Human Granulosa Cells-Stemness Properties, Molecular Cross-Talk and Follicular Angiogenesis.

The ovarian follicle is the basic functional unit of the ovary, comprising theca cells and granulosa cells (GCs). Two different types of GCs, mural GCs and cumulus cells (CCs), serve different functions during folliculogenesis. Mural GCs produce oestrogen during the follicular phase and progesterone after ovulation, while CCs surround the oocyte tightly and form the cumulus oophurus and corona radiata inner cell layer. CCs are also engaged in bi-directional metabolite exchange with the oocyte, as they form gap-junctions, which are crucial for both the oocyte's proper maturation and GC proliferation. However, the function of both GCs and CCs is dependent on proper follicular angiogenesis. Aside from participating in complex molecular interplay with the oocyte, the ovarian follicular cells exhibit stem-like properties, characteristic of mesenchymal stem cells (MSCs). Both GCs and CCs remain under the influence of various miRNAs, and some of them may contribute to polycystic ovary syndrome (PCOS) or premature ovarian insufficiency (POI) occurrence. Considering increasing female fertility problems worldwide, it is of interest to develop new strategies enhancing assisted reproductive techniques. Therefore, it is important to carefully consider GCs as ovarian stem cells in terms of the cellular features and molecular pathways involved in their development and interactions as well as outline their possible application in translational medicine.

Targeting the TLR2 Receptor With a Novel Thymopentin-Derived Peptide Modulates Immune Responses.

The innate and adaptive immune systems act in concert to protect us from infectious agents and other harmful substances. As a state of temporary or permanent immune dysfunction, immunosuppression can make an organism more susceptible to infection, organ injury, and cancer due to damage to the immune system. It takes a long time to develop new immunomodulatory agents to prevent and treat immunosuppressive diseases, with slow progress. Toll-like receptor 2 (TLR2) agonists have been reported as potential immunomodulatory candidates due to their effective activation of immune responses. It has been demonstrated that thymopentin (TP5) could modulate immunity by binding to the TLR2 receptor. However, the fairly short half-life of TP5 greatly reduces its pharmacological potential for immunosuppression therapy. Although peptide cathelicidin 2 (CATH2) has a long half-life, it shows poor immunomodulatory activity and severe cytotoxicity, which seriously hampers its clinical development. Peptide hybridization is an effective approach for the design and engineering of novel functional peptides because hybrid peptides combine the advantages and benefits of various native peptides. In this study, to overcome all these challenges faced by the parental peptides, six hybrid peptides (CaTP, CbTP, CcTP, TPCa, TPCb, and TPCc) were designed by combining the full-length TP5 with different active fragments of CATH2. CbTP, the most potent TLR2 agonist among the six hybrid peptides, was effectively screened through in silico analysis and in vitro experiments. The CbTP peptide exhibited lower cytotoxicity than either CATH2 or TP5. Furthermore, the immunomodulatory effects of CbTP were confirmed in a CTX-immunosuppressed mouse model, which showed that CbTP has increased immunopotentiating activity and physiological stability compared to the parental peptides. CbTP successfully inhibited immunosuppression and weight loss, increased immune organ indices, and improved CD4+/CD8+ T lymphocyte subsets. In addition, CbTP significantly increased the production of the cytokine TNF-α and IL-6, and the immunoglobulins IgA, IgM, and IgG. The immunoenhancing effects of CbTP were attributed to its TLR2-binding activity, promoting the formation of the TLR2 cluster, the activation of the TLR2 receptor, and thus activation of the downstream MyD88-NF-кB signaling pathway.

New Gene Markers Expressed in Porcine Oviductal Epithelial Cells Cultured Primary In Vitro Are Involved in Ontological Groups Representing Physiological Processes of Porcine Oocytes.

Changes that occur within oviducts after fertilization are dependent on post-ovulation events, including oocyte-oviduct interactions. Although general processes are well-defined, the molecular basis are poorly understood. Recently, new marker genes involved in 'cell development', 'cell growth', 'cell differentiation' and 'cell maturation' processes have been identified in porcine oocytes. The aim of the study was to assess the expression profile of genes in primary in vitro cultured oviductal epithelial cells (OECs), clustered in Gene Ontology groups which enveloped markers also identified in porcine oocytes. OECs (from 45 gilts) were surgically removed and cultured in vitro for ≤ 30 days, and then subjected to molecular analyses. The transcriptomic and proteomic profiles of cells cultured during 7, 15 and 30 days were investigated. Additionally, morphological/histochemical analyzes were performed. The results of genes expression profiles were validated after using RT-qPCR. The results showed a significant upregulation of UNC45B, NOX4, VLDLR, ITGB3, FMOD, SGCE, COL1A2, LOX, LIPG, THY1 and downregulation of SERPINB2, CD274, TXNIP, CELA1, DDX60, CRABP2, SLC5A1, IDO1, ANPEP, FST. Detailed knowledge of the molecular pathways occurring in the OECs and the gametes that contact them may contribute both to developments of basic science of physiology, and new possibilities in advanced biotechnology of assisted reproduction.

