Chapter 3.1.2 Generation of transgenic mice by pronuclear DNA injection

w.E. Crusio and R.T. Gerlai (Eds.) Handbook of Molecular-Genetic Techniquesfor Brain and Behavior Research (Techniques in the Behavioral and Neural Sciences, VoL 13) 9 1999 Elsevier Science BV. All rights reserved. CHAPTER

3.1.2

Generation of transgenic mice by pronuclear DNA injection Anthony Wynshaw-Boris, 1 Lisa Garrett, Amy Chen and Carrolee Barlow 2 Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, M D 20892, USA 1Department of Pediatrics, UCSD School of Medicine, La Jolla, CA 92093, USA 2Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA

Introduction

Transgenic mice can be produced in one of two ways: by direct pronuclear injection of cloned DNA into the male pronucleus of a fertilized mouse egg; or by targeted introduction of transgenes by homologous recombination using ES cells. These techniques have allowed for the stable transfer of normal, altered, or chimeric genes into the mouse germ line, thus providing powerful tools with which to study mechanisms underlying gene expression within a physiologic context. The previous chapter (3.1.1) outlined methods for producing knock-out mice using ES cell technology. This chapter will describe methods for generating mice by pronuclear injection. In particular we will discuss technical aspects of creating transgenic mice with emphasis on issues that impact the study of behavior and the brain. For further information about these techniques in general, the reader is referred to several excellent laboratory manuals describing techniques in transgenic manipulation and gene targeting (Hogan et al., 1994; Wasserman and DePamphilis, 1993). Construction of transgenes

Specific genetic alterations can be made in all cells or in specific populations of cells within an animal

using transgenic techniques. There are several unique requirements for the use of constructs to produce transgenic mice in comparison to those used for expression in cell lines. In both situations, a promoter and polyadenylation signal are required, to provide signals for initiating and terminating transcription, respectively. However, in transgenic mice, it has been shown that the inclusion of an intron in the construct leads to much higher levels of expression of the transgene. Inclusion of an intron, along with splicing sequences, may stabilize the resulting transgene mRNA after splicing. It has also been shown that plasmid sequences in commonly used cloning vectors interfere with the expression of the gene of interest and that linearized DNA integrates more readily. Therefore, the transgene can be surrounded by unique restriction sites in the plasmid construct. When preparing the transgene DNA for injection, the construct can be digested with these restriction enzymes. The transgene can then be separated from the plasmid sequences as described below. For this approach to be useful in vivo, the gene of interest must be expressed in the appropriate cell type within the mouse, and at an appropriate time in development to have a desired effect. In order to accomplish this, promoter and other regulatory elements must be included in the transgene

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construct. It is often difficult to determine a priori whether the chosen promoter will actually lead to expression of your gene of interest in the appropriate spatial and temporal pattern within the mouse. For example, not all promoters used in cell culture will result in predictable patterns of expression in analogous mouse tissues, and various promoters are sensiti~ to integration dependent misexpression. Several techniques have been employed in order to increase the chance that appropriate spatial and temporal transgenic expression will occur. As a first step, specific cell lines can be used to identify the smallest region of the promoter that gives tightly controlled and high levels of expression of the gene of interest. Fibroblast lines, such as NIH/3T3 cells, or differentiated neural cell lines such as PC12 cells, can be used to test for promoter activity. Neural cell lines should be the best ones to test neural-specific promoters. Once a promoter and other regulatory elements have been identified in cells, the defined region can be linked to a reporter gene, such as the /3-galactosidaseencoding LacZ gene. The transgene can be prepared and used for production of transgenic mice, and these mice can then be quickly screened for appropriate patterns of expressions. Such "reporters" are useful because the expression can be monitored in vivo, in whole embryos or tissues, by simple histochemical stains. This type of experiment has also been used to identify and study promoter elements in vivo. It often takes several modifications of endogenous promoters to identify sequences that will give appropriate levels of tissue specific transcription of transgenes in animals, particularly in nervous tissue. For example, the neurofilament nestin is expressed in the developing CNS. However, it is also expressed in the developing tooth and limb. Therefore, the endogenous promoter would not be appropriate for use in a CNS-specific experiment. By using the LacZ reporter gene assay in transgenic mice, researchers were able to identify regulatory regions (enhancer) in the second intron of the human nestin gene required for

