A detailed description of an economical setup for electroporation of chick embryos in ovo

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

Gene transfer to the embryo: strategies for the delivery and expression of proteins at 48 to 56 hours postfertilization.

BACKGROUND/PURPOSE: Although gene and protein transfer may potentiate the cure of genetic disease, current strategies involving fetal gene therapy remain nonfocal and confounded by the lack of imaging techniques and in vivo markers for precise gene transfer. METHODS: Fourteen white Leghorn chick eggs were incubated for 48 to 56 hours postfertilization until they reached stages 11 to 16, about 3 mm in size. In 7 chick embryos, a glass needle was placed at the midbrain/hindbrain level and 1 x 10(7) pfu of an adenovirus containing the green fluorescent protein (GFP) reporter gene was injected into the lateral head. In another 7 chicken embryos, colored agarose beads coated with Sonic hedgehog (Shh) protein were implanted at the level of the hindbrain under direct microscopy. The eggs were then sealed, incubated at 37 degrees C for 24 hours, and reimaged using fluorescent microscopy and confocal laser microscopy. RESULTS: At 24 hours postinjection, all embryos were alive and were imaged in vivo. Fluorescent microscopic imaging showed green fluorescence in the region of the injection site in all the embryos. In embryos that underwent bead placement, the beads were visualized under microscopy in the lateral hindbrain of all embryos, and the presence of the Shh protein was confirmed using fluorescein isothiocyanate (FITC)-conjugated secondary antibody. CONCLUSIONS: This study shows that embryonic 3-mm chick embryos survive adenoviral transduction or agarose bead implantation in a focal manner in vivo and that this delivery results in production of imageable levels of protein. This may be used in mammalian systems, including humans, to introduce genes and proteins.

Primary lymphoma of the breast.

Primary lymphoma of the breast is a rare finding. Two cases and a review of the literature are presented.

Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.

To visualize the movements of cells and their processes in developing vertebrates, we constructed replication-incompetent retroviral vectors encoding green fluorescent protein (GFP) that can be detected as a single integrated copy per cell. To optimize GFP expression, the CMV enhancer and avian beta-actin promoter were incorporated within a retrovirus construct to drive transcription of redshifted (F64L, S65T) and codon-modified GFP (EGFP), EGFP tagged with GAP-43 sequences targeting the GFP to the cell membrane, or EGFP with additional mutations that increase its ability to fold properly at 37 degrees C (S147P or V163A, S175G). We have used these viruses to efficiently mark and follow the developmental progression of a large population of cells in rat neocortex and whole avian embryos. In the chick embryo, the migration and development of GFP-marked neural crest cells were monitored using time-lapse videomicroscopy. In the neocortex, GFP clearly delineates the morphology of a variety of neuronal and glial phenotypes. Cells expressing GFP display normal dendritic morphologies, and infected cells persist into adulthood. Cortical neurons appear to form normal local axonal and long-distance projections, suggesting that the presence of cytoplasmic or GAP-43-tagged GFP does not significantly interfere with normal development.

Specification of the zebrafish nervous system by nonaxial signals.

The organizer of the amphibian gastrula provides the neurectoderm with both neuralizing and posteriorizing (transforming) signals. In zebrafish, transplantations show that a spatially distinct transformer signal emanates from tissues other than the organizer. Cells of the germring (nonaxial mesendoderm) posteriorized forebrain progenitors when grafted nearby, resulting in an ectopic hindbrain-like structure; in contrast, cells of the organizer (axial mesendoderm) caused no posterior transformation. Local application of basic fibroblast growth factor, a candidate transformer in Xenopus, caused malformation but not hindbrain transformation in the forebrain. Thus, the zebrafish gastrula may integrate spatially distinct signals from the organizer and the germring to pattern the neural axis.

Receptor-targeted co-transport of DNA and magnetic resonance contrast agents.

BACKGROUND: Ligand molecules conjugated to polylysine can be electrostatically bound to DNA and can bind receptors or antigens on the surface of cells, delivering the DNA into specific cells and tissues. Several researchers have used this approach to generate non-viral vehicles for the efficient delivery of DNA to specific cells. We have attempted to adopt this general approach to the cell-specific delivery of magnetic contrast agents for use in magnetic resonance imaging (MRI). RESULTS: We have synthesized a new class of agents capable of both transfecting genes into cells and enhancing the contrast of the targeted cells for MRI. DNA is used both to encode a marker gene and as a molecular scaffold, which electrostatically binds polylysine conjugated to transferrin, an iron uptake protein, and polylysine modified with gadolinium chelated to diethylenetriaminepetaacetic acid. When cells displaying the transferrin receptor are treated with these particles, high levels of gene expression are observed, higher than with control particles composed only of transferrin, polylysine and DNA. The treated cells show specific MRI contrast enhancement, which did not require expression of the marker gene. CONCLUSIONS: The development of this class of particles permits the use of novel protocols by which genes for genetic therapy and agents for MRI contrast are co-transported. These protocols may allow non-invasive MRI monitoring of DNA delivery for gene therapy in real time.

