A RAPID METHOD FOR mRNA DETECTION IN SINGLE-CELL BIOPSIES FROM PREIMPLANTATION-STAGE BOVINE EMBRYOS M.E. Collins, D.A. Stevens”. L.J. Jenne? and J. Brownlie Institute for Animal Health, Compton, Newbury, Berkshire, RG16 ONN, UK Received for publication : JuZy 18, 1394 Accepted : November 30, 1994 ABSTRACT Major questions concerning the control of development and gene expression at the cellular level are still unanswered. Nowhere is this more evident than during the earliest stages of development and embryogenesis. This study describes the detection of specific gene transcripts in single cells derived from bovine embryos. Following in vitro fertilization (IVF) and in vitro culture (IVC) of bovine embryos, small groups of cells and even single blastomeres from 32 to 64.cell embryos were micromanipulated into individual tubes for analysis of cytoplasmic RNAs. Reverse transcriptase-PCR was applied to cell lysates for the amplification of p-actin mRNA transcripts. Primers were designed to flank an intron expected to be present within genomic DNA sequences, thus allowing for simple differentiation between DNA- and RNA-derived amplification products. Using a 50-cycle amplification profile, a 260 bp band could be seen as a PCR product derived from a single blastomere following electrophoresis in an ethidium bromide-stained agarose gel. The identity of the band was verified by DNA sequence determination and diagnostic restriction digestion. Lysates derived from single blastomeres in this way have been used for simultaneously phenotyping multiple RNA products. This capability allows the spatial analysis of gene expression and development within embryos from the earliest stages of cellular differentiation. Key words: RT-PCR, mammalian development, single blastomere, mRNA phenotyping. INTRODUCTION The Polymerase Chain Reaction (PCR) has been used in many instances for Acknowledgments ’ Current address: Royal Brompton National Heart and Lung Institute, Dovehouse Street, London, UK. b Current address: Assisted Conception Unit, St. James’s University Hospital, Leeds, UK. Our thanks are due to Jill Ross for her assistance with the generation and culture of the embryos used and to Helen Prentice who additionally has demonstrated great skill in the micromanipulation of embryos and blastomeres, making this study possible. Stuart Kennedy is gratefully acknowledged for his assistance with the DNA sequencing. This work was funded by the AFRC. David Stevens was supported by a grant jointly funded by the Milk Marketing Board and the Meat and Livestock Commission. Theriogenology 43:1227-1238.1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas. New York, NY 10010 0093-691 X’95/$10.00 SSDI 0093-691 X(95)00094-0
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`1228 Theriogenology genotype analysis. The great power of this technique lies in its sensitivity which allows for the analysis of very small quantities of material. When applied at the single cell level, the PCR of pinched blastomeres from early embryos has allowed for successful differentiation between male and female embryos by amplification of Y-chromosome specific markers (3,8,15) and the determination of particular genotypes for markers such as milk proteins (1). While such analysis of the genome is of great importance in the selection of embryos of a desired genotype, particularly for breeding programs, it tells us nothing about the patterns of gene expression. In many cases it is the temporal or the spatial pattern of gene expression that is of critical relevance to the study of embryonic development, and simple PCR does not answer these questions. Much work has been done using in situ hybridization to investigate mRNA expression in tissue sections and even in whole mount Xenopus embryos (9) but these techniques cannot be easily applied to the study of mammalian embryos with the required degree of specificity and sensitivity. More recently, the application of reverse transcription-PCR (RT-PCR) has been developed as a sensitive technique for analysis of gene expression rather than genotype. In comparison with standard PCR of DNA templates the technique of RT-PCR requires the