Identification of Genes Cooperating with Oncogenes

Techniques have now been developed that allow for the identification and cloning of novel endogenous murine genes that cooperate with a transgenic oncogene to elicit tumorigenesis. These techniques, termed retrovirus-tagged mutagenesis, involve the infection of a transgenic animal with a reterovirus that lacks an oncogene, but that is able to promote tumorigenesis through the inadvertent insertion within or adjacent to a proto-oncogene, thereby increasing its expression or altering its...

Microinjection of Cloned DNA Fragments into Fertilized One Cell Mouse Eggs

Central to the process of making transgenic mice is the physical introduction of cloned DNA fragments into fertilized one-cell mouse eggs. First described 10 years ago by a number of investigators (1-5), microinjection remains the most popular and successful of the methods currently available for generating transgenic animals. Microinjection continues to be the method of choice, because the advantages of speed and reliability far outweigh the demands placed on the investigator for precise...

Notes

The vasectomy operation should be performed on young, healthy, sexually mature males of around 8 wk of age. Any strain with good sexual performance can be used. We have found that Swiss 3T3 mice perform well. 2. Following the operation, the mice should be caged individually and allowed to recuperate for at least 3 wk prior to the presentation of their first female. 3. Vasectomized males should be presented with females, at the most, every other day The performance of each male should be...

Toxicity Testing and Tumor Therapies

Cancer in humans is thought to be caused principally by exposure to environmental carcinogens that result in mutations. Transgenic mice are now being used to screen for such carcinogens. The MMTV-c-mjc mice developed by Leder are commercially available (marketed by Charles River, Wilmington, MA). The toxicity of particular substances can be tested by exposing the transgenic animals to it and asking if the pathology of the tumorigenesis is altered. Conversely, transgenic animals with...

Vaginal Smearing of

The various stages of the estrous cycle of the rat are most accurately determined by vaginal smearing. Vaginal smearing is carried out at approximately the same time each morning. The rats are iden- Changes in Cellular Content of Vaginal Smears Associated with an Ovulating Female Rat Stage of estrous cycle Cell characteristics Mostly squamous cornified cells Mostly leucocytes Epithelial cells and leulocytes Mostly round, nucleated cells tified either by ear-punches or tail-marks. Vaginal...

Immunohistochemistry Peroxidase DAB Reacted

The pretreatment of the slides consists of washing in isotonic buffer, blocking the endogenous peroxidase activity with H202, and blocking nonspecific binding with BSA and serum. The primary antibody is incubated overnight at 4 C. 1. Wash in PBS, pH 7.4, for 5 min. 2. Treat with 3 (v v) H202 in PBS containing 0.1 (w v) Na Azide for 3 min. 3. Wash in PBS containing 0.1 (w v) saponin, 1 (w v) BSA, and 2 (v v) blocking serum for 10 min. 4. Drain slides and pipet on 50-100 JL of first (primary)...

Recovery of Egg CylinderStage Embryos 55 d pc

Females are sacrificed by cervical dislocation, and the belly region swabbed with 70 alcohol. The abdominal cavity is then opened to reveal the reproductive tract as in Section 3.2.2. In turn, each uterine horn is cut just below the utero-tubal junction. 2. Grasping the end firmly with forceps, the uterus is pulled taut, and scissors used to tear away the mesometrium. Next, the uterine horn is opened by sliding the tip of a pair of iris scissors down along the antimesometrial wall of the...

Models for Gene Therapy

Although DNA introduced into mammalian somatic cells or injected into mouse eggs recombines with coinjected molecules quite readily, the foreign DNA typically integrates randomly within the chromosomes (1-3). Achieving targeted insertion therefore requires effective selection or screening strategies, or a method for reducing the nonhomologous events. One of the unique features of the MHC genes that make them of particular interest for insertion into mice is that inbred mice can be used that...

