Real Time Imaging of Transcriptional Activity in Live Mouse

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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1317-1321, February 1995 Developmental Biology Real time imaging of transcriptional activity in live mouse preimplantation embryos using a secreted luciferase (bioluminescence/luciferin/Vargula/transgene/development) ERIC M. THOMPSON*, PIERRE ADENOT*, FREDERICK I. TSUjIt, AND JEAN-PAUL RENARD* *Unite de Biologie du Developpement, Institut National de la Recherche Agronomique, 78352 Jouy-en Josas, France; and tMarine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093 Communicated by George E. Seidel, Jr., Colorado State University, Fort Collins, CO, October 3, 1994

ABSTRACT Study of gene expression kinetics during grated transgenes is enhanced relative to that from noninte- preimplantation mammalian development is difficult because grated DNA, the use of Vargula luciferase should permit rapid of the limited amount of material and the usually destructive, early detection of transgene integration. By avoiding the static nature of molecular analyses. We describe continuous, biopsy and amplification procedures of PCR-based diagnos- noninvasive monitoring ofgene expression in preimplantation tics, this approach may find important application in the embryos by using a secreted luminescent reporter, Vargula production of transgenic livestock. luciferase. Transgene expression profiles were followed by assaying aliquots of culture medium or by direct visualization MATERIALS AND METHODS of Vargula luciferase secretion from living embryos in real time through photon imaging. With this approach, it is Production ofTransgenic Mice. The 68- to 70-kDa family of possible to observe epigenetic modulations of gene expression murine heat shock proteins is expressed constitutively in the and to link this over time to the developmental capacity of two-cell embryo, repressed at the four-cell stage, and can be individual embryos. In addition, by developing a strategy induced in response to thermal stress in blastocysts (9). The where expression from integrated transgenes is enhanced 800-bp HSP70.1 promotor (10) was inserted into the Hincll relative to that from nonintegrated DNA, we provide evidence site of the Bluescript SK plasmid (Stratagene). Vargula lucif- that rapid detection of transgene integration prior to the erase cDNA (8) or, alternatively, firefly luciferase cDNA (11), blastocyst stage should be possible. Thus, imaging of Vargula linked to the small-t-intron donor-splice site acceptor and luciferase secretion may also be useful in the early screening simian virus 40 polyadenylylation signal, were then cloned into of embryos, for example, in the production of transgenic the HindIII-BamHI sites. These constructions were flanked by livestock scaffold attachment regions (SARs) isolated from the 5' and 3' boundaries of the human (3-interferon locus (12). A 3', 3-kb Embryonic development encompasses a complex interplay of Bgl II SAR fragment was inserted into the BamHI site, and a events ensuring, progressively, the spatial and functional or- 5', 2.2-kb EcoRI SAR fragment was ligated to EcoRI-Xho I ganization of a living organism. During preimplantation mam- adapters and cloned into the Xho I site, 5' of the HSP70.1 malian development, biochemical and molecular analysis of promotor (Fig. 1). this complexity is rendered difficult by the limited embryonic Three different types of transgenic mice were produced: material available. Reverse transcription-PCR techniques (1, those with the promotor and reporter, those with the promotor 2) have made the study of gene regulation more accessible and reporter flanked by plasmid sequences (i.e., Sca I linear- during this period, but the amplification process can limit the ization of the plasmid prior to SAR insertion), and those with interpretation of results. The transgenic approach, using sen- the promotor and reporter flanked by SARs. DNA fragments sitive reporter genes, such as firefly luciferase (3) and ,B-ga- were purified by the GENECLEAN procedure (Bio 101) and lactosidase (4), is also an important methodology in the injected at 2 ng/,ul into pronuclei of embryos produced