Differential Expression of Ped Gene Candidates in Preimplantation Mouse Embryos¹

^a Department of Biology, Northeastern University, Boston, Massachusetts 02115

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Ped (preimplantation embryonic development) gene influences the rate of mouse preimplantation embryonic development and subsequent survival. Four similar tandem genes in the Q region of the major histocompatibility complex—Q6, Q7, Q8, and Q9—were identified as Ped gene candidates. In this study, expression of these genes during preimplantation development was examined and quantitated by reverse transcription-polymerase chain reaction and single nucleotide primer extension assays in order to investigate their contribution to the Ped gene phenotype. The Q7/Q9 gene pair was found to be transcribed in preimplantation mouse embryos, whereas transcription of the Q6/Q8 gene pair was undetectable. Both Q7 and Q9 are expressed in embryos from one Ped fast strain, C57BL/6, while only the Q9 gene is expressed in another Ped fast strain, B6.K2. These results suggest that both the Q7 and Q9 genes can function as the Ped gene in the mouse. Interestingly, the expression pattern of the Q7 and Q9 genes in preimplantation embryos is the same as in splenic lymphocytes. However, the Q6 and Q8 genes are expressed in splenic lymphocytes but not in preimplantation embryos. Treatment of mouse preimplantation embryos with interferon gamma (-IFN) did not induce expression of the Q6/Q8 genes but enhanced expression of the Q7/Q9 genes. The mechanism of this differential transcription pattern is currently under investigation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Ped (preimplantation embryonic development) gene regulates preimplantation embryonic development and subsequent embryo survival [1–3]. Two alleles of the Ped gene have been defined: Ped fast and Ped slow. Ped fast mice have a faster rate of preimplantation development than Ped slow mice. In addition, Ped fast mice have larger litter sizes and are significantly larger at birth and at weaning.

The Ped gene maps to the Q region of the mouse major histocompatibility complex (MHC) [4]. The Qa-2 antigen is a family of nonclassical MHC class I molecules encoded by this region. The presence of Qa-2 antigen is directly correlated with the Ped fast phenotype, and the absence of Qa-2 antigen results in the Ped slow phenotype [5, 6]. It was thus shown that the Qa-2 antigen is the product of the Ped gene. Since the Qa-2 antigen is a family of isotypes encoded by four different tandem Q region genes—Q6, Q7, Q8, and Q9 [7–13]&; four genes were identified as Ped gene candidates.

The four Qa-2-encoding genes are highly homologous and seem to have evolved by the duplication of gene pairs [14, 15]. Since the Q7 gene is similar to Q9, and Q6 is similar to Q8, the Q7 and Q9 genes are jointly referred to as the Q7/Q9 gene pair, and the Q6 and Q8 genes as the Q6/Q8 gene pair. One of the Qa-2-encoding gene pairs, Q7/Q9, has been shown to be transcribed throughout the mouse preimplantation development period [16]. Studies with Q9 transgenic mice and antisense oligonucleotides that block Q7/Q9 transcription suggest that the Q7/Q9 genes are at least partially responsible for the Ped fast phenotype [17, 18]. Whether the other Qa-2-encoding gene pair, Q6/Q8, contributes to the Ped phenotype is still unknown, and this will be one focus of the research reported in this paper. Since the Ped gene functions during preimplantation development independent of maternal effects, any candidate for the Ped gene must be transcribed in preimplantation embryos. Therefore, in order to assess the functional importance of the four Qa-2 antigen-encoding genes, it is necessary to evaluate their individual contribution to total embryonic mRNA during mouse preimplantation embryonic development.

Previous studies have shown that interferon gamma (-IFN) has a direct effect on the Ped gene phenotype. -IFN treatment increases Qa-2 antigen levels and the rate of development of mouse preimplantation embryos [19]. All of the four Ped gene candidates contain interferon responsive sequences (IRS) in their promoter regions [15], and therefore they all have the potential to be up-regulated by -IFN.