Materials for Karyotype Analysis

Fixative 3 vol of absolute methanol to 1 vol of glacial acetic acid. 3. 2X standard saline citrate (SSC) 0.3M NaCl, 0.03M trisodium citrate. 4. Gurr's phosphate buffer, pH 6 (e.g., BDH Chemicals, Poole, UK). 5. 0.25 (w v) Trypsin (porcine Difco Laboratories Detroit, MI ) dissolved in Gurr's phosphate buffer. 6 5 (v v) Giemsa Gurr's R-66 stain (e g., BDH) in Gurr's phosphate buffer. 3. Methods 3.1. Preparation of Feeder Cells Historically, ES cells have been isolated and maintained on layers of...

References

1 Foulds, L. (1954) Tumor progression. Cancer Res. 17, 337-339 2. Foulds, L. (1969) Neoplastic Development. Academic, New York 3. Nowell, P. C. (1986) Mechanisms of tumor progression. Cancer Res. 46,22032207. 4. Weinberg, R. A. (1989) Oncogenes and multistep carcinogenesis, in Oncogenes and the Molecular Origin of Cancer (Weinberg, R. A., ed), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 307-326. 5. Cairns, J. (1981) The origins of human cancers Nature 269, 346-348. 6. Folkman, J....

Milk Proteins

The mammary gland is a useful target for the expression of transgenes that have been modified to produce foreign proteins of high value e.g., pharmaceutical and growth factors. The concept stems from the proposal of Palmiter et al. (6) that transgenic animals might be used for the manufacture of valuable proteins, and the use of the mammary gland itself for this purpose was pioneered by Clark and his colleagues, using the sheep P-lactoglobulin gene as a promoter source and the sheep as the...

Transgenesis Applied to Neurology

In the field of neuroscience an immediately apparent application of transgenic technology lies in the development of animal models designed to exhibit specific neurological syndromes and disease states. The current emphasis on neurodegenerative diseases, which reflects an increasingly evident incidence within aging populations, has provided a major impetus for such studies. In cases where characterized genes have been implicated in the etiology of brain disorders, it is possible to design...

Chloramphenicol Acetyl Transferase Assay

Tissue extracts are prepared by homogenization in 0.2 mL of 0.25M Tris-HCl, pH 7.8, followed by three rounds of freeze-thawing by immersion in liquid nitrogen (3 min) and in a 37 C water bath (3 min). Vortex the suspension thoroughly after the final thawing step, and then spin at 14,000 rpm in a microcentrifuge at 4 C for 10 min. Retain the supernatant for the analysis of CAT activity. Prior to assay, heat the extract to 65 C for 10 min to destroy endogenous CAT inhibitors see Note 1). 2 Set up...

Hybridization and Washing see Note

Identical filters are hybridized with equal counts corresponding to equal amounts of RNA synthesized in nuclei isolated from different tissues from the same animal or from equivalent animals, or from the same tissue taken from different animals at different developmental stages or subjected to different physiological conditions. 2. Filters are incubated for at least 60 h at 65 C with labeled RNA in as small a vol of hybridization buffer as possible (1-2 mL depending on the size of the filter)....

Choice of Transposon and Marker Gene

A large number of vectors suitable for constructing transposons have been described. We will consider here three of the more widely used ones. The transformation vectors based on rosy (ry) as a scorable marker were the first to be used. One of the most versatile versions of the ry-based vectors is pDM30 (4), which can be obtained from the Rubin laboratory. The major advantage of using ry-based vectors is that, since 1 of wild-type ry expression is sufficient to yield ry+ eye color, insertions...

Implantation of Microinjected Eggs Chiew Hun Phang

Microinjected eggs are normally incubated overnight in M16 microdrop culture at 37 C in C02 until 1400 h. By this time the eggs will have developed as far as the two-cell stage. The eggs are then implanted into surrogate mothers. As the membrane covering the oviduct of the rat is much tougher than the mouse, we normally cut it with a very fine pair of scissors rather than tearing it. For a female rat that had been successfully induced into pseudopregnancy, the infundibulum will be swollen and...