by analysis of developmental regulation. However, assay of these mating (C57BL/6 x CBA)F1 females (Iffa Credo) with reporters is usually not compatible with further development. (C57BL/6 x CBA)F1 males (13). Founder transgenic mice Thus, destructive molecular analyses make it difficult to follow were identified by slot blot hybridization or PCR. Copy the kinetics of early development in vitro and, to some extent, number was determined by densitometric analysis of quanti- negate the differential dynamics of simultaneous events on tative Southern blots. Lines were bred to homozygosity. which a developmental program is based (5). We demonstrate Vargula Luciferase Expression in Preimplantation Em- that the bioluminescent reporter Vargula luciferase (6) over- bryos. Heterozygotic transgenic embryos were obtained by comes these problems by allowing continuous noninvasive mating males, homozygous for the transgene, with superovu- monitoring of gene expression in preimplantation embryos. lated, (C57BL/6 x CBA)F1 females. Superovulation was by Vargula luciferase is responsible for the bioluminescence injection of 0.1 ml of physiological saline solutions containing (Ama. = 470 nm) of the marine ostracod Vargula hilgendorfii 10 units of pregnant mare serum gonadotropin (Intervet, (7). The cloned cDNA encodes the secretion signal peptide, Angers, France) followed 46-48 h later by 5 units of human and transfected mammalian cells secrete active Vargula lucif- chorionic gonadotropin (hCG; Intervet). Embryos recovered erase (8). Using image intensification, we now show that the from oviducts at the one-cell stage, 21-23 h after hCG transcriptional activation of genes can be directly visualized in injection, were placed in 40- to 50-Al drops of M16 medium situ in living embryos from the earliest stages of development. (13), under oil, at one embryo per ,ul of medium, and cultured Modulation of gene expression by external factors can be at 37°C and 8% CO2. At, intervals (Fig. 2), constitutive followed in time and linked to future developmental capacity. transgene expression was monitored by withdrawing 10-,l In addition, using a strategy in which expression from inte- aliquots of culture medium and replacing it with 10 ,ul of fresh preequilibrated medium. To measure induced expression in The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: SAR, scaffold attachment region; hCG, human cho- accordance with 18 U.S.C. §1734 solely to indicate this fact. rionic gonadotropin. 1317 Downloaded by guest on September 23, 2021 1318 Developmental Biology: Thompson et al. Proc. Natl Acad ScL USA 92 (1995) (SARI). The higher concentration of the SARI plasmid compensated for its larger size, yielding equivalent copy numbers. Constitutive expression was determined at various \ Apal stages by lysing embryos in 50 ,lI of extraction buffer [25 mM Apal AnlI BglI]/BamHI H3PO4/10 mM MgCl2/bovine serum albumin (0.1 mg/ ml)/1% Triton X-100/15% glycerol/i mM EDTA/1 mM FIG. 1. DNA elements used in the transgenic lines. The HSP70.1 dithiothreitol/0.2 mM phenylmethylsulfonyl fluoride]. Blasto- promotor-Vargula luciferase cDNA-small (Sm) t-intron-polyadeny- cysts were heat shocked and incubated for 5 h at 37°C prior to lylation signal fragment produced three lines: VI11, V163, and VI71, lysis in 50 ,ul of extraction buffer. The 50-,ul samples were with 53, 26, and 13 copies of the transgene, respectively. The same diluted with 50 pul of distilled water and assayed in an unmod- fragment flanked by pBluescript plasmid sequences generated four ified LUMAT LB 9501 by injection of 100 pul of extraction lines: PV3 (3 copies), PV13 (61 copies), PV14 (4 copies), and PV18 (29 buffer complemented with 0.3 mM firefly luciferin and 0.8 copies). With the Kpn I-Not I fragment shown, four lines were JIM produced with flanking human 03-interferon SARs: SVS1 (2 copies), ATP. Light output was integrated for 10 s at 18°C. Data SVS8 (1 copy), SVS10 (2 copies), and SVS11 (2 copies). Hatched analyses were performed with STATVIEW II software (Abacus portions represent polylinker sequences in the plasmid. Concepts, Calabasas, CA). blastocysts, culture dishes were sealed with parafilm and immersed in water at 43°C for 30 min. After