A highly efficient hybrid peptide ameliorates intestinal inflammation and mucosal barrier damage by neutralizing lipopolysaccharides and antagonizing the lipopolysaccharide-receptor interaction.

Intestinal inflammatory disorders, such as inflammatory bowel disease, are major contributors to mortality and morbidity in humans and animals worldwide. While some native peptides have great potential as therapeutic agents against intestinal inflammation, potential cytotoxicity, anti-inciting action, and suppression of anti-inflammatory activity may limit their development as anti-inflammatory agents. Peptide hybridization is an effective approach for the design and engineering of novel functional peptides because hybrid peptides combine the advantages and benefits of various native peptides. In the present study, a novel hybrid anti-inflammatory peptide that combines the active center of Cecropin A (C) and the core functional region of LL-37 (L) was designed [C-L peptide; C (1-8)-L (17-30)] through in silico analysis to reduce cytotoxicity and improve the anti-inflammatory activity of the parental peptides. The resulting C-L peptide exhibited lower cytotoxicity than either C or L peptides alone. C-L also exerted a protective effect against lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 macrophages and in the intestines of a mouse model. The hybrid peptide exhibited increased anti-inflammatory activity compared to the parental peptides. C-L plays a role in protecting intestinal tissue from damage, LPS-induced weight loss, and leukocyte infiltration. In addition, C-L reduces the expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1ß, and interferon-gamma (IFN-γ), as well as reduces cell apoptosis. It also reduced mucosal barrier damage caused by LPS. The anti-inflammatory effects of the hybrid peptide were mainly attributed to its LPS-neutralizing activity and antagonizing the activation of LPS-induced Toll-like receptor 4-myeloid differentiation factor 2 (TLR4/MD2). The peptide also affected the TLR4-(nuclear factor κB) signaling pathway, modulating the inflammatory response upon LPS stimulation. Collectively, these findings suggest that the newly designed peptide, C-L, could be developed into a novel anti-inflammatory agent for animals or humans.

A Novel Peptide Ameliorates LPS-Induced Intestinal Inflammation and Mucosal Barrier Damage via Its Antioxidant and Antiendotoxin Effects.

Intestinal inflammation is an inflammatory disease resulting from immune dysregulation in the gut. It can increase the risk of enteric cancer, which is a common malignancy globally. As a new class of anti-inflammatory agents, native peptides have potential for use in the treatment of several intestinal inflammation conditions; however, their potential cytotoxicity and poor anti-inflammatory activity and stability have prevented their development. Hybridization has been proposed to overcome this problem. Thus, in this study, we designed a hybrid peptide (LL-37-TP5, LTP) by combing the active centre of LL-37 (13-36) with TP5. The half-life and cytotoxicity were tested in vitro, and the hybrid peptide showed a longer half-life and lower cytotoxicity than its parental peptides. We also detected the anti-inflammatory effects and mechanisms of LTP on Lipopolysaccharide (LPS)-induced intestinal inflammation in murine model. The results showed that LTP effectively prevented LPS-induced weight loss, impairment of intestinal tissues, leukocyte infiltration, and histological evidence of inflammation. Additionally, LTP decreased the levels of tumour necrosis factor-alpha, interferon-gamma, and interleukin-6; increased the expression of zonula occludens-1 and occludin; and reduced permeability in the jejunum of LPS-treated mice. Notably, LTP appeared to be more potent than the parental peptides LL-37 and TP5. The anti-inflammatory effects of LTP may be associated with the neutralization of LPS, inhibition of oxidative stress, and inhibition of the NF-κB signalling pathway. The findings of this study suggest that LTP might be an effective therapeutic agent for treating intestinal inflammation.

In-depth LC-MS/MS analysis of the chicken ovarian cancer proteome reveals conserved and novel differentially regulated proteins in humans.

