[27] Application of LacZ gene fusions to preimplantation development

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[27] A p p l i c a t i o n o f L a c Z G e n e F u s i o n s to P r e i m p l a n t a t i o n D e v e l o p m e n t B y M U R I E L V E R N E T , CLAIRE BONNEROT, PASCALE B R I A N D ,

and JEAN-FRANqOIS NICOLAS Introduction The development of molecular genetics 1and transgenic mouse technology 2'3 offers new tools for studying regulation of genes. In particular, these tools include the introduction of recombinant genes back into the mouse via microinjection 4 or mouse embryonic stem cells. 5'6 However, there are difficulties associated with this approach when applied to the preimplantation stages of development. 7 The sensitivity of detection of almost all reporter genes achieved by measuring enzymatic activity is too low for the minute quantities of material obtainable from preimplantation embryos. Even when this is not the case (see [26], this volume), 8 information is lost concerning localization of the positive cells, an important issue from the time of allocation of cells to either the inner cell mass or trophectoderm. To alleviate these problems, reporter genes encoding enzymes whose activities can be visualized by staining with a chromogenic substrate have been used. The properties of bacterial fl-galactosidase encoded by the LacZ gene 9 make it an almost ideal candidate. The gene is developmentally neutral as proved by the normal developmental potential of fl-galactosidase-positive embryos; LacZ expression is cell autonomous,

i T. Maniatis, E. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 2 R. D. Palmiter, Annu. Rev. Genet. 20, 465 (1986). 3 B. Hogan, F. Costantini, and E. Lacy, "Manipulating the Mouse Embryo: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1986. 4 R. L. Brinster, H. Y. Chen, M. E. Trumbauer, M. K. Yagle, and R. D. Palmiter, Proc. Natl. Acad. Sci. U.S.A. 82, 4438 (1985). 5 B. Mintz and K. Illmensee, Proc. Natl. Acad. Sci. U.S.A. 72, 3585 (1975). 6 M. J. Evans and M. H. Kaufman, Nature (London) 292, 154 (1981). 7 j. Rossant and R. A. Pedersen (eds.), "Experimental Approaches to Mammalian Embryonic Development." Cambridge Univ. Press, Cambridge, 1986. 8 E. Martinez-Salas, E. Linney, J. Hassell, and M. L. DePamphilis, Genes Dev. 3, 1493 (1989). 9 K. Wallenfels and O. P. Malhotra, in "The Enzymes," Vol. 4, p. 409. Academic Press, New York, 1960.

METHODS IN ENZYMOLOGY, VOL. 225

Copyright © 1993 by Academic Press, inc. All fights of reproduction in any form reserved.

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thus allowing documentation of heterogeneity in cell responseS°; the sensitivity of detection is very high; staining for/3-galactosidase activity is easy and, with the appropriate conditions, is almost never affected by background activity; and, finally, quantitative methods to measure fl-galactosidase activity on limited numbers of cells are being developed (see below). There are several issues that can be addressed using these technical advantages: (1) determination of the activity of cis-acting control regions (promoters, enhancers, silencers) of transcriptional units in preimplantation embryos in transient expression assays 11-13 or in transgenic animals, (2) assays of engineered vectors for targeted expression in all cells or in subsets of cells of early embryos, (3) generation of an in situ transgenic e n z y m e marker for the preimplantation embryo, (4) identification of new genes specific to these stages by L a c Z enhancers or promoters traps, 14 and (5) determination of the pattern of expression of an endogenous gene with a L a c Z replacement vector in a mouse embryonic stem cell after transmission in the gene pool. 15 We summarize here techniques for the determination of gene activity in transient expression assays in oocytes, 1-cell embryos, and 2-cell embryos. H'16'17 These techniques are also relevant for analysis of eggs and embryos from L a c Z transgenic animals to gain insight into the molecular mechanism involved in regulation of integrated genes. Ways to obtain synchronized and unperturbed materials are emphasized. A section on inhibition of embryo development is included. L a c Z and Nuclear Locating L a c Z Reporter Genes

Cells of mammalian embryos can produce active bacterial fl-galactosidase. The E s c h e r i c h i a coli L a c Z structural gene is 3.1 kb long and its sequence is known. TM Sources of the L a c Z gene are pCHll0,19 l0 j. R. Sanes, J. Rubenstein, and J. F. Nicolas, EMBO J. 5, 3133 (1986). 11C. Bonnerot, D. Rocancourt, P. Briand, G. Grimber, and J. F. Nicolas, Proc. Natl. Acad. Sci. U.S.A. 84, 6795 (1987). 12H. R. Sch61er, A. K. Hatzopoulos, R. Bailing, N. Suzuki, and P. Gruss, EMBO J. 8, 2543 (1989). 13M. E. Stevens, J. J. Meneses, and R. A. Pedersen, Exp. Cell Res. 183, 319 (1989). 14C. Bonnerot, G. Grimber, P. Briand, and J. F. Nicolas, Proc. Natl. Acad. Sci. U.S.A. 87, 6331 (1990). ~5A. L. Joyner, BioEssays 13, 649 (1991). J6C. Bonnerot, M. Vernet, G. Grimber, P. Briand, and J. F. Nicolas, Nucleic Acids Res. 19, 7251 (1991). 17M. Vernet, C. Bonnerot, P. Briand, and J. F. Nicolas, Mech. Dev. 36, 129 (1992). 18A. Kalnins, K. Otto, U. Ruther, and B. Muller-Hill, EMBO J. 2, 593 (1983). 19C. V. Hall, P. E. Jacob, G. M. Ringold, and F. Lee, J. Mol. Appl. Genet. 2, 101 0983).