A dual embryonic origin for vertebrate mechanoreceptors.

Neuromasts, the mechanoreceptors of the lateral line system of fishes and aquatic amphibians, have previously been thought to develop exclusively from embryonic epidermal placodes. Use of fate mapping techniques shows that neuromasts of the head and body of zebrafish, Siamese fighting fish, and Xenopus are also derived from neural crest. Neural crest migrates away from the neural tube in developing vertebrates to form much of the peripheral nervous system, pigment cells, and skeletal elements of the head. The data presented here demonstrate that neuromasts are derived from both neural crest and epidermal placodes.

Structure and expression of the nicotinic acetylcholine receptor beta subunit of Xenopus laevis.

A cDNA encoding the beta subunit of the Xenopus muscle nicotinic acetylcholine receptor (AChR) was cloned from an embryonic Xenopus cDNA library. The predicted mature polypeptide has 469 amino acids and four membrane spanning regions corresponding to the M1-M4 regions identified in other AChR subunit clones. The polypeptide bears greater homology to beta subunits of Torpedo and mouse than to alpha, gamma or delta subunits of Xenopus. The earliest beta subunit transcripts were detected by RNase protection assays at the neural plate stage of development (stage 14) and the level of transcripts, as a fraction of total RNA, continued to increase through the age of hatching (stages 34-36). Co-injection of Xenopus alpha, beta, gamma and delta cRNAs into Xenopus oocytes led to expression of functional AChRs. Micromolar concentrations of ACh activated depolarizing AChR currents which reversed at -5 mV and were blocked by alpha bungarotoxin. Injection of alpha, gamma and delta subunits alone did not yield detectable ACh responses. With the cloning of the Xenopus beta subunit, structure/function relations of AChRs can now be studied using receptors composed entirely of Xenopus subunits.

Pattern regulation in the eyebud of Xenopus studied with a vital-dye fiber-tracing technique.

Evidence for pattern regulation in the developing Xenopus visual system has previously been obtained after surgical manipulations of the eyebud early in development. In one experimental paradigm, a "compound" eye is produced by combining a nasal (anterior) half-eyebud with normal dorsoventral polarity and a temporal (posterior) half-eyebud with inverted dorsoventral polarity. The adult retinotectal projection from such compound eyes, as assayed by electrophysiological mapping techniques, shows normal dorsoventral polarity in both halves, indicating an apparent reversal in the polarity of the surgically-inverted half. We have utilized a fluorescent vital-dye fiber-tracing technique to investigate the early events in this regulatory process. The results show that the change in dorsoventral polarity is not due to cell movements in the eyebud after surgery. Interestingly, the experiments also demonstrate that the pattern of connections initially formed by the developing eye does not reflect the pattern regulation observed in the adult retinotectal map; instead, the temporal half of the eye projects to the tectum with inverted dorsoventral order. Thus, the regulation observed in the adult does not become evident in the pattern of the projection until after early larval development.

Population education and China: prospects for the one child family.

PIP: With China's population approaching the billion mark much publicity has been given to promoting the 1 child family, including a massive educational campaign with a series of incentives and disincentives being introduced in some provinces. China needs to immediately achieve and maintain a growth rate of less than 1%. Demographic figures for 1978 suggested that 11 of China's most populous provinces had already reached the target of less than 1% growth rate per annum. Some incentives being recommended to encourage the 1 child family include: 1) working parents who promise not to have a 2nd child receive 6 yuan/month in child welfare subsidies until the child is 14 years old, 2) they will be entitled to an equal amount of living space as a family of 4, 3) the child will have priority of admission to schools and factories provided they meet the entrance requirements, and 4) medical benefits will be available free for the child in a single child family. Sterilization as a means of reducing fertility presents social and medical problems in China and is not being publicized to the same extent as the 1 child family. Incentives for 2 child families include: 1) priority in housing, 2) waiver of compulsory rustication, and 3) priority in getting city jobs for rural youth. There are also measures which discourage parents from trying to have a son when they already have 2 daughters since a daughter may replace her father in his job when he retires. Other incentives include subsidized schooling, priority to move from rural to urban areas and more equal pay for women for equal work. Traditional attitudes towards the transitory value of a daughter and the supposed enduring worth of a son is being challenged with daughters now being encouraged to persuade their husbands to become part of the daughters' households. Longterm implications of the new programs are more equitable food and job supply for the future. In addition, there are guarantees that the standard of living of the elderly will be high regardless of the fact that they have few children to support them. Population growth rates are falling as a result of these programs. The following are some of the economic sanctions levied against couples who produce a second child after being rewarded for only having one: deduction from welfare expenses, nonpayment of medical expenses, and no food coupons. China has taken a great social leap forward in promoting the 1 child family and the call for population education and family planning information dissemination has been established in public schools.^ieng