heat shock, the RESULTS AND DISCUSSION parafilm was removed, and the dishes were returned to the In this study the HSP70.1 promotor was used because the incubator. At 19-20 h after heat shock, the medium was expression profile of this gene has been characterized during collected; the embryos were rinsed in phosphate-buffered preimplantation mouse development (9). With the Vargula saline (PBS) and then placed in a tube containing 15 pll of 0.2 luciferase reporter, transgene expression in preimplantation M Tris HCl (pH 7.6), 0.2 M NaCl, 15% (vol/vol) glycerol, and embryos could then be continuously monitored by removing bovine serum albumin at 100 ,ug/ml (TNGB). Medium and aliquots of culture medium. The expected expression profile embryo samples were stored at -80°C until assay. was found in transgenic lines containing the promotor and The 10-plI aliquots of medium were diluted to 100 pll in 0.2 reporter alone (VI lines) or the promotor and reporter flanked M Tris HCl (pH 7.6), 0.2 M NaCl, and 15% glycerol (TNG) by plasmid sequences (PV lines). Constitutive transgene ex- and placed in a tube in a LUMAT LB 9501 photometer (EG pression was observed from two-cell embryos, and expression & G Berthold, Bad Wildbad, Germany). The 15-Al embryo was diminished during the four-cell stage (Fig. 3). In blasto- samples were passed through five freeze-thaw cycles and cysts, transgene expression was induced by heat shock (Fig. 4). diluted to 100 ,Al with TNG buffer prior to measurement. However, in transgenic lines with flanking SAR sequences Vargula luciferin, dissolved at 100 nM in sodium phosphate (SVS lines), the preimplantation expression profile was mod- buffer at pH 6.8, was placed in a glass vessel on ice with ified. continuous argon bubbling. This solution was connected to the SARs stimulate the expression of stably integrated genes in automatic injector of the photometer, and 100 plI was injected transformed cell lines in contrast to their neutral or negative into assay samples. Light output was integrated for 30 s. effect on expression in transient transfection experiments (15, Background was reduced by cooling the phototube to 7°C and 16). They are thought to act through alteration of chromatin inserting a 500-nm cutoff filter between the sample and the structure via interactions with a proteinaceous nuclear matrix photomultiplier. Vargula luciferin chemiluminescence was re- (17, 18) and/or proteins implicated in the degree of chromatin duced by culturing embryos in M16 without the phenol red compaction (19). We were interested in the effects of SARs on indicator. gene expression during the modification of chromatin struc- To visualize Vargula luciferase secretion, the Argus 50/ ture during early development. VIM-3 (Hamamatsu, Hamamatsu-city, Japan) imaging system SARs stimulated the per copy expression of the transgene at was used in a configuration similar to that described (14). A the two-cell stage and prolonged expression through the microscopic chamber with a series of 2-plI drops of 50 ,uM four-cell stage (Fig. 3). This indicates that repression of the luciferin in PBS covered with oil was maintained at 37°C on a endogene and the two other transgenic constructs is not Zeiss Axiovert 135 inverted microscope. Transgenic embryos related to the absence of specific transcription factors but is were cultured in vitro and at the two-, four-, eight-cell or instead mediated by changes in chromatin structure. Subse- blastocyst stages were rinsed in PBS and placed in the 2-plI quent studies using modifiers of chromatin structure support drops of PBS/luciferin. Imaging was done in a dark room with this conclusion (E.M.T. and J.-P.R., unpublished results). the microscope and camera further isolated in a dark box. SARs also stimulated heat shock-induced expression at the Transient Expression in Preimplantation Embryos. Promo- blastocyst stage (Fig. 4). Furthermore, in SARI lines, fairly tor-firefly luciferase and SAR-flanked promotor-firefly lu- consistent levels of transgene expression were observed on a ciferase fragments were prepared and microinjected at 2 ng/pld per copy basis. These same SAR effects were observed in into one-cell pronuclei. Supercoiled Bluescript plasmids con- transgenic mice in which the HSP70.1 promotor directed taining the promotor-reporter, with or without flanking transcription of firefly luciferase cDNA (SFS lines). In Vargula SARs, were injected at 25 ng/pl (SAR-) and 50 ng/pl transgenic mice, SARI lines had lower copy numbers than Fertilization hCG| 0 4-Lcell 8-cell 16-cell Morula Blastocyst a I ~~1-cell I 2-cell I II II II II 0 9 5 9 0 a I I 9 I 0 24 50 64 74 90 114 Hours post-hCG