isolation and enzymic reverse transcription of cellular RNAs prior to amplification. The technical challenge presented by this requirement is not trivial, particularly when applied to the analysis of embryonic mRNA expression. Cellular RNAs are notoriously susceptible to degradation, and due to their single stranded nature they can adopt a high degree of secondary structure, rendering them resistant to reverse transcription except in the presence of toxic denaturants such as methyl mercuric hydroxide. The potential benefits of RT-PCR arg significant, particularly for the study of embryonic development. If sufficient sensitivity can be developed RT-PCR will allow for the investigation of patterns of gene expression in cell lineages at the earliest stages of differentiation. The onset of embryonic transcription has been examined in many species. The effects of the transcriptional blocker cx-amanitin on patterns of protein expression in whole embryos has been investigated in the murine by Braude (4) and in the bovine by Barnes and First (2). In addition, Frei et al (6) showed changes in polypeptide synthesis and the beginning of incorporation of radiolabelled uridine into RNA to occur at the 8 to 16-cell stage in the bovine. These data indicate that in bovine embryos the onset of transcription occurs at this time, and it is likely that this increase in endogenous gene activity corresponding with the maternal-zygotic transition is important in the further development of the embryo. However, this does not allow for the identification of specific gene expression, nor does it provide information concerning the key events occurring at this stage in development. The RT-PCR has been used to investigate the expression of a variety of genes in early embryos (7,16,17,20). The possible role of various growth factors and growth factor receptors in the development and differentiation of early embryos has been investigated in both murine and bovine embryos (11 ,18,20), and significant differences in the patterns of gene expression were noted in bovine and murine embryos at the same stages of development. In these studies pools of in vitro cultured bovine embryos (50 to 150 embryos at each stage examined) were used for mRNA isolation and analysis. While this has provided more precise understanding of the temporal expression of specific genes, the use of whole embryo pools would not be suitable for investigating spatial
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`Theriogenology 1229 alterations in gene expression such as those that must occur during the differentiation of morulae and blastocysts. Methods have been described which detect multiple cDNAs from single cell- derived mRNA samples (10,12), but these utilize time-consuming RNA extraction procedures which are prone to the risk of sample loss and/or to multiple step reactions. Given the feasibility of micromanipulation of early embryos and blastomere pinching techniques, we have explored the possibility of using RT-PCR to investigate gene expression in single cells. The aim of this study was to produce a simple, rapid method which would be easily reproducible. To aid the development of this technique to the required degree of sensitivity, we chose p-actin as the target for amplification in the first instance. Thus we present a rapid method for the detection of specific mRNA in individual blastomeres from 16- and 32-cell stage embryos using RT-PCR. MATERIALS AND METHODS In-Vitro Production of Embryos Bovine embryos at the 16 to 32-cell stage were generated for this study by IVF of oocytes recovered from abattoir-derived ovaries. After collection the cumulus- oocyte-complexes were washed twice in TCM-199 with Earles’ salts and sodium bicarbonate containing 10% heat-treated adult bovine serum, 50 IU/ml penicillin G and 50 ug/ml streptomycin before being placed into 40 ul microdrops of maturation medium under silicone oil. The maturation medium was a bicarbonate buffered TCM-199 with Earles’ salts supplemented with 10% heat-treated estrus cow serum, 12.5 ug/ml estradiol-178, 2.5 IU/ml Folligon (PMSG; Intervet, Cambridge, UK), 0.4 mM L- glutamine, 0.2 mM pyruvate, 50 IU/ml