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pMC 1871, 2o or, more conveniently, pTZLacZ (C. Bonnerot and J.-F. Nicolas, unpublished results, 1992) (Fig. 1A). 21'22fl-Galactosidase cleaves the substrate X-Gal to yield a reaction product that can be precipitated, leading to a blue staining in the entire cytoplasm (Fig. IC). Cells with very low fl-galactosidase activity show a punctate pattern. The LacZ gene has been modified by an in-phase fusion with a sequence coding for a nuclear locating signal (nlsLacZ).11"21 In nlsLacZ, the 5' region up to the first four amino acids is a fragment of the early region of the simian virus (SV40) genome (nucleotides 5221 to 5133). 22 It contains the initiation translational AUG codon of the t antigens. This segment is linked to a segment containing the nuclear locating signal (amino acids 127 to 147 of large T antigen of SV40), fused to fl-galactosidase at the sixth amino acid. The source of nlsLacZ is L7RH fl-Ga111'21 or, more conveniently, pTZnlsLacZ (C. Bonnerot and J.-F. Nicolas, unpublished results, 1992) (Fig. 1B). The encoded fl-galactosidase yields, when using the substrate X-Gal, a blue staining in the nucleus 11 (Fig. 1D). One advantage of using nlsLacZ instead of LacZ for analysis in preimplantation embryo is that it permits direct visualization of the nuclei and, in 1-cell embryos, of the pronuclei. ~7 In addition, the chimeric nuclear protein is dispersed in the cytoplasm during mitosis and relocated to the nucleus at the next interphase.17 The nuclear staining is also compatible 2o S. K. Shapira, J. Chou, F. V. Richaud, and M. Casadaban, Genes 25, 71 (1983). 21 D. Kalderon, B. L. Roberts, W. D. Richardson, and A. E. Smith, Cell (Cambridge, Mass.) 39, 499 (1984). 22 j. Tooze, "Molecular Biology of Tumor Viruses," 2nd Ed., Part 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980.

FIG. 1. Reporter LacZ and nlsLacZ genes. (A) pTZLacZ is 6264 bp long. LacZ sequences are flanked by unique restriction enzyme sites. The 5' noncoding sequences and the first four amino acids (white box) are from SV40 (nucleotides 5122 to 5133). 22 It is followed by the linker sequence 5'-TTCCGGAGC-Y, coding for amino acids 5 to 7 (black box) linked at the sixth amino acid of LacZ 18(gray box). (B) pTZnlsLacZ is 6339 bp long. It is identical to pTZLacZ with the addition of the 5'-AAT TCC GCA AAA AAG AAG AGA AAG GTA GAA GAC CCC AAG GAC TTT CCT TCA GAA TTG CTA AGT TTT TTG AGT CCA3' sequence between the sixth and seventh codons of the linker sequence (white box flanked by black boxes). The sequence contains a nuclear locating signal. 11'21 (C) Expression of a LacZ gene and (D) of a nlsLacZ gene in a 2-cell embryo. (E) Control 2-cell-embryos. (F) Expression of nlsLacZ in an oocyte, stained in the germinal vesicle. Fixation and X-Gal staining of the cells in (C)-(F) were performed as described in the text. (G) The HPRTnlsLacZ construct. The HPRT promoter (white box) is a 1.5-kb fragment of a human HPRT genomic clone, nlsLacZ (gray box) is identical to the SalI-BamHI fragment of pTZnlsLacZ. The polyadenylation signal (black box) is from Moloney murine leukemia virus.

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with histological staining and immunochemistry (detailed in [28] in this volume). HPRTnlsLacZ 14(Fig. 1G) combines the promoter of the gene for hypoxanthine phosphoribosyltransferase (HPRT) that is expressed in all tissues and the nlsLacZ reporter (Fig. 1G). It is transcriptionally active in transient expression assays from the period of minor activation of the genome in early 2-cell embryos and in oocytes. 17 Therefore, it can be used as a positive control. Preparation of DNA for Microinjection DNA must be extensively purified to obtain reproducible results. Following large-scale preparation of plasmid DNA by techniques involving lysozyme-alkaline lysis or lysozyme-Triton X-100 lysis, two purifications by equilibrium ultracentrifugation in CsCl-ethidium bromide gradients are generally necessary.~ When linearized DNA molecules are tested, a purification step must follow the digestion by restriction enzymes and electrophoresis in agarose of the plasmid or inserts. Impurities from the agarose may interfere with egg development and may generate false-negative results. Glass bead purification 23 is recommended. State of Reporter Vector Constructs can be tested as supercoiled DNA (plasmids) or as inserts deleted of plasmid DNA sequences. Both forms of DNA are adequate substrates for transcription in 1-cell and 2-cell embryos. 16A7In contrast, linear DNA in oocytes is less active 24 than supercoiled DNA; therefore, before the injection, the inserts must be religated. Ligation is performed as follows. The DNA concentration is 1 to 6 nM; ligation buffer is 20 mM 1,4-dithiothreitol (DTT), 1 mM adenosine 5'-triphosphate (ATP), 10 mM MgC12 , 50/zg/ml bovine serum albumin (BSA), 50 mM Tris-HCl, pH 7.8. Ligation is conducted for 18 hr at 15° with 1 unit of T4 DNA ligase. DNA in the ligation buffer can be directly injected into cells without purification. It is generally diluted 2- to 10-fold in 10 mM Tris-HC1, pH 7.4, 0.1 mM ethylenediaminetetraacetic acid (EDTA) to bring it to the fight DNA concentration. The DNA sequences of bacterial plasmids are not all neutral. We notice promoter activity in embryos injected with pGEMl-nlsLacZ, a construct lacking eukaryotic promoter sequences. It is therefore important to delete all plasmidic bacterial sequences before testing for the expres23 B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. U.S.A. 76, 615 (1979). 24 L. E. Chalifour, D. O. Wirak, P. M. Wassarman, and M. L. DePamphilis, J. Virol. 59, 619 (1986).