Isolation of Embryonic Stem Cells from Blastomeres

The following is based on the method of Eistetter (33) for the establishment of ES cells from single blastomeres. (The author has attempted to repeat this work, but all initial ES-like colonies eventually differentiated into trophectoderm.) 1. To obtain single blastomeres from compacted 16- to 20-cell morulae, the zonae pellucidae must first be removed. This may be accomplished by treatment of embryos in either acid Tyrode's solution (pH 2.1) or with an enzymatic solution comprising 0.5 (w v)...

ES Cell Culture Media

Two media formulations are used for ES cell culture. For ES cell isolation, medium contains 20 (v v) serum (ES2o), whereas established ES cell lines may be maintained in medium with only 10 (v v) serum (ES10) and supplemented with recombinant DIA LIF (see Section 3.4.1. and Note 3). In both instances, serum comprises a 50 50 mixture of selected batches of fetal calf serum (FCS) and newborn calf serum (NCS). 1. Medium for murine embryonic stem cell isolation (ES20 medium)-64 mL Dulbecco's...

Isolation of Murine Embryonic Stem Cells

This section describes the typical procedures utilized to isolate ES cells from three different embryonic stages namely, from blastomeres of disaggregated morulae, from the ICM (inner cell mass) of delayed and nondelayed blastocyst-stage mouse embryos, and from d 5.5 pc primitive ectoderm. Emphasis is placed on the isolation of ES cells from blastocysts. However, apart from the timing of the disaggregation of the embryo outgrowth, the isolation procedures used are essentially identical for each...

Microinjection oDrosophila Embryos

Once the needle is lifted safely out of the way, the slide containing the embryos is placed on the microscope stage, so that the eggs have their posterior facing the needle. The micromanipulator is then used to bring the needle into the same plane as the line of eggs. 2. The tip of the needle should be brought level with the center of the first egg, and this is gaged by running the very end of the needle up and down the edge of the embryo. This ensures that the needle will not slide over the...

Modification of the Biochemistry of Domestic Animals

During the evolution of our major species of domestic animals, a significant number of metabolic pathways have been lost by irreparable damage to the genetic information that encodes them. The end products of these pathways must be supplied as essential nutrients in the diet, and there are instances where this source is insufficient for maximum productivity. Genetic engineering now provides the ability to transfer genes encoding these pathways from organisms where the pathways are functional,...

Materials see Note

Denaturing solution A (DSA) AM guanadinium thiocyanate, 25 mM sodium citrate, pH 7, and 0.5 (w v) sarkosyl. This solution is prepared by dissolving 472.8 g solid guanadinium thiocyanate in 500 mL DEPC-treated water (see Notes 2 and 3) followed by the addition of 100 mL of DEPC-treated 250-mM sodium citrate, pH 7, and 5 mL of 100 (w v) sarkosyl. The solutionis made up to 1 L with DEPC-treated water and purified by passage through a 0.45-pm Nalgene (Rochester, NY) filter. 2. Denaturing solution B...

Mating Mice David Murphy

In the process of creating and analyzing transgenic mice, matings between male and female animals are required for the following reasons 1. To produce fertilized one-cell eggs for microinjection. Natural matings between mature females (over 6-7 wk of age) and stud males (over 78 wk of age) can be used to supply the one-cell eggs. Such matings can provide around 10 F2 eggs mouse (from matings between CBA J x C57B1 6 or C57B1 10 F1 male and female mice). However, it is preferable to mate the stud...

The Glyoxylate Cycle

A second project designed to improve the general efficiency of feed utilization in sheep, and to increase specifically nutrient supply to sheep wool follicles involves the introduction of the glyoxylate cycle to sheep. The rationale for this research stems from the fact that the microorganisms that populate the rumen in sheep consume essentially all available carbohydrate in the ingested feed and produce a range of fermentation products, the most important of which, from an energy viewpoint,...

Safety

Ionizing radiation is both mutagenic and carcinogenic. Hence, care must be taken when handling radioactive isotopes, and in disposing of contaminated reagents and tubes. Each worker has a moral (and in many countries a legal) responsibility to ensure not only his or her own safety, but also the safety of his or her colleagues. In most countries, a system of licensing of radioactive workers exists, in which workers must have a basic familiarity with handling radioactivity, or work under a...

