogenes in chicken ovarian cancers to determine if these tumors have genetic alterations similar to those in human ovarian adenocarcinomas. Mutations in the p53 tumor suppressor gene and the H-ras and K-ras oncogenes were assessed by direct sequencing in 172 ovarian cancers obtained from 4-year-old birds enrolled at age 2 in two separate 2-year chemoprevention trials. Birds in trial B had approximately twice as many lifetime ovulations as those in trial A. Immunohistochemical staining for the HER-2/neu oncogene was done on a subset of avian ovarian and oviductal adenocarcinomas. Alterations in p53 were detected in 48% of chicken ovarian cancers. Incidence of p53 alterations varied according to the number of lifetime ovulations, ranging from 14% in trial A to 96% in trial B (P < 0.01). No mutations were seen in H-ras, and only 2 of 172 (1.2%) tumors had K-ras mutations. Significant HER-2/neu staining was noted in 10 of 19 ovarian adenocarcinomas but in only 1 of 17 oviductal adenocarcinomas. Similar to human ovarian cancers, p53 alterations are common in chicken ovarian adenocarcinomas and correlate with the number of lifetime ovulations. Ras mutations are rare, similar to high-grade human ovarian cancers. HER-2/neu overexpression is common and may represent a marker to exclude an oviductal origin in cancers involving both the ovary and oviduct.

Epidermal Growth Factor-Induced Proliferation of Chicken Primordial Germ Cells: Involvement of Calcium/Protein Kinase C and NFKB1

Abstract Epidermal growth factor (EGF) has been shown to stimulate survival in diverse cells in vitro. In the present study, the effects of EGF and the EGF-related signaling pathway on proliferation of chicken primordial germ cells (PGCs) were investigated. Results showed that EGF (10–100 ng/ml) increased the number and area of PGC colonies in a time- and dose-dependent manner. EGF also activated PKC, a process that was inhibited by AG1478 (an EGFR tyrosine kinase inhibitor) and ethyleneglycol-bis-(beta-aminoethyl ether)-N,N′-tetraacetic acid (EGTA; an intracellular Ca2+ chelator). In addition, the degradation of NFKBIA and NFKB1 (p65) translocation was observed after EGF treatment, which was significantly blocked by pretreatment with AG1478, EGTA, H7, or SN50 (NFKB1-specific inhibitor). Furthermore, we found that EGF-induced cell proliferation was significantly attenuated by AG1478, EGTA, H7, and SN50, respectively. On the other hand, inhibition of EGFR, Ca2+/PKC, or NFKB1 abolished the EGF-stimulated increase in the expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2, and BCL2, and restored the EGF-induced inhibition of BAX expression and caspase 3/9 activity, indicating that EGFR, PKC, and NFKB1 signaling cascades were involved in EGF-stimulated DNA synthesis and antiapoptosis action. In conclusion, EGF stimulated proliferation of chicken PGCs via activation of Ca2+/PKC involving NFKB1 signaling pathway. These observations suggest that EGF signaling is important in regulating germ cell proliferation in the chicken embryonic gonad.

Rapid sex determination of chick embryos using the polymerase chain reaction 1

Abstract The sex of early chicken embryos was determined by DNA dot‐blot hybridization and the polymerase chain reaction (PCR). An oligonucleotide probe, specific to the XhoI family of repetitive DNA of the W chromosome, was hybridized to crude and purified preparations of DNA obtained from blood or cells from embryos and detected using chemiluminescence. Complete agreement was observed in sexing 100 embryos using the dot‐blot assay and visual inspection of embryonic gonadal tissue. Although the dot‐blot system was useful for determining the sex of embryos prior to and throughout incubation, the time required for hybridization and detection did not allow for the immediate manipulation of embryos. Therefore, a PCR assay was developed using primers specific to the XhoI repetitive element and rapid thermocycling with an air thermocycler. A female‐specific amplification product was observed using DNA from as few as two cells in the PCR. Furthermore, female DNA was detected in mixtures of male and female DNA a...