The purpose of this study was to evaluate the individual contributions of the four Ped gene candidates to the Ped phenotype at the transcriptional level. Also, the effect of -IFN on the expression pattern of the four Ped gene candidates in mouse preimplantation embryos was tested in order to determine which candidate genes account for the increased level of Qa-2 antigen on the embryonic surface after -IFN treatment.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice and Embryos

The following mouse strains were used in this study: C57BL/6, B6.K2 and CBA/Ca. C57BL/6 and CBA/Ca mice were purchased from the Jackson Laboratory (Bar Harbor, ME). B6.K2 mice were originally provided by L. Flaherty (Wadsworth Center, Albany, NY) in 1985 and subsequently maintained in our laboratory. The mice were housed in an American Association for the Accreditation of Laboratory Animal Care-approved facility, in a day-night cycled room (lights-on 0400-1800 h eastern standard time), with food and water ad libitum. All experiments followed the National Institutes of Health (NIH) guidelines.

The MHC haplotypes, Q region genes, and Ped phenotypes of these three mouse strains are shown in Table 1. B6.K2 and C57BL/6 mice are congenic strains that are genetically identical except for the TL region of the MHC. CBA/Ca mice have different background and MHC genes from the other two strains. C57BL/6 and B6.K2 mice possess the Q6, Q7, Q8, and Q9 genes and are Qa-2-positive, whereas CBA/Ca mice have a deletion of the Q6, Q7, Q8, and Q9 genes and are Qa-2-negative (the deletion of the Q3 gene is unimportant since Q3 is a pseudogene [20]). C57BL/6 and B6.K2 mice have a faster preimplantation embryonic cleavage rate (Ped fast) than CBA/Ca (Ped slow).

Two- to six-month-old female mice were superovulated by injection of 5 IU eCG (Sigma, St. Louis, MO) at 1500 h standard time or at 1600 h daylight savings time, followed 48 h later by injection of 10 IU hCG (Sigma). Mice were mated individually and checked for vaginal plugs the next morning. The plug-positive female mice were killed by cervical dislocation, and embryos were collected into Whitten-Biggers (WB) medium at 17, 41, 53, 65, 77, and 89 h post-hCG injection, corresponding to the 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages of development. Fertilized one-cell embryos were treated with 0.3 mg/ml hyaluronidase (Sigma) for 5 min to remove cumulus cells.

Cells and Antibodies

WEHI-3 cells, a macrophage-like cell line, were purchased from the American Type Culture Collection (ATCC). This cell line was used as a positive control since it has been shown to increase MHC class I antigen expression on the cell surface in response to -IFN [21]. WEHI-3 cells were cultured in Iscove's modified Dulbecco's medium supplemented with 0.05% ß-mercaptoethanol, 0.1% antibiotics/antimycotics, and 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO₂ incubator.

Since splenic lymphocytes express Qa-2 antigen and are readily available, they were used to optimize reverse transcription-polymerase chain reaction (RT-PCR) conditions. Splenic lymphocytes were isolated using a Ficoll-Hypaque density gradient (density = 1.084) as previously described [15]. Activated splenic lymphocytes were obtained by culturing isolated splenic lymphocytes in RPMI 1640 with 10% heat-inactivated fetal bovine serum and 0.1% antibiotics/antimycotics in the presence of phytohemagglutinin (PHA) (Gibco BRL, Gaithersburg, MD) for 72 h.

A monoclonal antibody, 27-11-13, which is specific for the classical MHC class I molecules, H-2 D^b/d, was used to confirm that -IFN treatment had effectively induced MHC class I expression. This antibody was kindly provided by Dr. David Sachs (Massachusetts General Hospital, Boston, MA) [22]. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA).

Primers

Primers for RT-PCR and single nucleotide primer extension (SNuPE) assays were designed on the basis of the known DNA sequences of the Q6, Q7, Q8, and Q9 genes [14, 15] using the Oligo 5.0 program (National Bioscience Inc., Plymouth, MN) (Tables 2 and 3). Primers were synthesized by Genosys Biotechnologies, Inc. (The Woodslands, TX). Upstream and downstream primers for RT-PCR were designed in different exons so that the products amplified from mRNA could be distinguished from those that were amplified from possible contaminating genomic DNA or unprocessed RNA. The SNuPE primers were designed to bind adjacent to one base difference between the Q6 and Q8 genes, and the Q7 and Q9 genes.