expression in embryonic CNS stem cells and in the adult spinal cord after injury. By using this region as well as a portion of the promoter region, CNS specific expression could be obtained in the developing mouse (Lothian and Lendahl, 1997). The standard linear DNA fragments ranging from 5 kb to 50 kb for pronuclear microinjection are generally prepared by growing large cultures of bacteria containing the plasmid or cosmid and purifying the extrachromasomal DNA by double cesium chloride banding or commercial plasmid preparation columns such as Qiagen. However, we have had best success during microinjection with cesium chloride-prepared DNA. Nicked DNA, which reduces the efficiency of making transgenic animals, is removed by cesium banding. The plasmid DNA is then digested with restriction enzymes and the fragment for injection is purified away from any contaminating vector sequences either on a gel or on a sucrose gradient. If the DNA fragment is gel purified, the DNA can be either electroeluted or purified with glass milk beads. If the DNA is purified on a gradient, aliquots must be run on a gel to identify the sample without vector contamination. Vector-free aliquots are pooled and dialyzed against the microinjection buffer containing 10 mM Tris and 0.25 mM EDTA. Our laboratory protocol for purification of transgene DNA follows.

Plasmid DNA preparation Day 0

1. Begin overnight culture of bacteria in 35 ml of 2xYT or LB broth. 2. Mix up and autoclave 1-2 1 of LB broth per prep, cool overnight.

Day 1 1. Add 50 mg ampicillin per 1 liter LB broth for plasmids that contain the ampicillin resistance gene (amp is the most widely used antibiotic, but appropriate concentration of any other anti-

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biotic can be used to grow plasmids with other antibiotic resistance genes). 2. Add 10 ml overnight culture to the LB broth with ampicillin. 3. Incubate at 37~ until the culture reaches an optical density at 600 nm (OD) of ~0.8 (3-6 h). 4. Pellet bacteria in a Sorvall centrifuge, 20 min at 5000 rpm. 5. Resuspend pellet in 12.5 ml 25% sucrose-0.05 M Tris (pH 8.0). Mix and place in Oak Ridge centrifuge tubes (35-50 ml). 6. Add 1 ml 10 mg/ml lysozyme in 0.25 M Tris pH 8.0, incubate for 5 min at room temperature to digest the bacterial cell wall. 7. Add 1.5 ml 250 mM EDTA pH 8.0, keep at room temperature for 5 min, to inhibit lysozyme and DNase. 8. Add slowly 6 ml Triton X-100 solution. (1% Triton X- 100, 50 mM Tris pH 8.0, 62.5 mM EDTA), and shake vigorously to lyse bacteria. 9. Add 10 ml 10 mM Tris, 1 mM EDTA, pH 8.0 (TE) if necessary to bring up volume. 10. Centrifuge at 19 K rpm for 1 h at 4~ 11. Remove supernatnant (decant) into 50 ml plastic Corning tubes. Bring to 30 ml with TE. 12. Add 27 g CsC1 and dissolve (shake vigorously). 13. Add 2 ml 10 mg/ml ethidium bromide (EtBr), and bring to 42 ml final volume with 0.9 g/ml CsC1 in TE. 14. Load into ultracentrifuge tube (16 gauge needle and 30 cc syringe) and seal with caps. 15. Spin at 47,000 rpm for 18-24 h using a vertical ultracentrifuge rotor. Day 2 1. Visualize plasmid (lower band) and bacterial chromosomal DNA (upper band) with an ultraviolet light.