Ovarian cancer (OVC) remains the most lethal gynecological malignancy in the world due to the combined lack of early-stage diagnostics and effective therapeutic strategies. The development and application of advanced proteomics technology and new experimental models has created unique opportunities for translational studies. In this study, we investigated the ovarian cancer proteome of the chicken, an emerging experimental model of OVC that develops ovarian tumors spontaneously. Matched plasma, ovary, and oviduct tissue biospecimens derived from healthy, early-stage OVC, and late-stage OVC birds were quantitatively characterized by label-free proteomics. Over 2600 proteins were identified in this study, 348 of which were differentially expressed by more than twofold (p ≤ 0.05) in early- and late-stage ovarian tumor tissue specimens relative to healthy ovarian tissues. Several of the 348 proteins are known to be differentially regulated in human cancers including B2M, CLDN3, EPCAM, PIGR, S100A6, S100A9, S100A11, and TPD52. Of particular interest was ovostatin 2 (OVOS2), a novel 165-kDa protease inhibitor found to be strongly upregulated in chicken ovarian tumors (p = 0.0005) and matched plasma (p = 0.003). Indeed, RT-quantitative PCR and Western blot analysis demonstrated that OVOS2 mRNA and protein were also upregulated in multiple human OVC cell lines compared to normal ovarian epithelia (NOE) cells and immunohistochemical staining confirmed overexpression of OVOS2 in primary human ovarian cancers relative to non-cancerous tissues. Collectively, these data provide the first evidence for involvement of OVOS2 in the pathogenesis of both chicken and human ovarian cancer.

Epithelial cell tumors of the hen reproductive tract.

There is a paucity of preclinical models that simulate the development of ovarian tumors in humans. At present, the egg-laying hen appears to be the most promising model to study the spontaneous occurrence of ovarian tumors in the clinical setting. Although gross classification and histologic grade of tumors have been used prognostically in women with ovarian tumors, there is currently no single system that is universally used to classify reproductive tumors in the hen. Four hundred and one 192-wk-old egg-laying hens were necropsied to determine the incidence of reproductive tumors using both gross pathology and histologic classification. Gross pathologic classifications were designated as follows: birds presenting with ovarian tumors only (class 1), those presenting with oviductal and ovarian tumors (class 2), those with ovarian and oviductal tumors that metastasized to the gastrointestinal tract (class 3), those with ovarian and oviductal tumors that metastasized to the gastrointestinal tract and other distant organs (class 4), those with oviductal tumors only (class 5), those with oviductal tumors that metastasized to other organs with no ovarian involvement (class 6), and those with ovarian tumors that metastasized to other organs with no oviductal involvement (class 7), including birds with gastrointestinal tumors and no reproductive involvement (GI only) and those with no tumors (normal). Histopathologic classifications range from grades 1 to 3 and are based on mitotic developments and cellular differentiation. An updated gross pathology and histologic classification systems for the hen reproductive malignancies provides a method to report the range of reproductive tumors revealed in a flock of aged laying hens.

Evidence of a chemopreventive effect of progestin unrelated to ovulation on reproductive tract cancers in the egg-laying hen.

Epidemiologic, laboratory, and animal evidence suggests that progestins and vitamin D may be potent ovarian cancer preventives. Our objectives were to evaluate progestins as reproductive tract cancer chemopreventives in the chicken, determine whether restricted ovulation affected the incidence of reproductive tract tumors, and assess whether vitamin D would confer cancer protection either alone or in addition to progestin. A total of 2,400 two-year-old Single Comb White Leghorns were randomized into six groups (400 each) with hormonal and dietary manipulation for 2 years as follows: (i) no intervention, regular feed/caloric intake, (ii) control, (iii) vitamin D, (iv) the progestin levonorgestrel, (v) vitamin D plus levonorgestrel, and (vi) the progestin Provera (medroxyprogesterone acetate). Groups 2 to 6 were caloric restricted to inhibit ovulation. Our results indicated that caloric restriction decreased egg production by more than 60%, and was associated with a greater than 70% decrease in reproductive tract cancers. Ovulatory events did not differ among the caloric-restricted groups (groups 2-6), except for the group receiving levonorgestrel, which had fewer ovulatory events than controls (P = 0.046). After correcting for egg production, birds receiving progestins had significantly fewer reproductive tract cancers [OR, 0.61; confidence interval (CI), 0.39-0.95; P = 0.03], with similar proportionate reductions in tumors arising in either the ovary or oviduct. Vitamin D did not significantly affect cancer incidence overall, or add to the cancer preventive effect of progestins. This study suggests a protective effect of progestins against ovarian and oviductal cancers. These data support the concept that progestins provide a chemopreventive effect unrelated to ovulation.

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.

One-Year Plasma N-linked Glycome Intra-individual and Inter-individual Variability in the Chicken Model of Spontaneous Ovarian Adenocarcinoma.

Spontaneous epithelial ovarian cancer (EOC) in the chicken presents a similar pathogenesis compared with humans including CA-125 expression and genetic mutational frequencies (e.g., p53). The high prevalence of spontaneous EOC chickens also provides a unique experimental model for biomarker discovery at the genomic, proteomic, glycomic, and metabolomic level. In an effort to exploit this unique model for biomarker discovery, longitudinal plasma samples were collected from chickens at three month intervals for one year. The study described herein involved cleaving the N-glycans from these longitudinal chicken plasma samples and analyzing them via nanoLC-FTMS/MS. Glycans identified in this study were previously found in human plasma and this work provides a promising methodology to enable longitudinal studies of the N-linked plasma glycome profile during EOC progression. The structure, abundance, and intra-variability and inter-variability for 35 N-linked glycans identified in this study are reported. The full potential of the chicken model for biomarker discovery has yet to be realized, but the initial interrogation of longitudinally-procured samples provides evidence that supports the value of this strategy in the search for glycomic biomarkers.