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sion of the LacZ gene by the recombinant construct. In pTZLacZ- and pTZnlsLacZ-based vectors, useful restriction enzyme binding sites in the polylinkers flank the insert (Fig. 1A,B). Note that, in contrast to Xenopus and sea urchin e g g s , 25'26 replication of plasmid DNA does not occur in m o u s e eggs. 27

Glass Bead Purification of DNA 1. Electrophoresis of DNA is performed in 40 mM Tris-acetate, 1 mM EDTA. Cut the gel slice (preferably low-melting agarose) containing the fragment of DNA of interest under long-wavelength UV light (302 nm). Weigh and mince the slice. 2. Dissolve the gel slice in 6 M NaI, 20 mM NaESO3 in water. The solution must be filtered and saturated with 0.5 g/100 ml NazSO3 before use and be protected from light and stored at 4°. Use approximately 2 ml per gram of gel slice. Incubate at 37 ° until completely dissolved (about 15 min) in the dark (protect from light with an aluminum sheet). 3. Vortex the glass slurry until all the powder is in suspension. Glass slurry is prepared from silica 325 mesh as follows: Resuspend 100 ml of powder in 200 ml of distilled water; stir for 1 hr and let settle for 1 hr; take the supernatant and spin 10 min in a Sorvall centrifuge at room temperature; resuspend the pellet in 100 ml water; add nitric acid to 50% (v/v) in a chemical hood; bring close to boiling; and wash the pellet four times with water. Store as a 50% slurry in distilled water. 4. Add the slurry to the dissolved gel (1/zl for ! t~g of DNA). Leave for 2 to 5 hr in the tube on a rocker in the dark at 4°. 5. Spin for 20 sec in a microcentrifuge. Discard the supernatant. 6. Wash the pellet twice with 10 volumes of NaI, NazSO 3 solution in the cold. Resuspend the powder at each wash. 7. Wash the pellet twice with cold ethanol solution. Ethanol solution is 0.1 M NaC1, 1 mM EDTA, 50% ethanol, 10 mM Tris-HC1, pH 7.5 (stored at - 20°). Remove as much of the supernatant as possible. 8. Air-dry the pellet briefly. 9. Elute the DNA for 30 min in 3 volumes of 0.1 mM EDTA, 10 mM Tris, pH 7.5, at 37°.

Notes. This procedure is modified from Vogelstein et al. 23 Fragments from 200 bp to 18 kb can be purified with nearly complete recovery using 25 S. Rusconi and W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 78, 5051 (1981). 26 A. P. McMahon, C. N. Flytzanus, B. R. Hough-Evans, K. S. Katula, R. J. Britten, and E. H. Davidson, Dev. Biol. 108, 420 (1985). 27 D. O. Wirak, L. E. Chalifour, P. M. Wassarman, W. J. Muller, J. A. Hassel, and M. L. DePamphilis, Mol. Cell. Biol. 5, 2924 (1985).

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this method. DNA concentration can be determined by measuring the fluorescence of bisbenzimide H3325828 or comparing the fluorescence intensity after electrophoresis. DNA can be stored at 4° for months.

Preparation of Embryos When choosing the genetic origin of the cells, consider the ease of nuclear microinjection, the yield of eggs per female, and the ability to continue development in vitro. (C57BL/6J x DBA2)F 1 females mated with F~ males of the same strain are convenient. They give a high number of eggs when treated for superovulation (30 per 7- to 10-week-old female), and fertilized eggs pass with a high efficiency to the 4-cell stage in in vitro culture. Other F~ strains such as C57BL/6J x SJL/J and C57BL/6J × CBA are also convenient. Note that for the majority of mouse strains l-cell embryos arrest development in culture at the late 2-cell stage (2-cell block). There are two ways of looking at preimplantation embryos. In vivo material can be isolated at each stage (i.e., growing oocytes, fully grown oocytes, 1-cell, 2-cell, and 4-cell embryos) and analyzed immediately. Alternatively, the material can be isolated at one stage and cultivated in vitro until the eggs reach the desired stage. The efficiency of in vitro development of 1-cell mouse embryos to the morula or blastocyst stage is very high (80%), as is the developmental potential when transferred back into foster mothers (35%). However, it should be noted that in vitro cultivated material may not exactly represent the in vivo material. 17 Moreover, the times of ovulation, insemination, and fertilization vary considerably in vivo. In vitro fertilization reduces these variations, z9 Required Culture Media