2. Use a needle and syringe to remove lower plasmid band into 50 ml centrifuge tube. 3. Add 2 ml EtBr, and bring to 42 ml with 0.9 g/ml CsC1 in TE. 4. Load into ultracentrifuge tubes as above and spin at 47,000 rpm 18-24 h (second banding). Day 3 1. Remove plasmid band into 50 ml centrifuge tube as above. 2. Extract at least three times with an equal volume of isopropanol saturated with 9 g/ml Cs/C1 in TE. Spin at 3000 rpm for 5 min. Remove all but 1 ml of the top pink phase each time and discard. 3. After the top phase is no longer pink in color, place the bottom phase in a dialysis bag (BRLN 15961-022 3/4 inch 12,000-40,000 Da exclusion weight) equilibrated with TE. 4. Dialyse against 4 1 of TE, changing three times over 24 h at 4~ 24. Day 4

1. Quanitate plasmid DNA for restriction enzyme digestion by absorbance at 260 rim.

2. Digest 50-100 #g of plasmid containing the insert for microinjection, to liberate the transgene from the plasmid backbone. 3. Separate the transgene band from the plasmid band by electrophoresis on a low melting point ultrapure agarose gel. 4. Excise band from the gel and purify fragment by Gene Clean (glass mild beads) using the protocol of the manufacturer and TE for elution. It is important to remove all glass beads. 5. Quanitate DNA with a known standard (preferably a series of dilutions of a reference plasmid between ~ 2 ng-50 ng) on an agarose gel. 6. Dilute DNA to store for microinjection to 100 #g/ml in 10 mM Tris, 0.25 mM EDTA (pH 7.5).

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Construction of Transgenic Mice using Embryonic Stem Cells -I-

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Select for homologous recombinants using positive and negative selection

t endogenous gene homologous recombinant

Screen potential targeted clones by Southern analysis

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Electroporate establishedES cells with gene targetedconstructs

Expand targeted clones for microinjection...

...yield some blastocysts with incorporated stem cells.

... into the blastoc of 3.5 day blastocysts...

Surgically transfer -12 expanded blastocysts into uterine horns of a foster mother...

... after ~ 17 days gestation, many of the offspring are chimeric. Fig. 1. Producing transgenic mice by pronuclear injection. See text for details.

Pronuclear injection Transgenic mice are produced by microinjection of DNA directly into pronuclei of fertilized mouse eggs, resulting in the random integration of foreign DNA into the mouse genome (Fig. 1). Fertilized eggs are isolated the day after mating female mice (induced to superovulate by hormonal treatment) with fertile males. The male and female pronuclei do not fuse immediately after fertilization. The male pronucleus is large and, using micromanipulators to stabilize the egg, more than 100 copies of the purified DNA can be injected into the male pronucleus. The DNA generally integrates at the one cell stage and therefore foreign DNA will be present in every cell of the

"transgenic" animal. The manipulated embryo can be implanted at this time, or maintained in culture until the two cell stage, and is then transferred into the oviduct of a pseudopregnant female where the embryo will continue to develop. Pseudopregnant females are produced by mating females with vasectomized males. The act of mating initiates the hormonal cascade required to prepare the uterus for implantation of embryos, but since the male is sterile, the only embryos that develop and implant are those that have been injected and transferred. Once implanted, the manipulated embryo is carried to term, and delivered. Offspring are analyzed by Southern blotting or PCR, and transgenic animals which have integrated the gene into their DNA are

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referred to as founders. Several founders can be created during each day of injection. These founders, in which DNA has integrated into the germ cells, are able to transmit the transgene as a heritable trait and hence a transgenic line can be established. We produce fertilized eggs for microinjection by superovulating FVB/n females by the following protocol.

