Construction of Retroviral Vectors

For optimum expression of the inserted gene, there are several important points to consider when cloning First, the level of expression of the inserted gene is not only influenced by proviral sequences within the LTR, but also by sequences within the pol region. The more recently developed RCAS vectors containing the pol region from the Bryan high-titer strain of RSV, known as RCASBP (BP stands for Bryan pol), express introduced genes at higher levels (17) than do the original RCAS vectors and...

Neural Plate Explants

The neural plate can be excised cleanly from the underlying dorsal meso-derm as diagrammed in Fig. 14. The tip of the eyebrow hair should be pushed only through the neural plate, which consists of an epithelium and one layer of deep cells at these stages (stages 11-12.5), using the method described above (Fig. 4B). The depth of the neural plate is determined by experience, judging at what depth the eyebrow hair is two cells deep, staying on the shallow side. The eyebrow hair tip is used to...

Marysia Placzek and Kim Dale 1 Introduction

The characterization of molecular and antigenic markers that identify specific vertebrate cells has increased dramatically in recent years. As a result, patterns of cell differentiation and development can be observed in vivo, and subsequently, the tissue interactions and differentiation factors that may operate to establish these patterns can be examined in vitro. Three-dimensional collagen gels provide a culture environment in which in vitro assays can be established, and used to assess the...

Preparation of RNA

For the preparation of embryo tissue RNA, we routinely use a commercial reagent called RNA STAT-60. This procedure is based on the method described by Chomczynski and Sacchi (11), but the RNA STAT-60 reagent contains the guanidine thiocyanate together with the phenol for easier handling. Other manufacturers supply similar reagents yielding identical results (see Note 10). 1. Embryonic tissue should be collected and washed in ice-cold PBS before addition of the extraction reagent. If the tissue...

Photomicrography

Cameras used for photomicrography generally consist of either a standard single-lens-reflex (SLR) 35-mm camera body mounted via a tube to the microscope (which uses its own integral exposure meter) or consist of a specially designed unit that is integrated into a dedicated photomicroscope (which will generally have more sophisticated exposure controls and which may use either 35-mm or larger format film). Although film is still widely used, digital image acquisition is increasingly common....

Claudio D Stern 1 Introduction

Soon after Spemann and Mangold's (1) famous demonstration in 1924 that the dorsal lip of the blastopore of the gastrulating amphibian embryo has the unique ability to induce a second axis when grafted into an ectopic site in a host embryo, Waddington (2,3) showed that Hensen's node is its equivalent in amniotes. After transplanting this region into an ectopic site in interspecific combinations of rabbit, duck, and chick embryos, he found that a second axis developed, where the nervous system...

Notes

All sera (fetal bovine, calf, horse) are suitable for use in freezing media. 2. Chloroquine increases the retroviral titer (8). 3. Paraformaldehyde will dissolve in approx 20 min with heating and stirring. 4. K3Fe(CN)6 and K4Fe(CN)6 are commonly used at concentrations between 5 and 20 mM. The higher concentrations may result in increased staining intensity. 5. This pH range is optimal for detection of the bacterial lacZ activity. The use of higher pHs should be avoided so as to minimize the...

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...

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...

Early Cleavage Stages

It is important to note that early stages of Xenopus development rely completely on maternal stores of RNA and protein transcription does not begin until the so-called midblastula transition, about 7 h after fertilization and when there are 4096 cells (that is, after 12 cleavages) (7). The first Xenopus cell cycle occupies about 90 min at 21 C subsequent cycles last about 30 min. One of the greatest advantages of Xenopus as a tool for developmental biology is that cleavage planes are regular...

References

L. and Amaya, E. (1996) Transgenic Xenopus embryos from sperm nuclear transplantation reveal FGf signaling requirements during gastrulation. Development 122, 3173-3183. 2. Kroll, K. L. and Gerhart, J. C. (1994) Transgenic X. laevis embryos from eggs transplanted with nuclei of transfected cultured cells. Science 266, 650-653. 3. Leno, G. H. and Laskey, R. A. (1991) DNA replication in cell-free extracts from Xenopus laevis, in Methods in Cell Biology, vol. 36 (Kay, B. K. and Peng, H....

Methods

Two days before the IVF procedure, start the superovulation of the donor females as described in Subheading 4. 2. On the day before the IVF or at least 3 h before, preincubate one 35-mm sterile tissue-culture dish containing 1 mL FM without paraffin and six dishes containing 0.5 mL FM under paraffin oil at 37 C with 5 CO2. 3. Early on the morning of the IVF, prepare three tissue-culture dishes containing 2-3 mL M16 medium and one tissue-culture dish containing M16 microdrop cultures. Incubate...

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....

Analysis of Cultures

Subsequent to culture, the patterns of cell differentiation within explants can be examined either by immunohistochemical or in situ hybridization techniques (Fig. 2). In either case, explants can be sectioned directly in the gel and then analyzed, or processed for whole-mount labeling. The decision whether to section or process for whole-mount is largely probe-dependent. With a strong Fig. 1. Schematic diagram of a three-dimensional collagen gel culture. Side view, showing explants positioned...