Stock A solution (10 × concentrated) for M16 and M2: NaCI 950 mM (5.534 g/100 ml), KC1 48 mM (0.360 g/100 ml), KH2PO 4 11 mM (0.162 g/100 ml), MgSO 4 . 7H20 12 mM (0.294 g/100 ml), sodium lactate 60% syrup (3.2 ml/100 ml), glucose 55 mM (1 g/100 ml), penicillin (1 x 105 IU), and streptomycin (0.050 g/100 ml) M16: Stock A solution diluted 10-fold (1 ×) plus NaHCO3 25 mM (2.101 g/liter), sodium pyruvate 0.32 mM (0.036 g/liter), CaCI2 • 2H20 1.71 mM (0.252 g/liter), phenol red (0.01 g/liter), and BSA (4 mg/ml); adjust to pH 7.6 28 C. L a b a r c a a n d K. Paigen, Anal. Biochem. 101, 339 (1980). 29 S. K. H o w l e t t a n d V. N. Bolton, J. Embryol. Exp. Morphol. 87, 175 (1985).

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M2: Stock A solution diluted 10-fold (1 x ) plus NaHCO3 4 mM, sodium pyruvate 0.32 mM (0.036 g/liter), CaClz • 2H20 1.71 mM (0.252 g/liter), HEPES (5 mg/ml), and BSA (4 mg/ml); adjust to pH 7.4 T6: NaCl 97.84 mM (5.719 g/liter), KC1 1.42 mM (0.106 g/liter), MgC12 • 6H20 0.47 mM (0.096 g/liter), NazHPO 4 • 12H20 0.36 mM (0.129 g/liter), CaC12. 2H20 1.78 mM (0.262 g/liter), NaHCO 3 25 mM (2.101 g/liter), sodium lactate 24.9 mM (2.791 g/liter), sodium pyruvate 0.47 mM (0.052 g/liter), glucose 5.56 mM (1 g/liter), penicillin (1 x 105 IU), streptomycin sulfate (0.05 g/liter), and phenol red (0.01 g/liter) Phosphate-buffered saline (PBS): 138 mM NaCI, 2.7 mM KC1, 1.5 mM KH2PO 4 , 8.1 mM Na2HPO4, pH 7.3 Stock solutions can be stored at - 20° for months. Solutions are stored at 4 ° for no more than 2 weeks.

Isolation of Mouse Oocytes Fully grown oocytes are obtained from ovaries of 8- to 10-week-old F1 females. Such oocytes have acquired meiotic competence. 3° Each female yields approximately 20-30 oocytes. 1. The ovaries are dissected and placed in M2 medium. 2. The oocytes (80 /~m in diameter) are released from follicles by puncturing them with forceps. 3. Oocytes are washed three times (by transfer) in M2 containing 4 mg/ml BSA, an operation which eliminates follicular cells of the zona radiata and debris by pipetting. Keep the denuded oocytes. 4. Oocytes are cultured in M16 containing 4 mg/ml BSA in 5% CO2 in air. If the manipulations have been carried out in the absence of N 6 , 2 ' 0 dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP, Sigma), the breakdown of the germinal vesicle occurs within 1-2 hr. Therefore, nuclear injections are done immediately after preparation of oocytes. Only denuded oocytes are used because visualization of the germinal vesicle is very difficult with oocytes still in the corona radiata. At 18-20 hr after isolation in culture, this material yields 11% oocytes with intact germinal vesicles (GV), 37% oocytes at metaphase I, and 52% oocytes that had completed maturation as demonstrated by the emission of the first polar body.17 30 R. M. Schultz, in "Experimental Approaches to Mammalian Embryonic Development" (J. Rossant and R. A. Pedersen, eds.), p. 195. Cambridge Univ. Press, Cambridge, 1986.

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Notes. Injection in the cytoplasm does not disturb the maturation process. Injection in the germinal vesicle reduces the number of eggs which complete maturation (32% instead of 52%). To prevent spontaneous germinal vesicle breakdown, 100 tzg/ml dbcAMP is added in M2 medium from the step of the dissection of the ovaries. 31 Growing oocytes are obtained from ovaries of mice less than 15 days of age (usually 12- to 14-day-old females). They are smaller (40 to 60 ~m in diameter) than fully grown oocytes. They do not mature spontaneously and fail to resume meiosis when placed in a suitable culture medium. The ovaries are dissected manually in M2 medium and then treated exactly as the fully grown oocytes. The oocytes are also cultured in M 16 containing 4 mg/ml BSA in 5% (v/v) CO2 in air. DNA is injected 1-4 hr after isolation. In Vivo Fertilized 1-Cell and 2-Cell Embryos Superovulated females are obtained by injecting 6- to 8-week-old mice with 5 IU of pregnant mare serum gonadotropin (PMSG; folligon) diluted in 0.9% NaCI or in PBS (<100/zl total volume) in the abdominal cavity and 42-48 hr later with 5 IU of human chorionic gonadotropin (hCG; chorulon) diluted in 0.9% NaCI or in PBS (<100/zl total volume) in the abdominal cavity. The females are bred with males immediately after hCG injection. Vaginal plugs are observed the next morning (vaginal plugs are still visible 12 hr after mating). To obtain 1-cell embryos, fertilized oocytes are isolated from the ampulla tubae of superovulated females 20 hr posthCG injection. 1. The female is sacrificed by cervical dislocation and dissected. 2. Oviducts are placed in M2 containing 0.5 mg/ml hyaluronidase. The ampulla is manually dissected with forceps. This operation liberates the cumulus oophorus. Eggs are released from cumulus cells within 2 min. 3. Eggs are rinsed three times in M2, transferred to M16 medium, and incubated at 37°. Note that the male pronucleus starts forming 16-19 hr post-hCG and is clearly visible 20 hr post-hCG. The female pronuclei is visible after 19 hr post-hCG. Amphimixis occurs 30-32 hr post-hCG (Fig. 2). The 2-cell embryos are isolated from the oviducts of superovulated females 43 hr post-hCG. The oviducts are dissected in M2 and flushed with the same medium with a 5-ml syringe and a 30-gauge eroded needle. Both 1-cell and 2-cell embryos are incubated in M16 medium at 37° in 5% CO2 in air until microinjection. Dulbecco's modified Eagle's medium 31 W. K. Cho, S. Stern, and J. D. Biggers, J. Exp. Zool. 187, 383 (1974).