Plasmids

Four plasmid clones containing the Q6, Q7, Q8, and Q9 genes were described previously [15]. The plasmid DNA was isolated by using a Qiagen plasmid kit (Qiagen, Chatsworth, CA) according to the protocol supplied by the manufacturer.

RT-PCR Analysis

For optimizing conditions, mRNA isolated from splenic lymphocytes using TRISOLV reagent (Biotecx, Houston, TX) was used. After conditions were optimized, RT-PCR was performed on lymphocyte and embryo lysates by using a GeneAmp RNA PCR kit from Perkin Elmer (Branchburg, NJ). Briefly, lymphocytes and embryos were washed three times in diethyl pyrocarbonate (DEPC)-treated PBS and transferred in a minimum volume of PBS to a 0.5-ml thin-walled reaction tube. Two microliters of lysis buffer containing 10 mM dithiothreitol (DTT), 0.5% NP-40, and 0.5 U/µl ribonuclease (RNase) inhibitor were added. Then 1 µl of 50 µM random hexamer primers was added, and the total volume was brought to 8 µl with RNase-free water. RNA was denatured at 70°C for 5 min and then annealed at 30°C for 5 min. To obtain cDNA, 4 µl MgCl₂ (25 mM), 4 µl deoxynucleoside triphosphates (dNTPs; 10 mM mix), 2 µl 10-strength PCR buffer II, 1 µl MuLV reverse transcriptase (50 U/µl), and 1 µl RNase inhibitor (50 U/µl) were added to bring the total reaction volume to 20 µl; the reaction was incubated for 3 h at 37°C. At the end of incubation time, the reaction was heated at 99°C for 5 min to inactivate the reverse transcriptase. The amplification of the cDNA was performed in a 100-µl reaction mixture containing aliquots of the cDNA product described above (usually 5 µl), 6 µl MgCl₂ (25 mM), 8 µl dNTPs (10 mM mix), 10 µl 10-strength PCR buffer II, 2 µl each upstream primer and downstream primer (10 µM), 5 U AmpliTaq DNA polymerase (Perkin Elmer), and sterile water. The mixture was placed in a Perkin-Elmer Cetus thermal cycler (Norwalk, CT) and heated to 96°C for 1 min; this was followed by two cycles using a setting of denaturation at 96°C for 1 min, primer annealing at 58°C for 45 sec, and extension for 45 sec. Another 40 cycles were performed using the setting of 94°C for 1 min for denaturation, 58°C for 45 sec for annealing, and 72°C for 45 sec for extension, with a final extension at 72°C for 5 min. Amplification of both Q7/Q9 genes and Q6/Q8 genes was always performed on the same cDNA sample. The PCR products were analyzed by electrophoresis on a 6% polyacrylamide gel and staining with ethidium bromide. A 100-base pair (bp) DNA ladder (Gibco BRL) was used as a marker to determine the size of the PCR products.

Cloning and Sequencing

RT-PCR products were cloned into the PCRII vector using the TA cloning kit (Invitrogen, San Diego, CA). Plasmids were isolated using a QIAGEN plasmid kit (Qiagen Inc.). The inserts were sequenced by the dideoxy chain termination method with Sequenase version 2.0 (USB, Cleveland, OH) and [-³⁵S]dATP (specific activity 1000 mCi/mmol; Amersham, Arlington Heights, IL). Sequences were analyzed using DNASTAR software (DNASTAR Inc., Madison,WI).

Restriction Enzyme Digestion

RT-PCR products were purified using the QIAquick Gel DNA Extraction kit (Qiagen Inc.) and digested with Pst I. On the basis of a restriction fragment polymorphism pattern between the Q7 and Q9 genes, the expression of these two genes can be distinguished since there is an additional Pst I site in the RT-PCR product amplified from the Q7 cDNA compared to the Q9 cDNA.