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.

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.

Measuring the intra-individual variability of the plasma proteome in the chicken model of spontaneous ovarian adenocarcinoma.

The domestic chicken (Gallus domesticus) has emerged as a powerful experimental model for studying the onset and progression of spontaneous epithelial ovarian cancer (EOC) with a disease prevalence that can exceed 35% between 2 and 7 years of age. An experimental strategy for biomarker discovery is reported herein that combines the chicken model of EOC, longitudinal plasma sample collection with matched tissues, advanced mass spectrometry-based proteomics, and concepts derived from the index of individuality (Harris, Clin Chem 20: 1535-1542, 1974). Blood was drawn from 148 age-matched chickens starting at 2.5 years of age every 3 months for 1 year. At the conclusion of the 1 year sample collection period, the 73 birds that remained alive were euthanized, necropsied, and tissues were collected. Pathological assessment of resected tissues from these 73 birds confirmed that five birds (6.8%) developed EOC. A proteomics workflow including in-gel digestion, nanoLC coupled to high-performance mass spectrometry, and label-free (spectral counting) quantification was used to measure the biological intra-individual variability (CV(W)) of the chicken plasma proteome. Longitudinal plasma sample sets from two birds within the 73-bird biorepository were selected for this study; one bird was considered "healthy" and the second bird developed late-stage EOC. A total of 116 proteins from un-depleted plasma were identified with 80 proteins shared among all sample sets. Analytical variability (CV(A)) of the label-free proteomics workflow was measured using a single plasma sample analyzed five times and was found to be ≥CV(W) in both birds for 16 proteins (20%) and in either bird for 25 proteins (31%). Ovomacroglobulin (ovostatin) was found to increase (p < 0.001) over a 6 month period in the late-stage EOC bird providing an initial candidate protein for further investigation.

The impact of scheduled cage cleaning on older hens (Gallus gallus).

Researchers are increasingly using the domestic hen (Gallus gallus) as an animal model for ovarian cancer. The authors analyzed mortality rates of two large flocks of older hens that were being used for ovarian adenocarcinoma studies. All hens were fed the same maintenance diets, though some hens in each flock received experimental chemopreventive treatments. Per the request of a collaborating institution, partway through the study, the authors started to remove the hens in one of the flocks for cage changing once every 4 weeks. After the authors began cleaning some of the hens' cages, the mortality rate in this flock increased significantly. Throughout the study, within each flock, hens in the treatment and control groups had similar mortality rates. These results suggest that regularly cleaning the cages of older hens may not promote better welfare or improve flock mortality.

Epidermal growth factor-induced proliferation of chicken primordial germ cells: involvement of calcium/protein kinase C and NFKB1.

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 Ca(2+) 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, H(7), or SN50 (NFKB1-specific inhibitor). Furthermore, we found that EGF-induced cell proliferation was significantly attenuated by AG1478, EGTA, H(7), and SN50, respectively. On the other hand, inhibition of EGFR, Ca(2+)/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 Ca(2+)/PKC involving NFKB1 signaling pathway. These observations suggest that EGF signaling is important in regulating germ cell proliferation in the chicken embryonic gonad.

Avian multiple inositol polyphosphate phosphatase is an active phytase that can be engineered to help ameliorate the planet's "phosphate crisis".

Contemporary phytase research is primarily concerned with ameliorating the problem of inadequate digestion of inositol hexakisphosphate (phytate; InsP6) in monogastric farm animal feed, so as to reduce the pollution that results from the high phosphate content of the manure. In the current study we pursue a new, safe and cost-effective solution. We demonstrate that the rate of hydrolysis of InsP6 by recombinant avian MINPP (0.7 micromol/mg protein/min) defines it as by far the most active phytase found to date in any animal cell (the corresponding activity of recombinant mammalian MINPP is only 0.006 micromol/mg protein/min). Although avian MINPP has less than 20% sequence identity with microbial phytases, we create a homology model of MINPP in which it is predicted that the structure of the phytase active site is well-conserved. This model is validated by site-directed mutagenesis and by use of a substrate analogue, scyllo-InsP6, which we demonstrate is only a weak MINPP substrate. In a model chicken cell line, we overexpressed a mutant form of MINPP that is secretion-competent. This version of the enzyme was actively secreted without affecting either cell viability or the cellular levels of any inositol phosphates. Our studies offer a genetic strategy for greatly improving dietary InsP6 digestion in poultry.

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.