Peter D Currie Thomas F Schilling and Philip W Ingham 1 Introduction

Generation Flowchart

We describe a standardized mutagenic protocol and a methodology for small-scale directed screening of the zebrafish genome for mutations in specific developmental processes. The methods are based primarily on those developed for large-scale screens in Tubingen, Germany Boston, MA and Eugene, OR as well as our experiences with a smaller facility. By combining a marker-based screening protocol with both haploid and diploid screening methods, one can efficiently recover mutants in specific...

General Principles Guiding Cell Line Production from H2KbtsA58 Transgenic Mice

A number of general principles emerge from our experiments to date. One point of central importance is that we think it most appropriate to utilize heterozygotes for the generation of cell lines in most (and possibly all) circumstances. The successful derivation of cell lines from heterozygotes offers a number of practical advantages, not least of which is the fact that it is not necessary to maintain a homozygous breeding colony if one's intention is to use only animals from a limited series...

Stages and Major Features of Limb Regeneration

Figure 1 shows a series of drawings following amputation either through the midradius and ulna (the zeugopodium level) or the midhumerus (stylopo-dium level) of an adult newt limb. This emphasizes several principles whatever the level of amputation, the regenerate is a perfect copy of what was removed the stages are the same whatever the level of amputation regeneration from the lower arm is completed more quickly than from the upper arm. The latter occurs because there is less tissue to be...

Skeletal Staining

Introduction The retinoids comprise a large group of natural and synthetic compounds related to vitamin A (retinol). The family name is derived from the early observation of the necessity of vitamin A for normal vision, and the association of vitamin A deficiency with night blindness (1). With the exception of the visual cycle in the rod photoreceptor cells of the retina, in which protein-bound 11-cis-retinal is reversibly isomerized to free all-trans-retinal, retinoic...

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...

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 pg 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...

Marie Aimee Teillet Catherine Ziller and Nicole M Le Douarin 1 Introduction

The understanding of several mechanisms that are essential for embryonic development has greatly benefited from cell-marking techniques that allow tracing of definite cells and their progeny, and thus, the study of their behavior and fate. A cell marker must be precise and stable it must not interfere with normal development. The quail-chick labeling technique meets these requirements perfectly. The principle of the method (1) is based on the observation that in all embryonic and adult cells of...

Lineage Analysis by Single Cell Injection of Fluorescent Dextran

Labeling single cells in ovo and then studying their fate during subsequent development can be a powerful tool in the investigation of how embryos develop. It can be used to construct fate maps at the single-cell level, analyze morphogenetic movements, address issues of tissue specification, and assess the importance of lineage in the determination of regional or cellular identity. One way of labeling single cells is by infection with replication-deficient retroviral vectors. This method allows...

Cell Lines from Transgenic Animals

One way to make certain that all cell lines produced in an experiment contain the same site of integration of the immortalizing gene is to create cell lines from transgenic animals. Since all cells derived from such an animal will share the same insertion site, at least this source of variability in the biology of cell lines will be brought under more stringent control. The possibility of making cell lines from such transgenic animals was recognized from the earliest studies in which...

The Mouse as a Developmental Model

The laboratory mouse, Mus musculus, is the developmental biologist's mammal of choice for studies of development. Its embryology and genetics have been extensively studied for over 100 years. However, it is the advent of in vivo gene manipulation in the last few years that has established the mouse as probably the single most powerful animal system in vertebrate biology. Mouse developmental biology, as it exists today, has its origins in genetics and embryology. These days it is hard to...

Direct Derivation from H2KbtsA58 Transgenic Mice of Conditionally Immortal Myoblasts Able to Differentiate into

Transplantation of myogenic cells has been discussed as a treatment for genetic disease, in muscle and other tissues (75,76), and also holds promise as a tool for investigating the regulation of expression of muscle genes in vivo. However, there are two major obstacles to full exploitation of these techniques the finite mitotic capacity of primary myoblasts limits the cell numbers available from clones, whereas the tumorigenic tendency of established myogenic lines makes them difficult to use...

Methods and Protocols

Sharpe and Ivor Mason Molecular Embryology Methods and Protocols Edited by Paul T. Sharpe and Ivor Mason Dental and Medical Schools of Guy's, King's, and St. Thomas's Hospitals, King's College, London, UK Some images in the original version of this book are not available for inclusion in the netLibrary eBook. 1999 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system,...

Replacement of Blastoderms into the Ring Cultures and Completion of Setting Up

The antisense effect in this work is relatively transient within the time scale of development (one of its potential analytical advantages), so that it works best if little developmental pause in real time occurs immediately following administration of oligos. Chick blastoderms at streak headfold stages are very susceptible to a delay in resumption of development even at normal incubation temperature, following experience of a rapid downward temperature shift. Ideally, therefore, the return to...

Control of Limb Regeneration

Nerves Since the pioneering experiments of Todd (1), it has been known that a denervated limb will not regenerate. The incredibly detailed series of experiments by Singer and colleagues in the 1940s (16) in which he partially dener-vated limbs, counted the remaining nerve fibre numbers at various limb levels and then recorded the resulting frequency of regeneration, led to the hypothesis that a threshold number (between 30 and 50 of normal) was required for regen- Fig. 2. (continued) At the...