Superovulation of FVB/n mice and harversting fertilized eggs Day 3: 1. Inject 15-20 FVB / n donor females (3-4 wk) with 5 units of PMS (pregnant mares serum, Calbiochem 1000 I.U./vial). The time of hormone injection can be from 11:30 a.m. until 1:00 p.m. It is best to inject earlier in this interval. Day 1: 1. Inject 15-20 FVB / n females (previously injected with PMS) with 5 units of HCG (Organon, 10,000 units/vial) and mate with stud FVB/n males, one female per male. 2. On the same day, mate 20 CB6F1 female recipients (for the production of foster females for reimplantation of fertilized eggs) that are in estrus (moist, pink vagina, slightly gaping introitus) and mate with 20 vasectomized CB6F1 males. Vasectomized males can be purchased from Jacson laboratories, or made surgically in the lab of the investigator. Following induction of anesthesia (Avertin ~ see below) and sterilizing the surface of the skin with ethanol, a 1 cm midline incision is made through the skin, then through body wall. One testis is pushed up from scrotal sac into the abdomen and the testis fat pad is pulled through the incision. The vas deferens is identified and ligated in two places. A segment of vas between two ligatures is removed. This is repeated on the other

side. The body wall is sutured and the skin is clipped or sutured. Day 0: 1. Separate F V B / N females for egg harvest. Check for mating by the presence of a hard white copulation plug at the vaginal introitus. Separate "plugged" CB6F1 females from vasectomized males. 2. On the day of harvest, prepare six 35 mm dishes with 2 ml of M2 media (PGCcat. -r162 each. Add 0.75 ml of 10 mg/ml hyaluronidase in D M E M to two of the dishes. Place on 37~ slide warmer under a 150 mm dish with an extra 35 mm dish filled with water (for humidity). 3. Sacrifice mice by anesthesia and cervical dislocation and expose abdominal cavity. Remove both ovaries by cutting above the ovary and below the fallopian tubes and avoid taking as much fat as possible. Transfer the ovaries to the two dishes containing M2/hyaluronidase (divide the number of ovaries between the 2 dishes, 20 per dish). 4. Using a dissecting scope, release eggs from the swollen ampulla area and transfer torn ovary out of dish. (It is easiest to use Dumont # 5 forceps to tear open the ampulla). 5. Do not leave eggs in the media with the hyaluronidase for more than 10 min. Transfer the eggs to a new M2 dish without hyaluronidase. Transfer again to a second dish of M2 and get an approximate count. Eggs are now ready for injection. 6. To prepare D N A for microinjection, dilute construct D N A to approximately 2 ng/#l in a 200 ml total volume. Spin diluted D N A for 10 min at 14,000 rpm using Eppendorf microcentrifuge. Pull off all but 20 #1 and aliquot centrifuged D N A into 4 x 1.5 ml tubes. Place pulled needles

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into D N A solution and allow to backfill up to the tip of needle. 7. For microinjection, prepare a clean glass depression slide as follows: place a 25 #1 drop of M2 media in the center of the depression and cover the entire depressed area with light weight mineral oil. Transfer ~ 20-50 eggs into center of the drop. It is best to start out with a fewer number of eggs for injection. You do not want the eggs to sit under the microscope for more than 20 min at a time. Transfer injected eggs into a new dish of M2 media. If you are not going to implant the embryos on the same day (1-cell), transfer the unlysed, injected eggs into BMOC-2 media (PGC- cat. -CO-MR-013-D) which does not contain HEPES and incubate with CO2 at 37~ for over night culture.

concentration ranging from 1-5 ng//A, with a needle approximately 1-2 #m in diameter. The fertilized egg is held by the holding pipette to expose the two haploid pronuclei. The female pronucleus is smaller, while the male pronucleus, derived from the sperm, is larger. Because of the size difference, the egg is positioned to allow the injection pipette to penetrate to zona pelucida, and the male pronucleus. Injection of the D N A solution into the pronucleus causes swelling, a visual confirmation that D N A has been injected into the nucleus. Injected fertilized eggs are reimplanted into pseudopregnant foster females by the following protocol.