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(DMEM) containing 10% (v/v) fetal calf serum (FCS) is also adequate, but only for 2-cell embryos. A more complete description of these manipulations can be found in Hogan et al. 3 In Vitro Fertilization

Variation in the time of division of 1-cell embryos after in vivo fertilization is great (from 26 to 35 hr post-hCG, Fig. 2B). It is the consequence of variation both in the time of ovulation32 and in the interval between coitus and fertilization. 33'34 In contrast, the time of division of 1-cell embryos after in vitro fertilization is reduced (from 19 to 21 hr postinsemination, Fig. 2A) especially if the source of spermatozoa is limited to the cauda epididymides. 29Therefore, in experiments where timing is important, this kind of material is recommended. 1. Superovulated 6- to 8-week-old females are prepared by injection of 5 IU of folligon followed exactly 46 hr later by injection of 5 IU of hCG. Collection of the sperm is performed 12 hr after hCG injection, and oocytes are prepared exactly 14 hr after hCG injection. 2. The male is sacrificed by cervical dislocation, and sperm suspensions are prepared from the two cauda epididymides of an F1 male of at least 7 weeks of age. Sperm is collected in l ml of T6 medium by pressing the two cauda epididymides. 3. Sperm is next incubated for 20 min at 37° in preequilibrated warm T6 (in 5% CO2 in air) and counted. 4. Sperm is diluted in preequilibrated warm T6 containing 15 mg/ml BSA (Sigma, St. Louis, MO, fraction V) so as to give a sperm concentration ranging from 1 x 106 to 2 x 10 6 cells/ml. In general, one male yields 2 x 107 spermatozoa. The sperm is incubated for 1.5 hr to allow for capacitation. 5. Oviducts are prepared 14 hr after hCG injection from swollen ampulla of oviducts of the superovulated females. The cumulus masses are directly released in 1 ml of fully capacitated sperm in T6. This step is performed 2 hr after collection of the sperm. 6. Samples are incubated for 3-4 hr. Fertilized oocytes are washed three times by transfer in M16 containing 4 mg/ml BSA. 7. The fertilized oocytes are incubated in M16 containing 4 mg/ml of BSA at 37 ° in 5% in air until microinjection. 32 R. G. Edwards and A. H. Gates, J. Endocrinol. 18, 292 (1959). 33 H. Krzanowska, Folia Biol. 12, 231 (1964). 34 A. Nicol and A. McLaren, J. Reprod. Fertil. 39, 421 (1974).

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Notes. Timing of developmental events in mouse oocytes fertilized in vitro is summarized in Fig. 2A. DNA synthesis occurs from 11 to 17 hr

postinsemination. Cleavage is at 20 hr postinsemination. The fertilization protocol presented is slightly modified from Wood et al. 35 Microinjection of DNA Volume. It is possible to inject 1 to 2 pl in pronuclei (1-cell embryos), 0.4 to 0.8 pl in the nuclei of 2-cell embryos, and I to 4 pl in germinal vesicles. Injection volumes are visually estimated by the increase in nuclear diameter. The diameter of the nucleus expands by 20% of the original value. Exact measurement of injected volumes can be obtained using [3H]thymidine. For instance, a solution of [3H]thymidine at 2.5 x l09 disintegrations/min (dpm)/ml is microinjected and 3H is directly measured after transfer of the embryos in 1 ml of scintillator liquid (ACS2, Amersham). Radioactivity is assayed in spectrometer counter (Rack-fl 12II, LKB). With this protocol, we have estimated that we microinject 1 to 2 pl in male pronuclei, 6 to 10 pl in the cytoplasm of 1-cell and 2-cell embryos, and 2 to 3 pl in the cytoplasm of growing oocytes. Quantity. At 5 ng//zl a 5-kb construct is present at 1000 copies/pl. Up to 100 ng//xl DNA solutions are viscous, and the percentage of surviving embryos falls in consequence. DNA has toxic effects from approximately 0.1 to 1 /zg//zl. The DNA is diluted in 10 mM Tris-HC1, 0.1 mM EDTA at pH 7.4. In Vitro Culture of 1-Cell and 2-Cell Embryos

Incubation medium is M16 (containing 4 mg/ml of BSA) at 37° in 5% CO2 in air. Embryos are placed in plastic petri dishes treated for tissue culture. As embryos are particularly sensitive to low temperature or pH, manipulations are strictly performed with preequilibrated and warm medium at 37° . Inhibiting Embryo Development Inhibition o f D N A Replication. Inhibition of DNA replication in 1-cell embryos causes a failure of the first cleavage. In l-cell arrested embryos, synthesis of the 68-70 kDa complex normally detected in early 2-cell

3sM. J. Wood,D. G. Whittingham,and W. F. Rail, in "The Low TemperaturePreservation of Mouse Oocytes and Embryos in MammalianDevelopment"(M. Monk, ed.), p. 255. IRL Press, Oxford and Washington,D.C., 1987.