Microinjection of Fertilized One Cell Eggs

Remove 20 fertilized one-cell eggs from microdrop storage in the 37 C, 5 C02 incubator (see Note 4). While observing the eggs using a dissecting microscope, transfer the eggs into M2 medium contained in a 35-mm tissue culture Petri dish. Rinse the eggs in M2. Discharge the eggs into the injection chamber using a mouth-operated general eggtransfer pipet. Observe this procedure under the microscope using the 4X objective. Attempt to keep the eggs grouped together as they are discharged. Locate...

Introduction to Transgenesis David Murphy and David A Carter

Over the past decade, a number of techniques have been developed that allow the introduction of defined, cloned DNA sequences into animal germ lines. Once inserted, these sequences, now called transgenes, are stably passed on from generation to generation. In other words, the transgene becomes a part of the genetic make-up of that particular line of animal. Every individual of a particular line will carry the transgene in every cell of its body. Of fundamental importance has been the...

Materials

Molecular biology grade agarose (Gibco-BRL, Gaithersburg, MD). 2. 10X TBE 108 g Boric acid, 55 g Tris base, and 40 mL 0.5M EDTA, pH 8.0, to 1 L. Autoclave to sterilize. 3. 20 x 20 cm gel casting tray, running tank, and electrophoresis power pack. 4. 10 mg mL ethidium bromide in water (caution extremely carcinogenic). 5. 20X SSPE 3.6 M NaCl, 200 m M Na phosphate, pH 6.8,20 mM EDTA. 6. 10X loading dye 40 (v v) glycerol, 10X TBE, 0.1 (w v) bromophenol blue, 0.1 (w v) xylene cyanol. 7 Nylon...

Operation of the Automatic Injection System

With the output tap closed, switch on the Hitachi Bebicon air compressor, and allow the air tank to charge (around 2 min). 2. Switch on the Pico-Injector. Turn down the balance (Pbalance) and injection (Pinject) pressure taps to zero (Fig. 1). The output pressure of these modes is on the PRESSURE digital display when the PRESSURE METER SOURCE control is switched to the appropriate position. 3. Open the tap of the compressor air tank to allow pressurized air into the Pico-Injector 4. Set the...

Synchronization ofEstrus and Superovulation

For the provision of pronuclear stage embryos for microinjection and subsequent transfer to oviducts or uterus, three groups of ewes are treated as Donor Group, Recipient Group A, and Recipient Group B as in Table 1. There can be variations on this theme. The number of sheep entering this program depends on the yield of embryos, the experimental requirements, and the performance of the research team. Normally we would use 16 donor ewes and 14 recipients m the program described in Table 1 for...

Oncogenes and Tumor Progression Factors

An oncogene can be defined as a DNA sequence capable of disrupting normal growth controls resulting in pathological cellular proliferation. However, some of the genes involved in tumor development in the whole animal, for example, those activities involved in angiogenesis, invasiveness, and metastasis, would not fit into this classical notion of an oncogene. They are not directly involved in cellular proliferation, although they may indirectly promote proliferation by, for example, in the case...

Restriction FragmentPrimed Primer Extension

Combine together 24 pL of RNA in formamide, 3 pL of the DNA restriction fragment primer (at 20,000 cpm pL), and 3 pL of hybridization buffer B. 2. Place in a boiling water bath for 3 min to denature DNA. Incubate overnight at a temperature above the Tm of the DNA DNA duplex, but below that of the DNA RNA duplex (see Note 7). 3. Because formamide inhibits reverse transcriptase, it is necessary to purify the nucleic acids by precipitation. Add 170 pL of DEPC-treated water and 500 pL of cold (-20...

SI Nuclease Mapping

After incubation, 270 pL IX SI nuclease buffer, containing 200 U (see Note 8) SI nuclease, are rapidly added to each tube containing the hybridization reactions. The samples are briefly vortexed and placed on ice (see Note 16). After a short centrifugation to bring the liquid to the bottom of the tube, the samples are incubated at 37 C for 1 h. 2. The SI nuclease reactions are stopped by placing on ice. Each sample is extracted once with phenol chloroform, ethanol precipitated, washed once with...