SNuPE Analysis

SNuPE has been used to identify point mutations in genetic disorders and to quantitate the relative abundance of allele-specific sequences differing by one nucleotide [23–30]. A typical SNuPE assay is shown in Figure 1. The SNuPE reaction was performed in a total volume of 10 µl including 1 µl of 10-strength PCR buffer, 0.3 µl of 50 mM MgCl₂, 1 µl of 10 µM SNuPE primer, 1 µl of specific [³²P]dNTP (2 µCi/µl, diluted from 3000 Ci/mmol, 10 µCi/µl) (Amersham), 6.75 µl of the template and H₂O, and 0.15 µl of Taq DNA polymerase (Gibco BRL). The template used was the RT-PCR amplified product that had been purified using the QIAquick Gel DNA Extraction kit. The reaction mixture was overlaid with 20 µl of mineral oil. Then the reaction was incubated at 96°C, 1 min; 94°C, 1 min; 55°C, 2 min; 72°C, 2 min for 1 cycle in a Perkin-Elmer Cetus DNA Thermal Cycler. The samples were run on a 15% denaturing polyacrylamide gel. The gel was dried at 80°C, wrapped in a plastic membrane, and exposed to BioMax film (Kodak, Rochester, NY). The intensity of each band was scanned and analyzed by Alpha Imager 2000 (Alpha Innotech Co., San Leandro, CA).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Diagram of the SNuPE assay for Q7 and Q9 transcripts. A SNuPE assay consists of two steps. In the first step, RT-PCR is performed, and the segments containing one nucleotide difference between the Q7 and Q9 genes are reverse-transcribed and PCR-amplified proportionally in the same reaction. In the second step, the extension of the primer by only one nucleotide is performed in two separate reactions. In each reaction, the primer is complementary to one strand of amplified products from both the Q7 and Q9 genes, and the 3' position of this primer is adjacent to the position of the one nucleotide difference. The Q7 and Q9 genes can then be detected in these two separate reactions using the radiolabeled dNTP specific for only Q7 and only Q9. In the presence of excess primer, the incorporation of radioisotope represents the relative abundance of Q7 and Q9 transcripts in the original samples. The assay can be quantitated by establishing a standard curve with a template of known copy number.

-IFN Treatment

Mouse recombinant -IFN (Gibco BRL) treatment of WEHI-3 cells was performed to confirm the cytokine's ability to enhance the expression of mouse MHC class I molecules and to determine the optimal concentration for use in the mouse preimplantation embryo system. WEHI-3 cells were cultured with various concentrations of -IFN for 48 h. The expression of a classical class I MHC molecule, H-2 D^b, was determined by FACScan analysis (Becton Dickinson, San Jose, CA). Two-cell embryos from the three mouse strains were cultured in WB medium in the presence of -IFN for 48 h and then analyzed for changes in mRNA levels by the SNuPE assay.