penicillin G and 50 ug/ml streptomycin. After 24 to 26 h of maturation at 38.5”C in 5% CO, in air, the oocytes were inseminated for 24 h in a glucose-free IVF-TALP solution (Earles’ balanced salt solution containing 25 mM bicarbonate, 0.2 mM pyruvate, 20 ug/ml heparin, 10 mM caffeine, 7 0 uM hypotaurine, 10 mM lactate, 50 ug/ml gentamycin and 6 mg/ml BSA). After thawing the spermatozoa were washed twice using sperm-TALP (as IVF-TALP but containing 0.01 M Hepes buffer, 1.25 mM magnesium chloride, 21.5 mM lactate and 1 mM pyruvate) and adjusted to an insemination dose of 2x106/ml in IVF-TALP. After 24 h the inseminated oocytes were co-cultured on monolayers of granulosa cells for a further 5 to 6 d before removal for blastomere pinching. The co-culture drops were prepared 8 d prior to use and contained embryo culture medium (TCM-199 with Earles’ salts and sodium bicarbonate supplemented with 10% heat-treated adult bovine serum, 0.4 mM L-glutamine, 0.2 mM pyruvate, 10 mM lactate, 50 IU/ml penicillin G and 50 ug/ml streptomycin). Embryo Manipulation Bovine embryos (32 to 64-cell stage of development) were micromanipulated under oil in 40-ul drops of PBS containing 10% heat-inactivated bovine serum, 50 IU/ml penicillin G and 50 ug/ml streptomycin. The embryo was anchored firmly by suction onto a fine glass holding pipette. The zona pellucida was slit using a
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`Theriogenology microneedle before the introduction of a micropipette. Blastomeres were removed (“pinched”) by gentle suction and were transferred directly transferred into lOO-ul of medium (PBS containing 10% heat-inactivated bovine serum, 50 IUlml penicillin G and 50 ug/ml streptomycin) in 0.5-ml microtubes Extraction and Amplification of mRNA Pinched blastomeres were washed by serial transfer through 3 x 100 ul serum free PBS at 38.5”C and manipulated into 20 ul RT mix (15 mM (NH&SO,, 20 mM Tris HCI (pH 8.8) 2 mM MgCI,, 0.05% Tweet-r, 0.05% NP-40) with 0.25 ul RNAguard (Pharmacia Biotechnology, St Albans, UK) and 50 pmol primer A2 in 0.5-ml microtubes. Tubes were then heated in a thermal cycling block (PTC-100, MJ Research Inc., Massachusetts, USA) to 70°C for 5 min to denature the secondary RNA structures and to prevent potential mispriming of subsequent cDNA synthesis. The RT mix was completed with the addition of 5 units of Tth polymerase (Tet-Z, Amersham International, Little Chalfont, UK), 2.5 mM MnCl and 0.8 mM dNTP. The solution was covered with mineral oil and incubated at 60°C for 20 min to allow for reverse transcription of the RNA. The buffer composition was then optimized for DNA amplification by the addition of 80 ul of polymerase buffer (16 mM (NH&SO,, 67 mM Tris HCI (pH 8.8), 1.5 mM MgCI, and 0.01% Tween-20) containing 50 pmol of primer Al. The PCR regimen consisted of a denaturation step of 94°C for 2 min, followed by 50 cycles of 92°C for 1 min, 60°C for 2 min (combining both annealing and extension in a single step), and a final cycle of 92°C for 2 min and 60°C for 7 min. The final step held the tubes at a temperature of 6°C until they were removed from the block for analysis. As a positive control, total RNA extracted from tissue culture cells using the method of Chomcyzinski and Sacchi (5) was amplified in a parallel reaction. A variety of negative controls were included: reactions containing template RNA but no primers, or primers but no template. Particular care was taken to avoid sample contamination since the target (actin mRNA) is ubiquitously present. RT-PCR Primers Oligonucleotide primers were prepared using an ABI 392 DNA/RNA Synthesizer (Applied Biosystems, Warrington, UK.). Primer sequences were chosen on the basis of conserved regions in available 8-actin gene sequences (mammalian and avian). The sequence of the bovine 8-actin gene was not available for comparison. Two primers, A2 (5’-GAGAAGCTGTGCTACGTCGC-3’) and Al (5’- CCAGACAGCACTGTGTTGGC-3’) were designed to hybridize on either side of an intron expected to be present in the genomic DNA. It was expected that mRNA amplification products would be in the region of 260 bp, while products of genomic DNA amplification would be 318 bp in size.
