Materials

Pannett and Compton (PC) saline for bird embryos (original ref. given in 12) (PC saline). If filter-sterilized or autoclaved in the two parts when making up, this can be stored at room temperature for months. PC saline is needed in liter quantities per experiment dealing with 10-20 chick eggs, 500-mL bottles are thus convenient. 2. Hank's Balanced Salt Solution (BSS), made up with 0.1X the originally specified concentration of divalent cations CaMg2+. Use of the tissue-culture pH indicator dye...

Transgenesis by Sperm Nuclear Transplantation into Unfertilized Eggs see Notes

Inject two to four adult female frogs in the dorsal lymph sac with 500-800 U HCG, and incubate at 15 C for 12-16 h before transplantations. 2. Set up injection area Coat inside of transplantation needles with Sigmacote to prevent shearing of sperm nuclei flowing through the needle (needles can be coated 10 min to several months before use). Attach approx 1 cm Tygon tubing (R-3603 1 32 in. Fisher, cat. 14-169-1A) to the end of a plastic pipetman (200 L) tip, and use the pipetman to draw up...

Synthesis of cDNA and PCR

For cleaner results, polyadenylated RNA can be isolated as mentioned in the libraries chapter (Chapter 37). However, large amounts of RNA are required to obtain polyadenylated mRNA, so this choice is limited by the availability of the sample. Also, the time and effort employed in the extraction of more material do not necessarily correlate with an equal increase in performance. Therefore, total RNA will be the starting material used in these protocols. The choice of the arbitrary primer to be...

Claudio D Stern 1 Introduction

The somites are an intriguing invention of vertebrate embryos. They represent the most overtly segmented structures of the body plan, but they give rise to both obviously segmental (e.g., the axial skeleton) as well as not-so-obvi-ously metameric (dermis and skeletal muscle) elements. In addition, they play a key role in controlling several aspects of the organization of the central and peripheral nervous system of the trunk, and appear to participate in several different types of inductive...

Roller Bottle Culture After Oligo Treatment

Our laboratory has recently reported successful culture of chick embryos in a version of the roller-tube method used for mammal development (13). This permits more extended normal development (30 +somites), probably because the extraembryonic blood-vascular supply becomes as profuse as that in ovo, whereas this does not occur in ring culture. We now have evidence that phosphorothioate oligos simply added to these cultures can maintain interference with the same genes that have been successfully...

Cell Grafting and Labeling in Postimplantation Mouse Embryos

Trainor, and Patrick P. L. Tam 1. Introduction Fate mapping experiments provide direct information on the differentiation pathways normally taken by cells or tissues during embryogenesis. Systematic analyses of the developmental fate of cell populations localized in different parts of the embryo enables the construction of fate maps. A comparison of the expression pattern of lineage-specific genes and the fate map allows the identification of precursor tissue for...

Substage Condenser

The substage condenser contains a lens complex, an aperture diaphragm, and may also house prisms and filters used in contrast enhancement. The condenser can be focused to optimize the illumination of the specimen. The aperture diaphragm works independently of the field diaphragm to optimize contrast, depth of field, and resolution by altering the NA of the condenser lens (Fig. 2). The maximal NA of the condenser lens is usually engraved on its side. On some condensers, a swung-in lens can be...

Nuclear Transplantation Reagents and Equipment

1X MMR Prepared as described in Subheading 2.1., item 2. 2. 2.5 Cysteine in 1X MMR (titrate to pH 8.0 with NaOH). Make up fresh each day. 5. 0.4X MMR + 6 (w v) Ficoll (Sigma Type 400 F-4375) Sterilize by filtration. 6. 0.1X MMR + 50 g mL gentamycin (a 10 mg mL stock solution may be purchased from Gibco-BRL cat 15710-015). Add 6 (w v) Ficoll for culturing embryos prior to gastrulation. Culture embryos in 0.1X MMR without Ficoll after gastrulation. Sterilize by filtration. 7. Progesterone (Sigma...

San Ling SiHoe and David Murphy 1 Introduction

A pioneering experiment in the early 1980s demonstrated that microinjection of recombinant growth hormone into the pronuclei of fertilized one-celled mouse embryos resulted in inheritable changes in the growth of these mice (1). Mammalian transgenic experiments have since contributed tremendously to our understanding of numerous complex biological processes. The power of the technique lies in that it allows the function, and developmental and physiological regulation of almost any protein to be...