FACScan Analysis of MHC Class I Expression

FACScan analysis was performed to detect MHC class I protein expression on the cell surface of WEHI-3 cells. Titration of the first and second antibodies on WEHI-3 cells was first performed to determine the dilution of each antibody that produced the optimal signals. Cells were first washed and resuspended in PBSAZ (PBS + 0.1% NaN₃ + 1% BSA) at 2.5 x 10⁶ cells/ml. First antibody was diluted at 1:10 to 1:10⁶. For each dilution, three tubes were prepared which contained either cells only (tube A), or cells and secondary antibody only (tube B), or cells and first and secondary antibodies (tube C). Then 50 µl of first antibody at each dilution was added to 50 µl of cells at 2.5 x 10⁶ cells/ml in the proper tubes. For the cell control tube (A), and cell and secondary antibody control tube (B), 50 µl of PBSAZ was added instead of first antibody. After incubation at 4°C for 1 h, 300 µl of PBSAZ was added to each tube, and the tubes were spun at 1000 x g for 10 min. Cells were then resuspended in 50 µl PBSAZ. Then 50 µl of secondary antibody (FITC-conjugated rabbit anti-mouse IgG; Cappel, Westchester, PA), diluted in PBSAZ, was added to each tube except the cell control tube (A), to which 50 µl PBSAZ was added instead. Secondary antibody was titrated from 1:50 to 1:800 to determine the optimal dilution. Tubes were incubated at 4°C for 1 h. Then 300 µl PBSAZ was added to each tube, and cells were spun down and resuspended in 100 µl of PBSAZ to which 200 µl of 3% formaldehyde/PBSAZ was then added. These samples could be stored at 4°C for up to 1 wk. Samples were analyzed on a FACScan flow cytometer using Becton-Dickinson LYSIS II software (Becton Dickinson). Mean channel fluorescence of tube C was compared with that of tube A (cell autofluorescence) and that of tube B (background fluorescence from secondary antibody) to determine D^b antigen expression in each sample. In addition, two tube C's were compared to each other, one from before and one from after -IFN treatment.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
RT-PCR Detection of the Q7/Q9 and Q6/Q8 Gene Pairs Expressed in Splenic Lymphocytes

The Qa-2 antigen is expressed on the cell surface of splenic lymphocytes and preimplantation embryos. Since lymphocytes are readily available, while the supply of embryos is more limited, we first optimized reaction conditions on lymphocytes. We isolated RNA from PHA-activated lymphocytes and performed a sensitivity test. We found that the RT-PCR method we used is easily capable of detecting Q6/Q8 transcripts in 100 cells after ethidium bromide staining (Fig. 2). Faint bands in lanes 6 (50 cells) and 7 (5 cells) were also visible in the original picture. Thus, our RT-PCR method is very sensitive and can be used on a limited number of embryos. We were able to detect the expression of both the Q7/Q9 and Q6/Q8 gene pairs in unactivated and PHA-activated lymphocytes from both C57BL/6 and B6.K2 mice (Fig. 3).

View larger version (59K): [in this window] [in a new window]   FIG. 2. Sensitivity test on RNA isolated from C57BL/6 splenic lymphocytes using the primers for the Q6/Q8 gene pair. Lane MW: a 100-bp DNA ladder was used as a size marker; lane 1: 104 splenic lymphocytes from a CBA/Ca mouse were used as a negative control; lanes 2-8: the numbers of lymphocytes corresponding to the amount of RNA used in RT-PCR were 105, 104, 103, 102, 50, 5, and 0.5 cells, respectively.

View larger version (76K): [in this window] [in a new window]   FIG. 3. Transcription of the Q6/Q8 and Q7/Q9 gene pairs in splenic lymphocytes. Lane MW: a 100-bp DNA ladder; lanes 1 and 4: CBA/Ca (negative control); lanes 2 and 5: C57BL/6; lanes 3 and 6: B6.K2. Lanes 1-3 show RT-PCR products amplified using the Q6/Q8 primers, and Lanes 4-6 contain products using the Q7/Q9 primers. The Q6/Q8 gene product is 364 bp and the Q7/Q9 gene product is 380 bp (see Table 2).

Since the four Ped candidate genes are highly homologous, with 94% identity between the Q6/Q8 and Q7/Q9 gene pairs in the coding sequences and more than 99% identity within each gene pair [15], the specificity of the primer sets we used for RT-PCR needed to be evaluated. The RT-PCR products amplified from both Q6/Q8 and Q7/Q9 gene pairs were cloned, and then plasmids containing inserts were isolated. Sequencing data on inserts amplified with Q6/Q8 and Q7/Q9 primers verified the correct sequences after comparison with the known sequences for these four Qa-2-encoding genes. This verified that the primers we used can distinguish the expression of the Q7/Q9 versus the Q6/Q8 gene pairs.