FIG. 2. Protocol for monitoring preimplantation gene expression in transgenic embryos. The cell cleavage program, relative to the point of hCG injection, is shown above the time line. The moment of fertilization is roughly indicated but was not precisely controlled. At the intervals (in hours after hCG injection) indicated below the line, 10-,ul aliquots of culture medium were withdrawn and replaced with 10 ,A of fresh medium to monitor Vargula luciferase secretion. Downloaded by guest on September 23, 2021 Developmental Biology: Thompson et al. Proc. Natl. Acad. Sci. USA 92 (1995) 1319

3 For imaging experiments, the line SVS10, which contains A Expression -a -VI . -I.G- PV two transgene copies, was used. At the two-cell stage, the 2 instantaneous secretion rate was low, and only a few spots were seen at the blastomere surface (Fig. 5A). When transgenic 1 ~ ~t'\s ---ISV blastocysts were heat shocked and then returned to the incubator at 37°C, there was a lag of 3 h before Vargula luciferase .0 became detectable in the culture medium. At 4 h after heat 0 shock, the rate of luciferase secretion was greatly elevated and -M -14 the luminescent signal was easily detected. Under these con- 0 60 80 100 120 ditions, a clear signal was usually obtained from blastocysts in X~1.5 BExpression / Copy - -I 5-7 s, with some emitting sufficiently strongly for identifica- "'I''''''--T..... tion in a time lapse equivalent to the instrument readout (0.04 1.0 , A. Constitutive and induced expression 2-cell Rlastoevst 0.5 0 ! +1': -0.5 L- 60 80 100 120 Time after hCG, h FIG. 3. Constitutive preimplantation transgene expression. Var- gula luciferase was assayed in culture medium at the times indicated on the abscissa. Values are expressed as the change in relative light units (RLUs) per embryo between two measurement intervals. Mean values are plotted with the SE in the negative direction. VI, HSP70.1 promoter-Vargula luciferase reporter construct; PV, the same construct flanked by pBluescript plasmid sequences; and SVS, the promoter-reporter, flanked by human jB-interferon SARs. Data were B. Stimulation of expression by Trichostatin A analyzed using unpaired t tests. (A) Mean absolute expression. There 4-cell 8-cell was a significant (P < 0.05) increase in expression from SVS lines relative to VI lines at the eight-cell stage (74 h). (B) Mean expression per transgene copy. Expression from SVS lines was significantly higher than both VI and PV lines at the two-cell stage (50 h; P < 0.01), 4-cell stage (64 h; P < 0.01), and 8-cell stage (74 h; P < 0.05). In blastocysts (114 h), SVS expression was elevated compared to VI (P < 0.01) but not PV expression. Significant differences in expression were never observed between VI and PV lines. SAR- lines. Among firefly luciferase lines, this was reversed, with SARI lines having higher copy numbers. Therefore the low copy number in SVS lines, relative to VI and PV lines, would not appear to significantly bias interpretation of the data. These results then led us to investigate the possibility of FIG. 5. Imaging of photons produced when Vargula luciferase, se- in vivo visualization of Vargula luciferase secretion from trans- creted by transgenic embryos, reacts with Vargula luciferin. Photons are genic embryos. detected by an image intensifier, coupled to a charge-coupled device camera. Rapid readout (25 frames per s) of the digital information Blastocyst: Induced generated by the charge-coupled device allows spatial monitoring on a 0 video monitor in real time. Final processed images are obtained through [C1 RLU | T accumulation of photon counts over a desired time interval. (A) Imaging I RLU/copy of Vargula luciferase secretion from a two-cell embryo (constitutive t%2 expression) and a blastocyst (induced