Comparison of gene expression patterns between avian and human ovarian cancers

OBJECTIVES A putative model of spontaneous cancer has been described in the laying hen that bears significant similarities to human ovarian cancer. Our objective was to characterize and compare the patterns of gene expression in chicken and human forms of this disease. METHODS RNA from 20 localized and metastatic ovarian and oviductal chicken tumor samples was isolated, amplified using in vitro transcription, and hybridized against normal ovarian epithelium to a customized cDNA microarray constructed for these studies. Differentially expressed genes were identified for localized ovarian, metastatic ovarian, and oviductal (or tubal) cancer by class comparison using BRB-ArrayTools. Results were validated with semi-quantitative PCR. A gene list (prediction model) constructed with the class prediction tool was used in a human ovarian cancer microarray obtained from the GEO datasets (GSE6008) in order to compare these results across species. RESULTS Class comparison analysis between localized ovarian, metastatic ovarian and oviductal cancer yielded 41 different informative probes that coded for 27 unique genes. Localized ovarian samples clustered between metastatic ovarian and oviductal cancer samples. Using our chicken data as a training set and leaving oviductal samples out of the analysis, we created a prediction model that classified early stage and advanced stage human ovarian cancer gene expression arrays with 78% overall accuracy. CONCLUSIONS Gene expression of spontaneous ovarian cancer in the chicken is comparable to gene expression patterns of human ovarian cancer.

Reduction of Ovarian and Oviductal Cancers in Calorie-Restricted Laying Chickens

Epithelial ovarian cancer (OVAC) remains a highly lethal malignancy. It is a leading cause of cancer deaths among women in the United States causing more deaths than all other gynecologic malignancies combined. The pathogenesis of OVAC is not completely understood, but the process of repeated ovulation is believed to lead to genetic damage in the ovarian epithelium. As part of a prospective trial designed to evaluate OVAC chemopreventive strategies using the chicken model, caloric restriction (55% less energy) was used to inhibit ovulation in groups of hens receiving chemopreventives, thereby minimizing the impact of ovulation on the incidence of reproductive tract cancer. A separate group of chickens was maintained concurrently in the same environment, and managed similarly, except that caloric intake was not restricted. Among birds not receiving chemopreventive agents, we compared caloric versus noncaloric restricted birds to determine the relations between calorie restriction and risk of developing adenocarcinoma of the reproductive tract. Mortality in the calorie-restricted group was almost half that of those on full feed. Calorie-restricted chickens maintained body weights averaging 1.423 kg compared with the full-fed birds at 1.892 kg. Ovulation rate varied with the full-fed group producing 64% more eggs than the calorie-restricted group. Total reproductive cancers occurred in 57 (33.3%) birds for the full-fed group and 26 (10.3%) birds for the calorie-restricted group. On the basis of histopathology, 45 (26.3%) birds in the full-fed group had ovarian adenocarcinoma compared with 16 (6.3%) birds in the calorie-restricted group. Calorie restriction in laying hens resulted in a near five-fold reduction in OVAC. Cancer Prev Res; 4(4); 562–7. ©2011 AACR.

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 …

Multi-peptide nLC-PC-IDMS-SRM-based Assay for the quantification of biomarkers in the chicken ovarian cancer model

A novel form of ovomacroglobulin/ovostatin (OVOS2) predicted from EST data was previously identified in the chicken ovarian cancer model using a mass spectrometry-based shotgun label-free proteomics strategy. The quantitative label-free data from plasma showed a significant increase over time with the spontaneous onset and progression of ovarian cancer making it a potential protein biomarker for further study. Two other proteins of interest identified from this initial study included vitellogenin-1 (Vit-1), a lipid-transport protein tied to egg production, and transthyretin (TTR), a retinol binding transport protein currently used in the clinical management of ovarian cancer. A multiplexed protein cleavage isotope dilution mass spectrometry (PC-IDMS) assay was developed to quantify OVOS2, Vit-1, and TTR by selected reaction monitoring (SRM). A total of 6 stable isotope labeled (SIL) peptide standards were used in the assay with three tryptic peptides from OVOS2, one for Vit-1, and two for TTR. The assay was developed for use with un-depleted raw plasma combined with the filter assisted sample preparation (FASP) method and its use was also demonstrated for matched ovary tissue samples. The PC-IDMS data for the two TTR peptides did not correlate with each other with more than a 10-fold difference in concentration for all 5 time points measured. The PC-IDMS data from the longitudinal plasma samples correlated well for OVOS2 and Vit-1 whereas TTR was inconclusive. Interestingly, the absolute amount for one of the OVOS2 SIL peptides was 2-fold less compared with the other two SIL peptides. These data illustrate the successes and challenges of qualifying quantitative levels of proteins from an in-gel digestion sample preparation followed by LC-MS/MS (GeLC) label-free discovery-based approach to a targeted SRM-based quantitative assay in plasma and tissues.