RT-PCR Detection of the Q6/Q8 and Q7/Q9 Gene Pairs Expressed during Preimplantation Development

We next determined the temporal expression of the Q7/Q9 and Q6/Q8 gene pairs by performing RT-PCR on preimplantation embryos at the various stages of development. The amplification of both the Q6/Q8 and Q7/Q9 gene pairs was always performed from the same cDNA sample to ensure that comparisons were valid. The results show that the Q7/Q9 transcripts were detectable at the 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages in C57BL/6 and B6.K2 mice, but no transcripts for the Q6/Q8 genes could be detected at any stage of preimplantation development (Fig. 4). These results allowed us to eliminate the Q6/Q8 genes as Ped gene candidates since the ability to be expressed in preimplantation embryos is a prerequisite to qualify as a Ped candidate gene. Therefore, only Q7 and Q9 could be considered to be the Ped gene candidates.

View larger version (49K): [in this window] [in a new window]   FIG. 4. Differential expression of Ped candidate genes, Q6/Q8 (A and C) and Q7/Q9 (B and D), during preimplantation mouse embryo development. A and B are from C57BL/6 mice, and C and D from B6.K2 mice. RT was performed on 32 1-cell, 16 2-cell, and 8 4-cell embryos, 2 morulae, and 1 blastocyst. PCR reactions were performed on the cDNA equivalent of 8 embryonic cells. Lane MW: 100-bp DNA ladder was used as a size marker; lane 1 of A and C: RT-PCR products from lymphocytes from C57BL/6 mice were used as a positive control (C); lane 1 of B and D: RT-PCR assays on blastocysts from CBA/Ca mice were used as a negative control; lanes 2-7 in all four panels: 1C (1-cell), 2C (2-cell), 4C (4-cell), 8C (8-cell), M (morula), B (blastocyst).

Onset of the Activation of the Embryonic Q7/Q9 Genes

In order to determine at what stage of development the Ped gene candidates are turned on, RT-PCR assays were carried out on zygotes from crosses between C57BL/6 female mice, which are Q7/Q9-positive, and CBA/Ca male mice, which are Q7/Q9-negative. We were able to detect the Q7/Q9 transcripts in zygotes from this particular cross. However, the Q7/Q9 transcripts could not be detected from the reciprocal cross between the CBA/Ca females and the C57BL/6 males until the 2-cell stage (Fig. 5). Since CBA/Ca mice have a deletion of the Q7 and Q9 genes, the transcripts we detected in zygotes from crosses between C57BL/6 females and CBA/Ca males were most likely maternal RNA. We could not detect the Q7/Q9 transcripts in zygotes from the crosses between C57BL/6 males and CBA/Ca females since the C57BL/6 males do not contribute to any zygotic mRNA. The fact that we were able to detect the Q7/Q9 transcripts starting at the 2-cell stage from the crosses between the CBA/Ca females and C57BL/6 males suggests that the Q7/Q9 embryonic genes are turned on at the 2-cell stage, the time at which most mouse embryonic gene transcription begins [31]. These results confirm a similar study [16].

View larger version (86K): [in this window] [in a new window]   FIG. 5. The embryonic Q7/Q9 genes are turned on at the 2-cell stage. MW: 100-bp DNA ladder was used as a size marker; lane 1: RT-PCR was performed on zygotes from the crosses between C57BL/6 females and CBA/Ca males; lane 2: RT-PCR was performed on zygotes from the crosses between CBA/Ca females and C57BL/6 males; lane 3: RT-PCR was performed on 2-cell embryos from the crosses between CBA/Ca females and C57BL/6 males. Lanes 1 and 3 show expression of Q7/Q9 transcripts.

Differential Expression of the Q7 and Q9 Genes in Splenic Lymphocytes and Embryos

We next distinguished the expression of the Q7 and Q9 genes in splenic lymphocytes and embryos by utilizing a restriction fragment-length polymorphism between these two genes. One nucleotide difference between the Q7 and Q9 genes in exon 3 results in an additional Pst I restriction enzyme digestion site in the Q7 gene but not in the Q9 gene (Fig. 6). The Pst I enzyme digestion analysis of the RT-PCR products amplified from Q7/Q9 cDNA showed that both the Q7 and Q9 genes were expressed in lymphocytes and preimplantation embryos from C57BL/6 mice. However, only Q9, and not Q7 was expressed in lymphocytes and preimplantation embryos from B6.K2 mice (Fig. 6). Figure 6B has been published previously [15], but is included to emphasize the comparison with Figure 6A.