expression). (Upper) Transmission images ofthe embryos. (Lower) Luminescent images superimposed on the transmission images. A pseudocolor scale represents the intensity of photon accumulation from low (dark blue) to high (red) levels. Imaging cn of the two-cell embryo was performed 42 h after hCG injection with photon integration over 210 s. The blastocystwas cultured until 112 h after hCG injection at which point it was heat shocked. It was returned to the IL: L -1-- III incubator at 37C for 4 h, then rinsed in PBS, and imaged. The lumines- LFLi cent image after 120 s of photon counting was superimposed on the > > > transmission image. (B) Stimulation of transgene expression at the four- > >> * t t> > cell and eight-cell stages by trichostatin A. (Upper Left) Transmission Transgenic Line image of a four-cell embryo 64 h after hCG injection. This embryo was cultured to near the end of the two-cell stage (50 h after hCG) in M16. FIG. 4. Induced expression of the transgene from heat shocked At this point, 25 nM trichostatin A was added to the medium and culture blastocysts. Vargula luciferase was assayed in the culture medium after was continued until rinsing in PBS prior to analysis. (Lower Left) Lumi- recovery from a thermal stress. Mean relative light unit (RLU) ex- nescent image after 180 s of photon counting superimposed on the pression per embryo on an absolute and per transgene copy basis is transmission image. (Upper Right) Eight-cell embryo 73 h after iiCG shown with SE above each bar. The different types of transgenic lines injection. This embryo was cultured to near the end of the four-cell stage are, from left to right, promoter-reporter (VI), promoter-reporter (64 h after hCG), at which point 25 nM trichostatin A was added to the flanked by plasmid sequences (PV), and the promoter-reporter flanked medium and culture was continued until rinsing for analysis. (LowerRight) by SARs (SVS). Per copy transgene expression from SVS lines was Luminescent image after 180 s of photon counting superimposed on the significantly elevated (t test; P < 0.001) compared to both VI and PV transmission image. A Plan-Neofluar X40 immersion objective (Zeiss) lines. was used. Downloaded by guest on September 23, 2021 1320 Developmental Biology: Thompson et al. Proc. Natt Acad ScL USA 92 (1995)

that the signal was insufficient to attain the low end of the pseudocolor scale in the final processed image. The hatched portion of the blastocyst emitted an intense signal relative to the portion surrounded by the zona pellucida in either a 30- or 120-s integration (Fig. 6 Middle Left and Bottom Left). After 6 min of incubation in luciferin/PBS, the 30-s signal was more equitably distributed over the embryo, though it remained stronger over the hatched region (Fig. 6 Top Right). After rinsing in PBS, a 30-s integration without luciferin revealed virtually no luminescent signal over the hatched portion, with a weak signal remaining over the unhatched part of the embryo. Thus, the luminescent signal results from luciferase secreted into the medium, not from penetration of luciferin into cells, and suggests that the zona pellucida slows the rate of free diffusion of Vargula luciferin. An SFS line with four transgene copies was also used in imaging experiments. When 50 ZM firefly luciferin was injected into two-cell blastomeres, a luminescent image was obtained. However, when heat-shocked blastocysts were incubated in PBS/50 ,uM firefly luciferin, only a weak luminescent FIG. 6. Photons result from the secretion of Vargula luciferase. A signal was imaged from some blastocysts. Upon addition of time series of images was obtained from the same embryo. (Top Left) Triton X-100, a stronger signal was released into the surround- Transmission image of a hatching transgenic blastocyst. Superimposed ing medium. Enhanced in vivo luminescent signals can be on the transmission image are the luminescent image after 30 s of obtained by using esters offirefly luciferin that penetrate more photon counting in PBS/50 1lM Vargula luciferin (Middle Left), the easily into cells than native firefly luciferin (21, 22). Once luminescent image after 120 s of photon counting (Bottom Left), and inside, the esters are cleaved by