Immunohistochemistry Peroxidase Diaminobenzidene DAB Reacted

Primary antibody The selection of the primary antibody is the most important choice. Aim for one with high affinity and specificity for the antigen of choice. The specificity is most necessary because the tissue section provides an abundance of other proteins, and it is important that the antibody does not stick to them. It is best to use antibodies tested for immunohistochemistry or immunocytochemistry by the manufacturer. 2. Secondary antibody The secondary antibodies are anti-IgG to the host...

Major Histocompatibility Complex MHC 21 Introduction

The MHCs of the mouse (the H-2 complex) and of humans (the HLA complex) encode highly polymorphic cell surface and secreted glycoproteins that are important for immune regulation and function (5,6). Transplantation antigens that are important in allograft rejection are encoded by class I genes that map to the H-2K D L and HLA-A, -B, -C subregions in the mouse and humans, respectively. In addition, there are class I genes that map to the murine Qa and Tla subregions. Mature cell-surface class I...

Injection of Embryonic Stem Cells into Mouse Embryos

The procedure described here is a simplified version of the technique detailed by Bradley (20). 3.2.1. Blastocyst Injection Procedure 1. Embryos to be injected are transferred from the incubator into the injection chamber only in small groups to prevent them from being exposed to lower temperatures for prolonged periods. 2. The injection pipet is lowered to the base of the chamber, and 10-15 healthy ES cells for one injection are individually selected and gently drawn into the pipet by...

Induction of Immunological Tolerance

The immune system of an organism normally does not react to its own cells and tissues. Tolerance to self-antigens is a critical feature of the immune system. The repertoire for antigen bound to self-MHC proteins of peripheral T-lymphocytes in a given animal is controlled in several ways by the collection of germ-line genes available in the animal, by removal of self-reactive cells during tolerance induction, and by positive selection of cells bearing receptors biased for recognition of antigen...

And Mutant Mouse Strains

Transgenic mice constructed on an inbred background provide a novel approach to the study of class I gene expression and antigen function. In the transgenic system, a single well-defined gene can be placed on an inbred background in one generation. If the transgene is properly expressed and the locus carrying the transgene can be bred to homozygosity, the resulting mouse strains would be, in principle, analogous to congenic lines. These transgenic mice would be identical to the parental inbred...

The Modification of the Endocrine System of Domestic Animals

Manipulation of the endocrine status of domestic animals was an obvious early target for transgenesis following the pioneering research of Palmiter et al. (6,7) in laboratory mice and, as a consequence, has received considerable attention over the past few years. The results have been reviewed in detail several times in recent years (13-18), so in this section, only the overall conclusions from the work will be summarized, together with some evaluation of the commercial potential of the...

Loss of Function Experiments

Techniques are now available by which specific cell types can be ablated in transgenic animals. This powerful tool has been developed through the use of cytotoxic genes such as the A subunit of diphtheria toxin (DT-A) or ricin, which may be linked in transgene constructs to cell-specific regulatory elements (24,25). Cells expressing such constructs in transgenics are killed. Although the possibilities of this tech nique are exciting, a number of problems may limit its application in studies of...

Future Uses of Transgenic Fish

Fish are good candidates for transgenic studies. It now seems clear that our knowledge of fish genes and fish gene regulation will rapidly advance with the increased use of fish gene libraries and trans- genie induction with sequences derived from them. It also seems probable that, in the near future, transgenic fish with improved disease resistance, delayed sexual maturity, or increased growth rate will make a significant and valuable contribution to commercial aquaculture. Rainbow Trout These...

Injection of Zebra Fish Eggs

1 Fill the microinjection pipet with DNA solution see Section 2. . Place the pipet tip into about 50 pL of DNA solution on wax paper, and suck up into the pipet using the suction of a 6-mL syringe. 2. Inject the DNA using a continuously flowing pipet maintained with air pressure by an electrically driven automatic microinjector Fig. 3 , which maintains the pressure while injection is conducted. Precalibrate the amount injected by counting the number of seconds that the needle stays in the egg...