Transfer to recipients:

Equipment~materials needed The apparatus used for pronuclear injection of DNA solutions is similar to that used for microinjection of ES cells, as described in the previous chapter. A Zeiss Axiovert 135 inverted microscope with Narishige hydraulic manipulators or Eppendorf motorized manipulators is placed on an air table or vibration resistant table. One manipulator is attached to a holding pipette, which is linked to a syringe that controls gentle vacuum pressure to stabilize the fertilized egg for injection. The other manipulator is attached to the injection pipette, which is attached to an air supply to push the D N A solution out of the injection needle, or to an automatic microinjector (produced by Eppendorf, for example). Holding and injection pipettes are made from glass pipettes using a programmable pipette puller, to create glass needles of the appropriate diameters. Injection needles are made to a fine point, with a narrow opening to allow the DNA solution to be injected (shown schematically in the figure). To make the holding pipette, a microforge is used to break off the pipette to the appropriate size, and to polish the end to make it smooth, so as not to damage the egg. Plasmid or cosmid D N A is microinjected at a

Slide warmer for recipients and eggs (37~ 4r Dumont forceps (two) Scalpel ( # 11 or ~ 15) Serrafin clip Microdissecting forceps with teeth Microdissecting forceps with serrated, blunt tip 4-0 suture 9 mm surgical wound clips and applier Sterile drapes Avertin anesthetic agent. Avertin stock is a solution of 1.0 g 2,2,2-tribromoethanol dissolved in 0.5 g tertiary amyl alcohol. Store the stock solution in the dark in a glass container. Dilute the Avertin to the final working concentration by dissolving 1.2 ml of the stock in 100 ml of boiling PBS. Shake to dissolve. Aliquot and store at -20~ Syringe (1/2 to 1 ml) and needle (25-28 1/2 ga) Dissecting microscope with fiber optic light source Glass micropipettes Mouth pipetter 1. Following intraperitoneal injection of anesthetic agent into the pseudopregnant females

278 and induction of deep anesthesia, the back of the mouse is washed with ethanol. 2. Place the mouse on a 4 x 4 gauze pad on the dissecting scope platform with the right side towards you. Approximately one-third of the way of the back, make an incision in the skin approximately 1 cm in length and approximately 1 cm to the right of the spine. To locate the region of the ovary, identify a vessel and a nerve running diagonally across the ovarian fat pad (the fat pad should appear easily as a white mass just below the surface of the peritoneum). The ovary is just caudal to the fat pad. Grasp the peritoneum with the micro-dissecting forceps with teeth and make an incision approximately 0.5 cm in length above the fat pad. With blunt forceps, pull out the fat pad with the ovary attached through the incision and place on a sterile drape. Anchor the fat pad and ovary in place with a Serrafin clip. Do not grasp the ovary or oviduct, but only the fat pad. 3. Adjust the magnification on the dissecting microscope so that the ovary and the oviduct are visible in the field of view. 4. Focus so that the interior of the infundibulum is visible. In the infundibulum, look for the oviduct opening at the end of the oviduct. With -r162 Dumont forceps, carefully tear the mesovarium away from the ovary and oviduct, without tearing any blood vessels. 5. Collect 15-20 one cell embryos in the glass transfer pipette with an absolute minimum of M2 liquid. Rate of loading and volume can be controlled by the introduction of air bubbles. Insert the pipette into the infundibular opening and gently blow the embryos into the oviduct. Also blow a small bubble of air into the oviduct to be sure that all the embryos have been expelled. 6. Gently push the ovary and uterus back into the peritoneal cavity with the blunt forceps. 7. Place one suture into the musculature and close the skin with 2 wound clips. 8. When transfer is complete place the recipient in a warm recovery cage.

9. When recovery is complete transfer the recipients to a clean cage. Implanted pseudopregnant females give birth after 19-21 d, depending upon the strain used. Once the animals are old enough, a tail biopsy is performed to isolate DNA, and founder lines containing the transgene are identified. Under ideal conditions, the efficiency for generating transgenic founder mice containing 5-50 kb fragments is between 20-30%. In general, the transgene integrates as 1-50 copies arranged in a head-to-tail orientation (tandem repeats). Founders must then be screened for expression of the transgene RNA or protein, using the various techniques described above, as sequences surrounding the integration site may result in undesired patterns of expression or may even prevent the gene from being expressed. In general, the expression pattern from integrated transgenes remains stable over many generations.