endogenous esterases, and the the luminescent image obtained from 30 s of photon counting after the substrate is free to react with firefly luciferase. The enhance- blastocyst had been in 50 ,uM luciferin in PBS for 6 min (Top Right). ment of esterified luciferins relative to native luciferin is most The hatching blastocyst was then removed by mouth pipette, rinsed in 1 ml of PBS, and returned to the microscopic chamber into PBS without pronounced at low luciferin concentrations (6-fold at 10 ,uM) luciferin. (Middle Right) Transmission image of the rinsed hatching blas- and is less important at higher concentrations (2-fold at 100 tocyst. Note that after pipetting, the blastocyst no longer had the same ,uM) (21). At 50 ,uM luciferin, a 2- to 6-fold enhancement orientation in the camera field. (Bottom Right) Luminescent image after would not have resulted in significant signal gain relative to 30 s of photon counting, superimposed on the transmission image. that observed with Vargula luciferase. Thus, under equivalent substrate concentrations, Vargula luciferase gave much more s). Effects of perturbations of the chromatin environment on satisfactory luminescent images. Compared to other biolumi- transgene expression were also observed (Fig. SB). Constitu- nescent enzymes and photoproteins, Vargula luciferase has a tive expression at the four-cell stage is lower than that of relatively high turnover rate of 1600 molecules of luciferin per two-cell embryos but can be stimulated by incubating embryos min (23), therefore generating brighter point sources when in 25 nM trichostatin A (20). This compound inhibits histone integrated over time. This may account in large part for the deacetylases; its action results in hyperacetylation of core improved spatial imaging. Firefly luciferin is commercially histones and more accessible DNA around the nucleosome. In available, and the possibility of using firefly luciferase for SARI lines, there was a considerable increase in expression of screening transgenic embryos has been discussed in a recent the transgene in the presence of this molecule. Stimulation of abstract (24); however, the chemical synthesis of Vargula lu- transgene expression by trichostatin A was reduced at the ciferin has been known since 1966 (25). eight-cell stage but remained detectable. These results suggest that imaging of Vargula luciferase The luminescent signal was due to luciferase secreted into secretion could be useful for early screening of embryos in the the medium as shown in a time series of images from a hatching production of transgenic livestock. At present, the cost of blastocyst (Fig. 6). In considering this series, it should be noted production of transgenic domestic species is high, mainly be- that bit range selection for the superimposed images resulted cause, among these species, <1% of embryos injected with a in the elimination of lower intensity signal to avoid oversatu- transgene construct yield a transgenic newborn (26). Use of ration at higher intensities. Therefore, when maximum inten- this strategy to produce transgenic livestock would involve sity was achieved in one region of the embryo, a lack of signal coinjecting the reporter construct with the transgene of inter- elsewhere did not indicate undetectable luminescence, only est. In the mouse, this is compatible with specific expression of Table 1. Transient expression from microinjected DNA constructs in preimplantation embryos Preimplantation firefly luciferase expression* Injection One cell Two cell Four cell Blastocyst Construct Formt ng/al RLU* %/n§ RLUt %/n§ - RLU* %/n§ RLU* %/n§ SAR- Insert 2 3.8 ± 3.7 18.2/11 2.5 ± 1.6 7.0/57 23.6 ± 12.7 16.7/36 0.02 ± 0.02 1.9/53 sc plasmid 25 ND ND 52,127 ± 8509 98.0/49 1038 ± 351 100/23 ND ND SAR+ Insert 2 12,040 ± 2670 96.9/32 1917 ± 412 75.0/40 1953 ± 771 32.0/28 2993 ± 886 46.7/30 sc plasmid 50 ND ND 63,959 ± 7710 96.2/52 1205 ± 223 100/12 ND ND ND, not done. *Injection was into one-cell pronuclei. Constitutive expression was measured in one-, two-, and four-cell embryos, and induced expression was measured in blastocysts. Expression at the one-cell stage was from blocked embryos, harvested 41-42 h after hCG injection. tDNA was injected as SAR- or SAR+ insert fragments or as supercoiled pBluescript plasmids (sc plasmid) containing these inserts. tMean expression ± SE in relative light units (RLU). §Percentage of responding embryos over the total number of embryos analyzed. Downloaded by guest on September 23, 2021 Developmental Biology: Thompson et al. Proc. NatL Acad Sci USA 92 (1995) 1321