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[27]

embryos 36'37occurs, but the synthesis of the polypeptide chains indicative of the major activation of the zygotic genome normally detected in late 2-cell embryos 36'37 does not occur. 38 Therefore, arrested 1-cell embryos are not indicative of normal late 1-cell embryos. It remains to be analyzed to what extent they correspond to "normal" early 2-cell embryos. If 1-cell embryos are maintained in the presence of aphidicolin, an inhibitor of DNA polymerase Or,39 o r 5-fluorodeoxyuridine4° (FUdR), a thymidine analog, at any time between 8 and 16 hr postinsemination (i.e., during the S phase), they fail to divide. The effect is reversible if the embryos are cultured in the presence of the drug for less than 2 hr. Inhibition of DNA synthesis is obtained by culturing eggs in M16 containing 4 mg/ml BSA and either 2/xg/ml aphidicolin or 100/zM FUdR. Stock solutions of aphidicolin are at 2 mg/ml in dimethyl sulfoxide (DMSO) stored at - 20°, and those of FUdR are at 10 mM in water stored at - 20°. Two-cell embryos maintained in aphidicolin of FUdR from 21 to 25 hr postinsemination fail to cleave. The effect is reversible. Similarly, inhibition of DNA synthesis in 4-cell and 8-cell embryos prevents the following cleavage. 41 Inhibition of Cleavage. Methyl [5-(2-thienylcarbonyl)]- 1,4-benzimidazole 2-carbamate (nocodazole) causes arrest of 1-cell embryos in metaphase by inhibiting spindle formation. 42 This microtubule-disrupting drug causes arrest in metaphase in second meiosis when eggs are fertilized in presence of the drug and in metaphase at first mitosis when the drug is added to fertilized eggs from 15 hr postinsemination. The effect is reversible when the drug is removed. It is used at 10/xM in the culture medium of the egg. The stock solution of Nocodazole is 10 mM in DMSO. Cytochalasin D inhibits cytokinesis but not karyokinesis. Late 1-cell embryos treated with cytochalasin D (from 15 hr postinsemination) fail to divide into 2-cell embryos, but the nuclei divide and the DNA replicates. The embryos synthesize late 2-cell polypeptides. 37 Therefore, the drug has no effect on transcriptional activation. Cytochalasin D is used at 0.5 to 1/xg/ ml in the culture medium of the egg. 36 G. Flach, M. H. Johnson, P. R. Braude, R. A. S. Taylor, and V. N. Bolton, EMBO J. 1, 681 (1982). 37 V. N. Bolton, P. J. Oades, andM. H. Johnson, J. EmbryoI. Exp. Morphol. 79, 139 (1984). 38 S. K. Howlett, Roux's Arch. Dev. Biol. 195, 499 (1986). 39 S. Ikegami, T. Taguchi, M. Ohashi, M. Oguro, H. Nagano, and Y. Mano, Nature (London) 275, 458 (1978). 4o N. R. Cozzarelli, Annu. Rev. Biochem. 46, 461 (1977). 41 R. K. W. Smith and M. H. Johnson, J. Embryol. Exp. Morphol. 89, 133 (1985). 42 j. Hoebeke, G. Van Nigen, and M. De Brabander, Biochem. Biophys. Res. Commun. 69, 319 (1976).

[27]

PREIMPLANTATION STAGES AND L a c Z REPORTERS

447

Survival and Development of Injected Embryos Uninjected control embryos develop to 2-cell embryos in culture at a frequency of 90 to 98% and to fetuses (after transfer to foster mothers) at a frequency of 75%. DNA-injected 1-cell embryos develop to 2-cell embryos at a frequency of 70 to 82%. Similarly, 80% of the blastomeres which survive injection at the 2-cell stage cleave normally. 4,43 The timing of cleavage of injected 1-cell embryos and of injected blastomeres is retarded a few hours relative to that of controlsJ v'43 This is an important parameter to take into account during the interpretation of results, as biochemical events may be independent of morphological events and, in particular, of cleavage.