Fate Mapping with Lipophilic Membrane Dyes

Human Meridian Imaging

The lipophilic membrane dye DiI is the first-choice dye for following the fate of relatively small groups of neighboring cells in ovo. It is expensive at first glance ( 190 for 100 mg), but 100 mg does go a long way. DiI, like the other carbocyanine membrane dyes, such as DiO and DiA (Molecular Probes D-275 and D-291), is lipophilic, and thus following application to a tissue, it readily and preferentially diffuses into cell membranes rather than remaining in and diffusing through the aqueous...

Intraventricular Injection of Retrovirus

The procedure for making injections of retrovirus into the telencephalic ventricles of embryonic mice and rats will be described. The basic procedures can be adapted to inject retrovirus into other structures, such as the eye (15) or olfactory epithelium (16,17). Intraventricular injections of retrovirus can be made most readily between embryonic d 11 and 15 of mice and between embryonic d 13 and 17 of rats. Injections earlier and later than the stated times are difficult because the uterine...

Preparation of Culture Media for Fertilized One Celled Eggs

Two types of media are required for the in vitro manipulations of the eggs (12). 1. M16 for maintaining the eggs in microdrop cultures in a 37 C incubator gassed with 5 CO2. M16 is buffered with bicarbonate alone and unsuitable for maintaining the eggs outside of the incubator, since the eggs are very susceptible to pH changes. 2. M2 essentially similar to M16, except that the bicarbonate is partially replaced by HEPES buffer to facilitate the survival of the eggs outside the CO2 incubator....

Generation of Conditionally Immortal Cell Lines by Genetic Modification In Vitro

There are two major approaches that can be used to generate cell lines of interest. The first of these, and still the most frequently used, is to transduce an immortalizing gene construct into cultures of dividing cells. This can be accomplished by transfection or by use of retroviruses to insert the immortalizing gene randomly into the cellular genome. Despite the undoubted importance of in vitro gene insertion in the creation of cell lines, there remain certain drawbacks intrinsic to this...

Neuronal Tracing Using Lipophilic Membrane Dyes Fluorescent Dextrans and Horseradish Peroxidase HRP

The organization of neurons and their axons in the CNS and peripheral nervous system can be examined using anterograde and retrograde tracing techniques with fluorescent or nonfluorescent dyes. The lipophilic membrane dyes, such as DiI, have the advantage that they may be used on fixed as well as living tissue, whereas the intracellular dyes, such as fluorescent dextrans and HRP, rely to some extent on the mechanisms of axonal transport and thus work best on living tissue. The principle is the...

Organ Culture in the Analysis of Tissue Interactions

Tissue Culture Trowell Method

Introduction Interactions between epithelial and mesenchymal tissues constitute a central mechanism regulating the development of most embryonic organs. Studies on the nature of such interactions require the separation of the interacting tissues from each other and the follow-up of their advancing development in various types of recombined explants. The tissues can be either transplanted and their development followed in vivo, or they can be cultured as...

Reporter Transgene Design

When designing a transgenic reporter construct, careful planning is of the utmost importance. The production and analysis of transgenic animals is both labor- and time-intensive, and a well-designed construct will give you a good base on which to plan your subsequent experiments. Although the exact layout of a given reporter depends on the aims of the experiment in question, certain general considerations should be addressed. These will be discussed below. 1. A reporter transgene requires the...

Notes

A 20- imol aqueous solution of PMSF at pH 8.0 has a half-life of 35 min, and therefore, this inhibitor must be added just prior to use 12 . The inclusion of 10 mM iodoacetamide is also useful to prevent crosslinking of proteins owing to the presence of free sulfhydryl groups during immunoprecipitation. 2. The dissociation buffer is usually made reducing by the addition of 0.1 M dithiothreitol or 1.4 M P-mercaptoethanol prior to boiling, under which conditions disulfide-linked chains will...

Isolation of Primordial Germ Cells

Primordial germ cells can be obtained from different stages of embryos. Subheading 3.1.1. will describe how to dissect early embryos 8.5 d pc in order to obtain early migratory PGCs, whereas Subheading 3.1.2. will describe how to isolate the genital ridge from later embryos 11.5 d pc and 15.5 d pc in order to obtain later PGCs. Subheading 3.1.3. describes how these isolated tissues should be processed for in vitro culture. Further information about the numbers of PGCs and their location in the...