Analysis of transgene expression in founder lines and offspring The expression of most transgenes is not as easily detectable as the lacZ marker gene. Consequently, it may be difficult to confirm that the transgene is expressed in the appropriate tissue. This can be particularly important when studying transgene expression in the brain, as it is likely necessary to be able to precisely identify subsets of neuronal populations that express a transgene. It may be possible to develop an antibody that is specific for the transgene product and does not cross react with any endogenous form of the protein in the mouse. Such an antibody could be used to perform immunohistochemistry on sections, but it is rare that such antibodies can be made with that degree of specificity to the transgene relative to homologous endogenous mouse genes. R N A in situ hybridization can also be used if there are unique sequences in the transgene that are not present in any endogenous gene, such as the junction between the polyA tail and the cDNA.

279 As an alternative approach, several types of modifications of transgenic constructs have been employed to allow expression of the transgene to be followed carefully in vivo. It is possible to link the expression of the gene of interest to a "reporter" gene to mark the cells where the transgene is expressed. For example, one can create a fusion protein by linking the gene of interest to a reporter fragment or gene. Unique protein domains, or "tags," such as hemagluttinin or c-myc, have been placed at the amino or carboxy terminus of transgenic proteins. These domains often do not alter the activity of the protein, and good antibodies have been developed to detect tagged proteins by Western blotting or immunohistochemistry. One disadvantage of this approach is that these mutant fusion proteins may not have the same biological properties as unmodified proteins. An alternative approach is to take advantage of various viral internal ribosome-entry site (IRES) which permit the effective internal initiation of translation in mammalian cells by directing efficient mRNA cap-independent entry of the translation apparatus. The promoter/regulatory region is linked to a transgene that includes the gene of interest, followed by the IRES sequence and finally a reporter gene. A single mRNA is produced, but two proteins are coded for, and the IRES allows both to be translationally initiated. Several sensitive histological markers, such as the aforementioned /~-galactosidase encoding lacZ gene and the human placental alkaline phosphatase (hpAP) gene, have been used (for a recent review of the technique see Li et al., 1997). In addition, use of the IRES system eliminates the need to construct a transgene fusion that has the correct translational reading frame for efficient production of the reporter. By using transgene tags, fusion proteins or dicistronic IRES-containing constructs, one can identify precisely which cells express the transgene by evaluation of the presence of the reporter using standard immunohistochemistry techniques which are especially important when studying the brain.

Transgenic mice produced with large DNA fragments Recently, it has become possible to create transgenic mouse strains from the injection of large DNA vectors, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) (Peterson et al., 1993; Peterson et al., 1995; and Gnirke et. al., 1993; for additional examples see Chapters 5.8 and 5.9). Although technically more difficult than the injection of smaller transgenes, the introduction of large DNA fragments raises the possibility of doing functional genomics in the mouse (Smith and Rubin, 1997). For example, mice that are trisomic for a region of human chromosome 21 have been created by injecting YAC clones (Smith et al., 1997). These mice have been used to examine sequences which influence learning and memory when present in three copies. YAC transgenesis has also been used to rescue a specific neurologic phenotype and verify that the transgenic fragment contains the mutant gene. For example, the mouse mutant gene vibrator (vb) results in an early-onset progressive action tremor, degeneration of brain stem and spinal cord neurons, and juvenile death. This gene was cloned using an in vivo positional complementation strategy. The vb locus was mapped to a region of the mouse genome, and the region containing the gene was narrowed to a 76 kb region by transgenic complementation of the phenotype by injecting YACs from the region. Once the region was narrowed, the vb mutation was found to be an intracisternal A particle insertion in intron 4 of the phosphatidylinositol transfer protein alpha gene (Hamilton et al., 1997). We have found that there are several important points to consider when preparing and microinjecting DNA over 100 kb in size: mechanical shearing and denaturation of the DNA; DNA concentration; and purification of the DNA to avoid clogging the microinjection needle. YAC DNA is especially large and can