20 is time consuming, demands precise manipulation, and may (4) compromise further development. In routine practice, there 0 (6) are false positives, due to contamination, and false transgenics 15 (28), because of the amplification of injected but noninte- 5) grated transgenic DNA. In contrast, by linking or coinjecting ~D 10 the Vargula luciferase reporter gene with a desired transgene, 0 (4) it should be possible to rapidly screen embryos by adding O_D (3) Vargula luciferin to the culture medium and selecting embryos 5 emitting a bioluminescent signal. This approach may also (2) permit the selection of more effective insertion sequences or 0 vectors for the generation of transgenic organisms. 0 20 40 60 80 100 Thus, secretion of Vargula luciferase permits noninvasive Cummulative Percentage of Embryos analysis of preimplantation gene expression by assaying aliquots of culture medium or by direct in situ imaging of live FIG. 7. Cumulative percentage of embryos versus expression level embryos. This opens additional perspectives for kinetic studies in heat-shocked blastocysts. The SARI construct was microinjected of an important period of development in which experimental into one-cell pronuclei at 2 ng/,ul. At the blastocyst stage (112 h after approaches are, at present, constrained by the limited amount HCG injection), embryos were heat shocked and returned to a 37°C of cellular material available. incubator for 5 h. They were then lysed, and firefly luciferase expression was determined. Individual embryos are plotted in order of We thank Dr. J. Bode (Gesellschaft fiir Biotechnologische For- increasing activity with the cumulative percentage shown on the ab- schung, Braunschweig-Stockheim, Germany) for the ,3-interferon scissa. On the right hand ordinate, the mean expression levels of five SARs, Dr. 0. Bensaude (Ecole Normale Superieur, Paris) for the lines transgenic for the same construct are indicated with copy num- HSP70.1 promotor, Dr. M. Yoshida (University ofTokyo) for a sample bers in parentheses. RLU, relative light units. of trichostatin A, Mr. M. Pontoizeau (Hamamatsu) for loan of the Argus 50, and Mr. B. Masson (EG & G Berthold) for loan of the each transgenic construct (27). Alternatively, the reporter modified LUMAT LB 9501. M.-G. Stinnakre (Institut National de la construct could be linked in tandem with the desired trans- Recherche Agronomique) assisted in transgenic mouse production, gene. The HSP70.1 promotor used here is probably not opti- and F. Fort (Institut National de la Recherche Agronomique) assisted mal for screening because it involves submitting embryos to a with the photographs. Partial support was provided by Grant MCB91- thermal thus 04684 from the National Science Foundation (F.I.T.) and contract stress, compromising embryo survival. Nonethe- Rh6ne-Merieux-INRA-MRT 90T0968 from the Ministere de la Re- less, the use of a promotor that can be induced at a specified cherche et de la Technologie (J.-P.R.). moment for diagnosis would be advantageous over a consti- 1. Rappolee, D. A., Brenner, C. A., Schultz, R., Mark, D. & Werb, Z. (1988) tutively expressed promotor. Science 241, 1823-1825. Data from microinjection experiments support the feasibil- 2. Hou, Q. & Gorski, J. (1993) Proc. Natl. Acad. Sci. USA 90, 9460-9464. ity of the luminescent screening approach (Table 1). SAR- 3. DiLella, A. G., Hope, D. A., Chen, H., Trumbauer, M., Schwartz, R. J. & and SARI constructs were injected as linear inserts at 2 ng/,ul, Smith, R. G. (1988) Nucleic Acids Res. 16, 4159. to that in 4. Goring, D. R., Rossant, J., Clapoff, S., Breitman, M. L. & Tsui, L.