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`Theriogenology 1231 General DNA Techniques Purification and digestion of PCR fragments, gel electrophoresis and other general DNA manipulations were performed according to standard methods (14) or according to protocols supplied by the manufacturer of the reagents. Following amplification, 15 ul of the 100-ul amplification reaction was electrophoresed on a 1.5% or 2% agaroseflBE gel containing 0.5 ug/ml ethidium bromide. To estimate the size of amplified products, 0.5 ug of Haelll digested PhiX174 DNA molecular weight markers (Promega, Southampton, UK) were also electrophoresed. The fragment sizes of these markers were 1,353, 1,078, 872, 603, 310, 281, 271, 234, 194, 118 and 72 base pairs, respectively. Before restriction digestion, PCR fragments were column purified using the Magic PCR purification system (Promega) according to the manufacturers protocol. The DNA was eluted in 50-ul water, and an appropriate volume (containing approximately 50ng DNA) was digested with Plel (New England Biolabs, distributed by Cambridge Bioscience, UK) at 37°C before gel electrophoresis. To confirm that amplification of the 260 bp band was RNA-dependent, an aliquot of the template solution was incubated with 1 ul of RNAase A (10 mg/ml, Pharmacia Biotech.) prior to amplification. Similarly, to confirm that amplification was DNA- independent, an aliquot of the template material was incubated with 1 ul DNAase I (10 u/ul, Pharmacia Biotech.) at 37°C for 2 h followed by inactivation of the enzyme at 70°C for 10 min prior to amplification. DNA Sequencing A 50-ng sample of Magic PCR purified DNA (see previous section) was used for DNA sequence analysis using the primers described above with the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit and the model 373A Automated DNA Sequencer according to the manufacturers instructions (Applied Biosystems). Data generated in this way were analyzed using the programs in the GCG DNA sequence analysis package (Genetics Computer Group, Wisconsin, USA, 1991). RESULTS Initial attempts at amplification of actin-specific sequences from total cellular RNA extracted from tissue culture cells confirmed the efficacy of both the primers and the amplification protocol. The size of the amplimer and the persistence of specific amplification following pretreatment of the template with DNAase I confirmed the amplimer as being RNA derived. Conversely, similar treatment of the template with RNAase A abolished amplification (Figure 1). The first attempts at amplification of actin from small numbers of blastomeres were unsuccessful. Similar reactions on comparable dilutions of total RNA derived from known numbers of tissue culture cells produced bands of the expected size, indicating that the interference was due to the blastomeres rather than to a lack of sensitivity of the RT-PCR methodology. Since the blastomere samples were lysed directly in the RT buffer rather than undergoing extensive RNA purification, it was possible that other cellular factors present in the lysate inhibited amplification.
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`1232 Theriogenology Figure 1. Amplification of 8-actin following pretreatment of the cell lysate with either DNAase I or RNAase A. Lane 1, DNAase I; Lane 2, RNAase A; Lane 3, no pretreatment; Lane 4, no template control; Lane 5, PhiX174 Haelll molecular weight markers. The size of the PCR product and of selected marker fragments is indicated in base pairs (see Materials and Methods for full details of marker sizes). Treatment of the template with RNAase A was found to abolish amplification while DNAase treatment has no effect, indicating that the amplification product is RNA-derived. Figure 2. Detection of specific amplification products from defined numbers of blastomeres. Lane 1, 1 blastomere not washed in serum/antibiotic-free PBS priorto amplification; Lane 2, 1 blastomere, but washed in serum/antibiotic-free PBS prior to amplification; Lanes 3, 4 and 5, respectively, 2, 9 and 1 washed blastomeres; Lane 6, no template control; Lane 7, positive control, amplification product derived from tissue culture cell RNA; Lane 8, PhiX174 Haelll digested molecular weight markers. The size of selected marker fragments is indicated in base pairs (see Materials and Methods for full details of marker sizes).
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`Theriogenology 1233 When the blastomeres were washed free of serum and antibiotics prior to RT- PCR, the expected, specific amplification signals were obtained (Figure 2). The single blastomere samples in Figure 2, Lanes 2 and 5 show the expected amplification products. A similar, but unwashed, sample in Lane 1 failed to give any amplification product. The presence of serum or antibiotics in the PBS inhibits subsequent amplification. Washing of the blastomere in serum and antibiotic-free PBS removes this inhibition, allowing for the detection of amplification products from a single blastomere. Following this success, a smaller number of blastomeres was micromanipulated into individual tubes for amplification. Finally, single blastomeres from 32 to 64-cell embryos were individually lysed and amplified using the 50-cycle protocol described. In all cases, the amplification product from single cell-derived RNA was readily detectable on an ethidium bromide stained agarose gel without recourse to more sensitive detection techniques such as Southern blotting or nested PCR. 1234567 Figure 3. Amplification of actin from decreasing numbers of blastomeres. Lane 1, PhiX174 Haelll molecular weight markers (the size of selected marker fragments is indicated in base pairs); Lane 2, total cellular RNA from approximately 50 tissue culture cells; Lanes 3, 4, 5, 6, 7, respectively, 12, 8, 5, 3 and 2 blastomeres. Because no specific efforts were made to remove DNA from this RNA preparation both the genomic DNA- and mRNA-derived amplification products can be seen (indicated by the marginal arrows) in Lane 2. When greater numbers of cells were used, a larger amplification product could be detected in addition to the authentic cDNA product (Figure 3). This was presumed to be derived from genomic DNA. The amplimer was larger than expected, indicating that the bovine gene may have a larger intron than has been described for other species. Generally, the presence of this additional band was not problematic since the size was easily distinguished from that of the mRNA amplimer, and the aim of
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`1234 Theriogenology these experiments was to amplify mRNA from smaller cell numbers where the genomic DNA amplification product was presumably present in quantities too small to be directly visualized on an ethidium bromide stained gel. In both Figures 2 and 3, higher molecular weight smears of DNA were seen in the blastomere lysate-derived samples. This is probably due to random priming of DNA amplification by sheared genomic DNA, since it does not occur in the positive control samples where the RNA preparation is of a higher purity than the whole cell lysates.