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easily be sheared in solution. Therefore, yeast containing a YAC to be microinjected are generally embedded in agarose plugs. Enzymatic digestion by yeast lytic enzymes within the agarose plug breaks open the yeast cell wall, releasing the YAC and yeast chromosomes, and plugs are treated with detergents for deproteinization. The YAC DNA is purified on pulse field gels in order to separate the YAC from contaminating yeast chromosomal DNA. It is important to have a high concentration of yeast per agarose plug to purify the YAC DNA. YAC D N A is carefully excised from the pulse field gel and treated gently with agarase, concentrated by ultrafiltration and quantified. High molecular weight D N A must be handled carefully to avoid shearing. It is usually manipulated with large bore pipette tips and diluted with a microinjection buffer that contains additional NaC1 to stabilize the DNA. For microinjection, the YAC is injected at a concentration of 1 ng/#l. The microinjection needle is prepared with a much larger opening than a needle used for plasmid or cosmid injection, and injection of YAC D N A generally requires frequent needle changes. The efficiency for production of intact YAC transgenics ranges from 5-20%. BAC or PAC D N A can be grown in large bacterial cultures and purified similarly to plasmid DNA (Yang et al., 1997). Chromosomal D N A is separated from extrachromasomal D N A by cesium chloride banding or sucrose gradients and then dialyzed against the microinjection buffer. BAC and PAC D N A is usually microinjected as circular DNA, but linear D N A can be injected as well, and careful handling is required as described above for YACs. Sepharose chromatography can be used to separate circular from linear DNA. It is not necessary to remove the plasmid backbone D N A from BAC or PAC DNA, since these sequences have not been found to interfere with transgene expression. Larger needles are used as well as large bore pipet tips for transferring the DNA. The efficiency of gener-

ating transgenic founder animals with BAC or PAC D N A is 10-20%.

Use of chicken retroviral receptors to target gene transfer and expression It has recently been reported that by using a combination of transgenics and retroviral infection, high rates of delivery of target genes into adult nervous tissue has been observed (Holland et al., 1998). In these experiments a transgene encoding a specific receptor for an avian retrovirus (A avian leukosis virus ALV-A) under the control of an astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was used to make a line of transgenic mice expressing the retrovirus receptor in glial tissue. These transgenic mice were then infected with a replication incompetent ALV-A virus that expressed a particular gene of interest, in this case a basic fibroblast growth factor by direct injection of virus into the ventricles of newborn mice. These viruses were made with a retroviral expression vector (RCAS), and virus was produced in chicken viral packaging cell lines. The only tissues infected were those that express the retroviral receptor, namely glial cells, sparing all other tissues from the consequences of infection. The authors were able to demonstrate glial specific infection and concomitant expression of the retrovirally delivered gene. Multiple viruses can infect the same cell, since transgenically expressed chicken receptor is not blocked by ALV-A infection. Consequently, it is possible to infect each cell with different viruses, each containing a different gene, to assay the effects of multiple genes simultaneously on a defined cell population in vivo. Using this type of methodology, one could envision that several different types of promoters, such as nestin, or other neuron specific promoters, could be used to allow for infection of neurons as well as glia. This would allow for testing the effects of overexpression of a wide variety of products directly in the nervous system of adult animals,