-C. (1987) equivalent used generating transgenic animals. Al- Science 235, 456-458. ternatively, supercoiled SAR- and SARI plasmids were in- 5. Gurdon, J. B. (1992) Cell 68, 185-199. jected at concentrations that we have found to be optimal in 6. Thompson, E. M., Nagata, S. & Tsuji, F. I. (1989) Proc. Natl. Acad. Sci. studies of transient expression in preimplantation embryos. USA 86, 6567-6571. Injection of plasmids revealed no significant differences be- 7. Harvey, E. N. (1952) Bioluminescence (Academic, New York), pp. 297-331. 8. Thompson, E. M., Nagata, S. & Tsuji, F. I. (1990) Gene 96, 257-262. tween the SAR- and SARI groups both in expression level (t 9. Bensaude, O., Babinet, C., Morange, M. & Jacob, F. (1983) Nature (Lon- test: two cell, P = 0.305; four cell, P = 0.748) and the per- don) 305, 331-333. centage of responding embryos. Thus, SARs did not act as 10. Hunt, C. & Calderwood, S. (1990) Gene 87, 199-204. enhancers in the transient assay, in 11. de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R. & Subramani, S. simple agreement with (1987) Mol. Cell. Biol. 7, 725-737. previous results in transformed cells (16). However, injection 12. Bode, J. & Maass, K. (1988) Biochemistry 27, 4706-4711. of inserts at 2 ng/,ul revealed significantly greater expression 13. Hogan, B., Costantini, F. & Lacy, E. (1986)Manipulatingthe MouseEmbryo and percentages of responding embryos in the SARI group. (Cold Spring Harbor Lab. Press, Plainview, NY). Results obtained with the to 14. Inouye, S., Ohmiya, Y., Toya, Y. & Tsuji, F. I. (1992) Proc. Nat!. Acad. Sci. SAR- insert compared the SAR- USA 89, 9584-9587. plasmid suggest that the insert was not stable or that it was a 15. Phi-Van, L., von Kries, J. P., Ostertag, W. & Stratling, W. H. (1990) Mol. poor template for transcription. In this regard, it was of interest Cell. Biol. 10, 2302-2307. to compare frequencies of transgenesis obtained with SAR- 16. Klehr, D., Maass, K & Bode, J. (1991) Biochemistry 30, 1264-1270. flanked and to the 17. Gasser, S. M., Amati, B. B., Cardenas, M. E. & Hofmann, J. F.-X. (1989) promotor-reporter fragments (SVS SFS) Int. Rev. Cytol. 119, 57-95. promoter-reporter fragments alone (VI and FI). The number 18. Getzenberg, R. H., Pienta, K. J., Ward, W. S. & Coffey, D. S. (1991)J. Cell. of transgenics over the number of individuals analyzed were Biochem. 47, 289-299. VI, 5 of 75; FI 5 of 73; SVS, 13 of 57; and SFS 9 of 40. Pairwise 19. Zhao, K, Kas, E., Gonzalez, E. & Laemmli, U. K. (1993) EMBO J. 12, 3237-3247. tests showed that of were X2 frequencies transgenesis signifi- 20. Yoshida, M., Kijima, M., Akita, M. & Beppu, T. (1990) J. Biol. Chem 265, cantly higher using SARI fragments and that there were no 17174-17179. significant differences within the SARI or SAR- groups. 21. Craig, F. F., Simmonds, A. C., Watmore, D., McCapra, F. & White, M. R. Finally, at the diagnostic blastocyst stage, the cumulative per- (1991) Biochem. J. 276, 637-641. 22. Yang, J. & Thomason, D. B. (1993) BioTechniques 15, 848-850. centage of embryos was plotted against expression level for the 23. Shimomura, O., Johnson, F. H. & Masugi, T. (1969) Science 164, 1299- SARI injection at 2 ng/,ul (Fig. 7). This was compared to mean 1300. expression levels of five SFS lines. Fifty percent of the embryos 24. Matsumoto, K., Anzai, A., Nakagata, N., Takahashi, A., Takahashi, Y. & were eliminated as nonresponders. By setting the expression Miyata, K. (1994) Theriogenology 41, 250 (abstr.). 25. Kishi, Y., Goto, T., Inoue, S., Sugiura, S. & Kishimoto, H. (1966) Tetra- level at that of the lowest expressing transgenic line, 70-80% hedron Lett., 3445-3450. of the embryos would be rejected. This corresponds well with 26. Pursel, V. G. & Rexroad, C. E., Jr. (1993)J. Anim Sci. 71 (Suppl. 3), 10-19. the 22-23% frequency of transgenesis actually observed with 27. Overbeek, P. A., Aguilar-Cordova, E., Hanten, G., Schaffner, D. L., Patel, SARI constructs. P., Lebovitz, R. M. & Liberman, M. W. (1991) Transgenic Res. 1, 31-37. luminescent offers over PCR 28. Horvat, S., Medrano, J. F., Behboodi, E., Anderson, G. B. & Murray, J. D. Therefore, imaging advantages (1993) Transgenic Res. 2, 134-140. diagnoses, which have been used to screen morula and blas- 29. Bowen, R. A., Reed, M. L., Schnieke, A., Seidel, G. E., Jr., Stacey, A., tocysts (28, 29). Using PCR, embryo biopsy is required, which Thomas, W. K. & Kajikawa, 0. (1994) Biol. Reprod. 50, 664-668. Downloaded by guest on September 23, 2021