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`Figure 4. Cleavage of the actin-derived amplimer with Plel. Lane 1, PhiX174 Haelll digested molecular weight markers; Lane 2, Plel digestion (incomplete); Lane 3, undigested amplimer. Cleavage of the 260 bp amplimer with Plel was expected to give approximately 178 bp and 82 bp products. These can be seen in addition to a small quantity of the starting material, which failed to digest to completion in this sample. The size of the cleavage products and of selected marker fragments is indicated in base pairs (see Materials and Methods for full details of marker sizes). The identity of the amplimer was confirmed by DNA sequence determination or by restriction digestion. The DNA sequence generated was compared to other actin sequences using the computer programs MAP, GAP, TRANSLATE and PILEUP. The bovine sequence displayed 98.6% similarity to the human actin cDNA sequence in the region considered. This sequence information confirmed the presence of a Plel site which was then used for diagnostic restriction digestion. A 50-ng aliquot of purified PCR product was digested with Plel (New England Biolabs); this cleaved the 260 bp fragment to give 178 bp and 82 bp products (Figure 4). Given the ease and rapidity
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`Theriogenology 1235 of the technique, restriction digestion was routinely used in preference to sequencing. Using this protocol mRNA from a single blastomere pinched from a 32-cell embryo can be successfully amplified and detected by direct visualization following agarose gel electrophoresis. DISCUSSION The genotype of embryos from various sources has previously been determined by PCR amplification of pinched blastomere samples (3,8,15), and could be used as the basis for selective breeding programs. A further extension of this procedure has been used for the mRNA phenotyping of preimplantation embryos, and has identified mRNA profiles of growth factor gene expression that may be species-specific. It has been concluded that some growth factors are expressed from de novo synthesised mRNAs in murine embryos, while the equivalent protein in early bovine embryos is synthesised from maternally-derived mRNA (11,20). The use of pooled embryos for RNA extraction in these trials while facilitating the detection of low copy number transcripts limits the potential for examination of mRNA profiles in differentiating cell lineages during these early stages of embryonic development. If we are to investigate gene expression during these first stages of embryonic organization, then it is imperative that we have the capacity to analyze either small numbers of isolated cells or even individual cells. The generation of embryos of suitable stages of differentiation by in vitro fertilization and in vitro culture are prerequisites for this. The micromanipulation of developing morulae and blastulae are essential skills for analysis of cell lineages following microdissection or disaggregation of embryos. Methods for the isolation, analysis and quantification of low levels of cellular RNAs have now been developed to the point at which single cells can be subjected to this rigorous analysis (10,12). The results described above prove the feasibility of examining mRNA expression in single cell biopsy samples from preimplantation embryos using a rapid, single-enzyme amplification system. In this method, the opportunities for sample loss or degradation are minimized, while the use of a relatively high temperature for reverse transcription permits the use of primers with a high melting temperature, thereby increasing specificity. Actin mRNA was deliberately chosen as the target for amplification both because of its sequence conservation (the bovine actin sequence was not available in the computer database) and because of its relative abundance. Primer sequences were selected based on estimates made from the alignment of mammalian and avian p-actin cDNA sequences. It was reasoned that regions conserved in both mammalian and avian sequences would be more likely to be similarly conserved in the bovine genome. Estimates of the expected size of mRNA and genomic DNA amplification products and diagnostic restriction enzyme sites were achieved in a similar manner. Subsequent analysis of bovine cellular RNA revealed that the mRNA-derived amplimer was of the expected size, approximately 260 bp, given the limits of resolution of the agarose gel. The DNA sequence determination of this amplimer confirmed that the bovine actin sequence was 99% homologous to the human cDNA sequence and that cleavage with Plel generated fragments which would have been predicted. The bovine