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...

Culture of Postimplantation Mouse Embryos

A major disadvantage of working with postimplantation mammalian embryos is their relative inaccessibility to experimentation while they develop within the maternal uterus. Two techniques allow us to get around this problem to a large extent. The first, which is the subject of this chapter, can be used for mouse embryos explanted between 7.5 d of gestation (E7) and 12.5 d of gestation (E12), and involves dissecting embryos from the uterus and culturing them in roller bottles (1). In this way,...

Fish Raising and Embryo Manipulation

Tanks and water (see Note 2) Systems for rearing and maintaining zebrafish range from commercially available modular systems to small-scale, self-built facilities (8,12). What is of most importance, however, is water quality. To manage a diploid screen efficiently, fish must lay in a high percentage of pair matings. This seems to relate, at least in part, to the water quality. Tap water quality can be greatly improved by the use of filtration systems. Charcoal filters seem to be neccesary with...

Ring Culture Procedure Modified After ref [12 with Preincubation in Oligos

Part 1 is carried out in the dish (see Subheading 2., item 12) with a black area on the bottom, in a layer of PC saline covering the yolks, at between 25 and 30 C (where they rapidly cool to 20 C see Notes 3 and 4). Initially, process 6 yolks maximum 25-cm diameter dish, though with skill (speed in this case), 15 or more can be set up from such a dish. This is a naked-eye operation with good overhead lighting, but many workers could also use an engineers anglepoise lamp, which is in a ring...

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...

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...

Chemicals and Solutions

100 Hank's (1) 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3. It is made from stock solutions that can be kept for several months. Sodium bicarbonate solution is made fresh. Solution 1 8.0 g NaCl, 0.4 g KCl in 100 mL water. Solution 2 0.358 g Na2HPO4, 0.6 g KH2PO4 in 100 mL water. Solution 3 0.72 g CaCl2 in 50 mL water, Solution 4 1.23 g MgSO47H2O in 50 mL water. Hank's premix combine the following in order, 10 mL solution 1, 1 mL solution...

Sperm Nuclei Preparation

We have generally followed the standard protocol of Murray (4), but have omitted the protease inhibitors leupeptin and phenylmethylsulfonyl fluoride from many steps to avoid transfer into the final mixture, which is diluted for egg injections. 1. Dissect and isolate the testes from a male a. Anesthetize a male in a bucket containing a liter of 0.1 Tricaine (MS222, aminobenzoic acid ethyl ester, Sigma A-5040) and 0.1 sodium bicarbonate for at least 20 min (immersion of the animal in ice water...

Microinjection of Fertilized One Celled Mouse and Rat Eggs

Remove about 20 one-celled eggs from storage in M16 medium at 37 C using a general-transfer pipet. 2. Wash the eggs twice in M2 medium. Load them in as small a volume as possible in the transfer pipet, and discharge them into the injection chamber. Observe the entry of the eggs into the chamber using a 4x objective. Try to keep the eggs in a group positioned below the holding and injection pipets. Avoid releasing any air bubbles into the chamber eggs become obscured or even lost, necessitating...

Choice of Objectives

A typical compound microscope will be set up with a range of objectives of increasing magnification, which will all be approximately parfocal (see Note 2) (a standard range would be x2.5, x10, x25, x40, x60 or x100). Parfocality means that it is possible to switch objectives without adjustment to the focus. Where eyepieces have an adjustable focus collar (diopter adjustment), parfo-cality will also depend on whether the diopter of the eyepieces has been correctly set (see Note 3). Low-power...

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

Culture Bead Method Preservation

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...

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...

Fate Mapping with Lipophilic Membrane Dyes

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...

Jane Brennan and William C Skarnes 1 Introduction

Gene trapping in mouse embryonic stem ES cells offers a method to create random developmental mutants with a direct route to cloning and defining the expression pattern of the disrupted gene 1 . Gene trapping involves the use of reporter gene constructs that are activated following insertion into endogenous transcription units. A number of plasmid- and retroviral-based vectors have been developed, which differ in their requirements for reporter gene activation reviewed in refs. 2 and 3 ....

Preparation of Culture Media

It is important that the rat and cord blood are spun as soon as possible after collection, preferably before clotting commences. The serum obtained by squeezing the fibrin clot is known as the immediately centrifuged serum. 2. Serum used as medium supplement should have a low lipid content clear instead of cloudy . Hemolyzed serum does not support normal culture of mouse embryos. 3. The medium in the first well of the NUNC slide is used to wash the embryo once before transferring to the...

Killing Mice and Rats

The recommended way of killing mice is by breaking the neck cervical dislocation . This is quick, and causes the animal the minimum of distress. Pick the mouse up by the base of the tail, and place it on the top of the cage. Allow it to run away such that the animal is stretched out with its hindlegs almost in the air and its forelimbs gripping the cage bars firmly. Using a blunt instrument, press down firmly on the base of the skull and pull on its tail. The stretching action breaks the neck,...