X-Galactoside Staining

1. Cultured oocytes or 1-cell, 2-cell, or 4-cell embryos are rinsed in PBS and then transferred in the fixative medium for 5 min. Fixative medium is 1% (v/v) formaldehyde and 0.2% (v/v) glutaraldehyde in PBS containing 1% (v/v) serum. 2. Cells are then rinsed three times (2 min each, by successive transfer) in PBS containing 1% serum. 3. Cells are transferred to the histochemical reaction mixture. The histochemical reaction mixture is 1 mg/ml 4-chloro-5-bromo-3-indolylfl-galactoside (X-Gal), 4 mM K4Fe(CN)6 • 3H20 (potassium ferrocyanide), 4 mM K3Fe(CN)6 (potassium ferricyanide), and 2 mM MgC12 in PBS. Stock solutions are 40 mg/ml in DMSO for X-Gal (storage at - 2 0 °) and 0.2 M in water for potassium ferricyanide and potassium ferrocyahide. Stock solutions must be protected from light and made flesh every 2 weeks. 4. Cells are incubated at 37° in an humidified chamber (usually a petri dish). The histochemical reaction proceeds slowly. In general, the first positive eggs can be seen after I-3 hr of incubation, but the positive cells are scored only after 20 hr of incubation. There is no background activity in control cells (see Fig. 1E). Notes. For best results, it is important to use a fixative medium with both formaldehyde and glutaraldehyde and to respect the 5-min fixation time. Using only one aldehyde or a shorter fixation time is inadequate. The eggs appear ghostly in the histochemical reaction mixture. To minimize the stickiness of the embryo to glass pipettes during transfer, it is essential 43 K. Ueno, Y. Hiramoto, S. Hayashi, and H. Kondoh, Dev. Growth Differ. 30, 61 (1987).

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GENE EXPRESSION" REPORTER GENES

[27]

to have proteins (provided by the serum) in all media and to transfer the cells rapidly in groups of no more than 5 to 10. Siliconized pipettes can also be used. To avoid background activity, the pH (7.3) of the reaction mixture is important. Also note that enzyme activity decreases rapidly in acidic media.

Reference Scale of X-Galactoside Stained Embryos The generation of quantitative data from experiments with LacZ reporter genes is complicated by several factors. Visual observation does not allow precise quantification of the reaction, and, in particular, it is not possible to see differences above a certain level of precipitate. In addition, there are large variations in the response of an individual egg to a given construct. Nevertheless, it is possible to estimate roughly semiquantitatively the strength of constructs by visual observation of the eggs in reference to a scale of X-Gal staining. The reference scale is best generated by microinjection of known quantities of purified /3-galactosidase (Sigma). Differences in the intensity of staining are observed in eggs microinjected with increasing amounts of/3-galactosidase from 2 x 10 -1° to 2 x 10 -8 units and stained with X-Gal for 18 hr at 37°. One /3-galactosidase unit corresponds to the hydrolysis of 1/xmol of o-nitrophenyl-/3-o-galactopyranoside to o-nitrophenol and o-galactose per minute at pH 7.3 and 37 °. Under these conditions, we estimate the lower limit of sensitivity of/3-galactosidase detection at approximately 103-104 molecules per egg. 1. Dissolve 1000 units of/3-galactosidase (EC 3.2.1.23) from E. coli (Sigma) in 1 ml of 50 mM Tris-HC1, pH 7.3. Prepare serial 10-fold dilutions. 2. Prepare siliconized micropipettes for microinjection as follows. Place the micropipettes in a 15-ml Falcon tube and fill it with Sigmacote (Sigma); discard the Sigmacote and let dry for 1 hr. Rinse 6 times with distilled water. Dry and autoclave the pipettes. 3. Prepare oocytes or 1-cell or 2-cell embryos as described above. 4. Inject the solution (from 1 to 4 pl) into the nucleus. Injection into the nucleus allows better control of the delivered volume (judged by swelling of the nucleus) compared to injection into the cytoplasm. 5. Let the eggs recover for 30 min in M16 containing 4 mg/ml BSA at 37 ° in 5% CO2 in air. 6. Stain the eggs with X-Gal. /3-Galactosidase-positive eggs are obtained at 2 x 10 -~° to 2 x 10 - 9 units per nucleus after an overnight incubation. The staining is in the nucleus.

[27]

PREIMPLANTATION

STAGES

AND

A

LacZ

449

REPORTERS

B

3

T

10

2

y

E

o

control

-Aph

+Aph

10 v

10 •

10 ~

10 J

10 *

10 5

Fluorescence units 6

C

5

E

4 3

H

Z

1 0 10

Fluorescence units

FIG. 3. Assay of/3-galactosidase with MUG. (A) Fertilized eggs were microinjected with 4000 copies of SVnlsLacZ inserts and cultured for 20 hr in the absence ( - Aph) or in presence ( + Aph) of 2 tzg/ml aphidicolin, a drug which blocks the first segmentation of the eggs. Control corresponds to noninjected eggs. Values are the mean per embryo calculated from the measure of the /3-galactosidase activity of pooled eggs. (B, C) Enzymatic reaction measured on individual eggs. The experiment was the same as in (A), on control embryos (B) and aphidicolin-treated embryos (C).

Quantitative Analysis: 4-Methylumbelliferyl-fl-D-galactoside Assay 4-Methylumbelliferyl-/3-D-galactoside (MUG) is a fluorogenic substrate of/3-galactosidase. The reaction product, 4-methylumbelliferone, can be measured by its emission at 355 nm (excitation at 480 nm). M U G permits detection of/3-galactosidase in single embryos. Quantitative indication of the e n z y m e activity can be obtained by measuring methylumbelliferone fluorescence by a Fluoroskan fluorimeter (Flow Labs, M c L e a n , VA). Figure 3 illustrates the effect of blockage of D N A replication on expression of SVnlsLacZ, a construct in which the promoter of SV40 drives nlsLacZ. Fertilized o o c y t e s are injected with 4000 copies of the insert and incubated