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and for following their effects on neurologic function and behavior. Strain issues The use of specific strains for the production of transgenic animals has implications in their use for studies of behavior (e.g. see Chapter 3.1.10). The most commonly used mouse strains for isolating fertilized eggs are C57BL/6J, FVB/n, or F1 hybrids such as C57BL/6J • C3H. These mice, especially FVB/n, have practical advantages for pronuclear injection, such as large egg yield, large and easily visualized pronuclei, and resistance to lysis after injection. However, there are no absolute technical limitations to using virtually any mouse strain for producing transgenic mice, since the pronuclei of all mouse fertilized eggs can be visualized microscopically. Of particular relevance to the topic of this book, there is a wide range of neurologic and behavioral phenotypes displayed by different strains of mice (Crawley et al., 1997). The choice of mouse strain for a particular transgenic experiment should be guided by the phenotypes that one wishes to address. Conclusions It is now feasible to manipulate the mouse genome to make precise genetic alterations, and to create gain-of-function and loss-of-function alleles of specific genes. In these two chapters, we have briefly reviewed the techniques required to create genetically manipulated mice using either pronuclear injection or targeted mutagenesis in ES cells Other chapters in this book will provide further details and examples of the use of these techniques to study neurologic and behavioral function in the whole animal. References Crawley, J.N., Belknap, J.K., Collins, A., Crabbe, J.C., Frankel, W., Henderson, N., Hitzemann, R.J., Maxson, S.C., Miner, L.L., Silva, A.J., Wehner, J.M.,

Wynshaw-Boris, A. and Paylor, R. (1997) Behavioral phenotypes of inbred mouse strains. Psychopharmacology, 132: 107-124. Gnirke, A., Huxley, C., Peterson, K. and Olson, M.V. (1993) Microinjection of intact 200-500 kb fragments of YAC DNA into mammalian cells. Genomics, 15: 659-667. Hamilton, B.A., Smith, D.J., Mueller, K.L., Kerrebrock, A.W., Bronson, R.T., Van Berkel, V., Daly, M.J., Kruglyak, L., Reeve, M.P., Nemhauser, J.L., Hawkins, T.L., Rubin, E.M. and Lander, E.S. (1997) The vibrator mutation causes neurodegeneration via reduced expression of PITP alpha: positional complementation cloning and extragenic suppression. Neuron, 18: 711-722. Hogan, B., Beddington, R., Costantini, F. and Lacy E. (1994) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Holland E.C. and Varmus, H.E. (1998) Basic FGF induces cell migration and proliferation after glia-specific gene transfer in mice. Proc. Natl. Acad. Sci. USA, 95: 1218-1223. Lothian C. and Lendahl U. (1997) An evolutionarily conserved region in the second intron of the human nestin gene directs gene expression to CNS progenitor cells and to early neural crest cells. Eur. J. Neurosci., 9: 452-462. Li, X., Wang, W. and Lufkin, T. (1997) Dicistronic LacZ and alkaline phosphatase reporter constructs permit simultaneous histological analysis of expression from multiple transgenes. Biotechniques, 23: 874-878, 880, 882. Peterson, K.R., Clegg, C.H., Huxley, C., Josephson, B.M., Haugen, H.S., Furukawa, T. and Stamatoyannopoulos, G. (1993) Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human fl-globin locus display proper developmental control of human globin genes. Proc. Natl. Acad. Sci. USA, 90: 7593-7597. Peterson, K.R., Li., Q.L., Clegg, C.H., Furukawa, T., Navas, P.A., Norton, E.J., Kimbrough, T.G. and Stamatoyannopoulos, G. (1995) Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of fl-globin locus YAC mice carrying human globin developmental mutants. Proc. Natl. Acad. Sci. USA, 92: 5655-5695. Smith, D.J., Stevens, M.E., Sudanagunta, S.P., Bronson, R.T., Makhinson, M., Watabe, A.M., O'Dell, T.J., Fung, J., Weier, H.U., Cheng, J.F. and Rubin, E.M. (1997) Functional screening of 2 Mb of human chromosome 21q22.2 in transgenic mice implicates minibrain in learning defects associated with Down syndrome. Nat. Genet., 16: 28-36. Smith, D.J. and Rubin, E.M. (1997) Functional screening and complex traits: human 21q22.2 sequences affecting learning in mice. Hum. Mol. Genet., 6:1729-1733. Wasserman, P.M. and DePamphilis, M.L. (1993) Guide to techniques in mouse development. Methods in Enzymology, Vol. 225. Academic Press, San Diego. Yang, X.W., Model, P. and Heintz, N. (1997) Homologous recombination based modification in Esherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotech., 15: 859-865.