View larger version (40K): [in this window] [in a new window]   FIG. 6. Differential expression of the Q7 and Q9 genes in splenic lymphocytes and preimplantation mouse embryos from B6.K2 (A) and C57BL/6 mice (B). RT-PCR was performed using Q7/Q9 primers on different stages of preimplantation embryos from B6.K2 and C57BL/6 mice. Then RT-PCR products were purified and subjected to Pst I digestion. The diagram shows the expected products after digestion of Q7 and Q9 with Pst I. U, Uncut RT-PCR products; C, products cut with Pst I. Panel B was previously published [15] and appears with permission of the publisher, Springer-Verlag GmbH&Co. KG.

Detection of mRNA for the Q6, Q7, Q8, and Q9 Genes in Lymphocytes by the SNuPE Assay

The SNuPE assay is a quantitative method to distinguish Q7 from Q9 and Q6 from Q8 mRNA expression. First, the specificity of the SNuPE primers was tested by using individual plasmid clones for the Q6, Q7, Q8, and Q9 genes as templates (Fig. 7). The Q7/Q9 SNuPE primer was specific for the Q7 or Q9 plasmid DNA in the presence of the specific radiolabeled dNTP. The Q6/Q8 primer was specific only for Q6 plasmid DNA, not for Q8, because there were also signals for the Q7 and Q9 plasmid DNA in the presence of [³²P]dGTP. However, because in our experiments the Q7/Q9 and Q6/Q8 gene pairs were specifically amplified before being subjected to the SNuPE assay, the fact that the Q6/Q8 SNuPE primer has some cross-reaction with Q7/Q9 does not compromise the interpretation of the results. Before the SNuPE assay was applied to embryos, the assay was tested on splenic lymphocytes. We performed SNuPE assays on the RT-PCR products of both the Q7/Q9 gene pair and the Q6/Q8 gene pair from splenic lymphocytes from CBA/Ca, C57BL/6, and B6.K2 mice. The results confirmed the expression pattern shown by our previous RT-PCR results. In addition, the results revealed that both the Q6 and Q8 genes were expressed in splenic lymphocytes from C57BL/6 and B6.K2 mice but not from CBA/Ca mice (Fig. 8).

View larger version (32K): [in this window] [in a new window]   FIG. 7. Autoradiogram showing specificity of SNuPE primers. Plasmid clones containing Q6, Q7, Q8, and Q9 genes were used to confirm the specificity of the SNuPE primers. A) Q7/Q9 SNuPE primer was used. B) Q6/Q8 SNuPE primer was used. Plasmid templates across bottom apply to lanes in both A and B.

View larger version (28K): [in this window] [in a new window]   FIG. 8. Detection of Q6, Q7, Q8, and Q9 gene transcripts in splenic lymphocytes from CBA/Ca, C57BL/6, and B6.K2 mice by the SNuPE assay. The gel-purified templates corresponding to 20 µl of RT-PCR-amplified products were used for a SNuPE primer extension with [32P]dCTP, or [32P]dGTP, or [32P]dATP. Lane 1: Q7/Q9 RT-PCR products amplified from splenic lymphocytes from CBA/Ca mice plus [32P]dCTP. Lane 2: Q7/Q9 RT-PCR products amplified from splenic lymphocytes from C57BL/6 mice plus [32P]dCTP. Lane 3: Q7/Q9 RT-PCR products amplified from splenic lymphocytes from B6.K2 mice plus [32P]dCTP. Lane 4: Q7/Q9 RT-PCR products amplified from splenic lymphocytes from CBA/Ca mice plus [32P]dGTP. Lane 5: Q7/Q9 RT-PCR products amplified from splenic lymphocytes from C57BL/6 mice plus [32P]dGTP. Lane 6: Q7/Q9 RT-PCR products amplified from splenic lymphocytes from B6.K2 mice plus [32P]dGTP. Lane 7: Q6/Q8 RT-PCR products amplified from splenic lymphocytes from CBA/Ca mice plus [32P]dATP. Lane 8: Q6/Q8 RT-PCR products amplified from splenic lymphocytes from C57BL/6 mice plus [32P]dATP. Lane 9: Q6/Q8 RT-PCR products amplified from splenic lymphocytes from B6.K2 mice plus[32P]dATP. Lane 10: Q6/Q8 RT-PCR products amplified from splenic lymphocytes from CBA/Ca mice plus [32P]dGTP. Lane 11: Q6/Q8 RT-PCR products amplified from splenic lymphocytes from C57BL/6 mice plus [32P]dGTP. Lane 12: Q6/Q8 RT-PCR products amplified from splenic lymphocytes from B6.K2 mice plus [32P]dGTP. RI, Relative intensity.