450

GENE EXPRESSION: REPORTER GENES

[27]

for 20 hr in the presence or absence of 2/zg/ml aphidicolin./3-Galactosidase is then measured in individual embryos with MUG (Fig. 3C). Noninjected control embryos have a mean of 50 fluorescence units (FU), whereas microinjected 2-cell embryos cultured in the absence of aphidicolin have a mean of 300 FU. The blockage of egg development at the l-cell stage results in a 10-fold increase of/3-galactosidase activity (Fig. 3A). The result is consistent with the idea of DePamphilis and co-workers that some negative regulatory factor first appears as a component of zygotic nuclear structure in 2-cell e m b r y o s : MUG Assay

1. Cultured eggs are transferred individually in 50 ~1 of lysis buffer in a 96-well microplate. Lysis buffer is 60 mM NazHPO4,40 mM NaHzPO 4 , 10 mM KC1, 1 mM MgSO4, pH 7.2, and 0.5% Triton X-100. It is a modified Z buffer. 2. The reaction is started by the addition of 0.5 mM MUG. MUG stock solution is 5 mM in Z buffer. Solubilization is achieved at 90 ° for a few minutes. Incubation of the eggs is allowed at 37° for 20 hr. 3. The reaction is stopped by the addition of 50/zl of glycine I00 mM, pH 10. 4. Fluorescence of methylumbelliferone is quantitated by a Fluoroskan fluorimeter. N o t e s . Another useful substrate for/3-galactosidase is a phenylgalactose-substituted dioxetane. Chemiluminescence of the enzymatic reaction product is measured with a luminometer. Conditions of the luminometric assay are described by Beale et al. 44

Transgenic Mice Expressing LacZ in Preimplantation Embryos To date only two mouse transgenic lines that express L a c Z at the 2-cell stage have been reported in the literature. HPRTnlsLacZ-4 has been obtained by microinjection of a position-dependent transgcne. 14Therefore, its expression at the preimplantation stages is probably due to cis complementation by a genomic control element of a transcriptional unit active at this stage. CMZ12-LacZ was obtained by microinjection of a L a c Z construct driven by the promoter of cytomegalovirus. 45 Its expression is subject to variegation and is dependent on maternal inheritance. E. G. Beale, E. A. Deeb, R. S. Handley, H. Akhavan-Tafti, and A. P. Schaap, BioTechniques 12, 320 (1992). 4s M. A. Surani, R. Kothary, N. D. Allen, and P. B. Singh, Development (Cambridge, UK) Suppl., 89 (1990).

[28]

POSTIMPLANTATION STAGES AND LacZ REPORTERS

451

T h e s e and o t h e r L a c Z transgenic mice derived f r o m position-dependent or p o s i t i o n - i n d e p e n d e n t c o n s t r u c t s will certainly p r o v i d e 2-cell emb r y o s with m a r k e r s indicative o f the m i n o r and m a j o r activations o f the z y g o t i c g e n o m e . In the near future, this material will be o f help in nuclear t r a n s p l a n t a t i o n e x p e r i m e n t s 46'47 and in studies designed to c o m p a r e in vivo material and in vitro cultured e m b r y o s . Species Other T h a n M o u s e C o m p a r a t i v e analysis o f the b i o c h e m i s t r y o f egg d e v e l o p m e n t in several species could indicate the i m p o r t a n c e o f o b s e r v a t i o n s m a d e in one species. It is relatively e a s y to p r e p a r e and to microinject e m b r y o s f r o m rabbits, pigs, and cattle. Details and references for induction o f s u p e r o v u lation and collection and in vitro culture o f rabbit, 48-5° p i g : 1 and b o v i n e 52'53 e m b r y o s h a v e b e e n published. Acknowledgments We thank Iris Tong for comments and C. Tran for typing the manuscript. C.B., P.B., and J.F.N. are from Institut National de la Sant6 et de la Recherche M6dicale. 46 M. A. H. Surani, S. C. Barton, and M. L. Norris, Cell (Cambridge, Mass.) 45, 127 (1986). 47 D. Solter, J. Aronson, S. F. Gilbert, and J. McGrath, Cold Spring Harbor Syrup. Quant. Biol. 50, 45 (1985). 48 C. Delouis, C. Bonnerot, M. Vernet, and J. F. Nicolas, Exp. Cell Res. 201, 284 (1992). 49 M. Techakumphu, S. Wintenberger-Torres, and C. Sevellec, Anim. Reprod. Sci. 12, 297 (1987). 5oA. K. Voss, A. Sandm611er, G. Suske, R. M. Strojek, M. Beato, and J. Hahn, Theriogenology 34, 813 (1990). 51 A. J. Clark, A. L. Archibald, M. McClenaghan, J. P. Simons, C. B. A. Whitelaw, and I. Wilmut, Proc. N. Z. Soc. Anim. Prod. 50, 167 (1990). 52 K. Saeki, M. Hoshi, M. L. Leibfried-Rutledge, and N. L. First, Biol. Reprod. 44, 256 (1991). 53T. Greve and V. Madison, Reprod. Nutr. Dev. 31, 147 (1991).

[28] Application of LacZ Gene Fusions to

Postimplantation Development B y CLAIRE BONNEROT and JEAN-FRAN~;OIS NICOLAS

Introduction L a c Z c a n be u s e d as an in situ e n z y m e r e p o r t e r gene for the visualization o f gene activity or as a m a r k e r o f cells during e m b r y o g e n e s i s . T h e

METHODS IN ENZYMOLOGY, VOL. 225

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