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`1236 Theriogenology genomic DNA amplification product was approximately 550 bp in size, greater than would have been predicted from the known sequences of homologous genes, suggesting the presence of a larger intron in the bovine genomic DNA. When total RNA extracted from tissue culture cells (without any specific DNAase I treatment to remove contaminating DNA) was amplified, both genomic and cDNA amplimers were detected. As the quantity of template RNA was reduced either by dilution, or, as fewer cells were used, it was no longer possible to directly visualize the larger amplimer. This confirms that it is often easier to detect mRNA transcripts, even of single copy genes, than the actual gene itself, particularly when low numbers of cells are used. The relative abundance of actin mRNA proved particularly useful in the development of the technique, but this fact should not limit the usefulness of the procedure as, should sensitivity be a concern, additional methods of signal amplification could be applied such as nested PCR or Southern blotting of PCR products. The RNA from a single cell can be divided and probed for the presence of a variety of different mRNAs (unpublished observations; 12) and, if required, levels of expression of different genes could be quantified. Several methods exist for quantification of templates in PCR reactions and could easily be adapted to suit these procedures (13,19). In this study no attempt was made to quantify product or template, but it was observed that the use of lysates from pooled blastomeres gave rise to (subjectively) larger amounts of product and that the mRNA amplimer was always more readily detectable than that derived from genomic DNA, suggesting that amplification may be limited by template availability. In this system, it is possible that the presence of both genomic DNA and cDNA templates could complicate quantification, but pretreatment of the lysate with DNAase I, which could be inactivated prior to RT-PCR, would eliminate this problem. This DNAase I digestion was successfully completed in the single tube and buffer system described above (Materials and Methods and Figure 1). The routine use of DNAase I would also eliminate the higher molecular weight DNA smears observed in the lysed blastomere samples in figures 2 and 3 but as this is does not obscure the specific amplification it would not seem essential for routine analyses. Obviously, if a higher degree of specificity were essential or if there was the requirement to make a whole cell cDNA library by using random oligonucleotides to prime reverse transcription then the DNAase I treatment could easily be incorporated into the procedure. The selected PCR protocol makes use of the enzyme Tth polymerase (Tet-Z, Amersham International) for both
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`of RNA and amplification of first strand cDNA. One major advantage of this is the ability of the enzyme to reverse transcribe RNA in the presence of manganese ions. The thermostable nature of the enzyme allowed for the reaction to proceed at elevated temperatures (60°C in this case). This necessitated the use of oligonucleotide primers with a relatively high annealing temperature and decreased the risk of mispriming of the reactions, thus increasing specificity. The use of 60°C for reverse transcription of RNA templates has also reduced frequently encountered problems associated with RNA secondary structure (M. Collins, 1992, unpublished observations). In other circumstances the use of hot-start PCR has been shown to reduce nonspecific background which could otherwise mask low-level but specific amplification products. Since this work was
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`Theriogenology 1237 completed, Perkin Elmer have made available a single tricine-based buffer, which is capable of supporting both reverse transcription and DNA amplification by the enzyme Tth polymerase. We have not tested this for amplification of the blastomere biopsy samples, but this obviously has the potential for further simplifying our method to create a single tube, single enzyme, single buffer system. In conclusion, in this paper we describe the use of a rapid, reliable technique for the analysis of RNA derived from micromanipulated single blastomere biopsy samples using RT-PCR. Using the methodology described, blastomere mRNA analysis can be completed within 5 hours, making this a very rapid procedure. The subsequent application of other amplification/detection schemes that may be necessary for the detection of low copy mRNAs or multiple product phenotyping should not prolong the period between the initial biopsy and the successful detection of specific amplimers beyond 1 to 2 d. The application of these techniques will permit the rapid analysis of developmental gene expression in embryos at a level of resolution not previously described. 1, 2. 3. 4. 5. 6. 7. 8. 9. 10. 14. 12. REFERENCES Agrawala PL, Wagner VA and Geldermann H. Determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR. Theriogenotogy 1992; 38:969-978. Barnes FL and First NL. Embryonic transcription in in vitro cultured bovine embryos. Mol Reprod Dev 1991; 29:117-123. Bradbury MW, lsola LM and Gordon JW. Enzymatic amplification of a Y chromosome repeat in a single blastomere allows identification of the sex of preimplantation mouse embryos. Proc Natl Acad Sci USA 1990; 87:4053-4057. Braude PR. Time-dependent effects of a-ammatin on blastocyst formation in the mouse. J Embryo1 Exp Morph 1979; 52:193-202. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-l 59. Frei RE, Schultz GA and Church RB. Qualitative and quantitative changes in protein synthesis occur at the 8-16-cell stage of embryogenesis in the cow. J Reprod Fertil 1989; 86:637-641. Gaudette MF and Crain WR. A simple method for quantifying specific mRNAs in small numbers of early mouse embryos. Nucl Acids Res 1991; 19:1879-1884. Handyside AH, Penketh RJA, Winston RML, Pattinson JK, Delhanty JDA and Tuddenham EGD. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1989; i:347-349. Hemmati-Brivanlou A, Frank D, Bolce ME, Brown BD, Sive HI and Hartand RM. Localization of specific mRNAs in Xenopus embryos by whole mount in situ hybtidisation. Development 1990; 1 lo:325330. Lambolez B, Audinat E, Bochet P, Crepe1 F and Rossier J. AMPA receptor subunits expressed by single Purkinje cells. Neuron 1992; 9:247-258. Rappolee DA, Brenner CA, Schultz R, Mark D and Werb Z. Developmental expression of PDGF, TGF-a, and TGF-8 genes in preimplantation mouse embryos. Science 1988; 241:1823- 1825. Rappolee DA, Mark D, Banda MJ and Werb Z. Wound macrophages express TGF-a and other growth factors in vivo: analysis by mRNA phenotyping. Science 1988; 241:708-712.
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`1238 Theriogenology 13. 14. 15. 16. 17. 18. 19. 20. Robinson MO and Simon Ml. Determining transcript number using the polymerase chain reaction: Pgk-2, mP2, and PGK-2 transgene mRNA levels during spermatogenesis. Nucl Acids Res 1991; 19:1557-i 562. Sambrook J, Fritsch EF and Maniatis T. “Molecular cloning. A laboratory manual.” 1989; 2nd edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Schroder A, Miller JR, Thomsen PD, Roschlau K, Avery B, Paulsen PH, Schmidt M and Schwerin M. Sex determination of bovine embryos using the polymerase chain reaction. Anim Biotech 1990; 1 :121-l 33. Serrano J, Shuldiner AR, Roberts CT Jr, LeRoith D and de Pablo F. The insulin-like growth factor (IGF-I) gene is expressed in chick embryos during early organogenesis. Endocrfnology 1990; 127:1547-l 549. Shuldiner AR, de Pablo F, Moore CA and Roth J. Two nonallelic insulin genes in Xenopus @&are expressed differentially during neurulation in prepancreatic embryos. Proc Natl Acad Sci USA 1991; 88:7679-7683. Telford NA, Hogan A, Franz CR and Schultz GA. Expression of genes for insulin and insulin- like growth factors and receptors in early postimplantation mouse embryos and embryonal carcinoma cells. Mol Reprod Dev 1990; 27:81-92. Vandenheuvel JP, Tyson FL, Bell DA. Construction of recombinant RNA templates for use as internal standards in quantitative RT-PCR. Biotechniques 1993; 14:395-398. Watson AJ, Hogan A, Hahnel A, Weimer KE and Schultz GA. Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo. Mol Reprod Dev 1992; 31:87- 95.
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