Quantitation of mRNA of the Q7 and Q9 Genes in Preimplantation Embryos by the SNuPE Assay

The real benefit of the SNuPE assay is that it allows the quantitation of mRNA levels in individual embryos. Because only Q7 and Q9 were transcribed in mouse embryos, and not Q6 and Q8 (Fig. 4), we used the SNuPE assay to quantitate the abundance of Q7 and Q9 transcripts at different stages of preimplantation development in C57BL/6 and B6.K2 mice. First, SNuPE assays were performed on different dilutions of Q7 and Q9 plasmid DNA, and a standard curve was plotted (Fig. 9). Next, we established conditions so that the signal from embryos would fall on the standard curve and also at the logarithmic amplification phase of PCR. These conditions entailed use of the equivalent of 32 embryonic cells for the RT reaction, and the cDNA equivalent of 8 embryonic cells (e.g., one 8-cell embryo or one quarter of a 32-cell embryo) for 35 cycles of PCR. We determined the relative expression of the Q7 and Q9 genes at different stages of development in preimplantation embryos from C57BL/6 and B6.K2 mice (Fig. 10). By performing SNuPE assays on known copy numbers of Q7 and Q9 plasmid DNA side by side with the Q7/Q9 RT-PCR products from the embryos, we were able to estimate the copy number of Q7 and Q9 transcripts in the preimplantation embryos from C57BL/6 mice, and the copy number of the Q9 transcript from B6.K2 mice (Table 4).

View larger version (25K): [in this window] [in a new window]   FIG. 9. Quantitation of SNuPE assay. Lane 1: 1012 copies of Q9 plasmid DNA. Lane 2: 5 x 1011 copies of Q9 plasmid DNA. Lane 3: 2.5 x 1011 copies of Q9 plasmid DNA. Lane 4: 1.25 x 1011 copies of Q9 plasmid DNA. Lane 5: 1012 copies of Q7 plasmid DNA. Lane 6: 5 x 1011 copies of Q7 plasmid DNA. Lane 7: 2.5 x 1011 copies of Q9 plasmid DNA. Lane 8: 1.25 x 1011 copies of Q7 plasmid DNA. The error lines represent the standard error of the mean. RI, Relative intensity.

View larger version (28K): [in this window] [in a new window]   FIG. 10. Quantitation of Q7 and Q9 gene transcripts at different stages of mouse preimplantation development. In A, the SNuPE assay was performed on preimplantation embryos from C57BL/6 mice. In B, the SNuPE assay was performed on preimplantation embryos from B6.K2 mice. RT reactions were performed on 32 1-cell, 16 2-cell, and 4 8-cell embryos, and 1 blastocyst. PCR reactions were performed on the cDNA equivalent to 8 embryonic cells. PCR conditions were the same except the number of PCR cycles was reduced to 35. Eighty microliters of RT-PCR products were purified and eluted in 20 µl. Then 5 µl of purified RT-PCR products were used for each SNuPE reaction. Lane 1: Q7/Q9 RT-PCR products amplified from 1-cell embryos plus [32P]dCTP. Lane 2: Q7/Q9 RT-PCR products amplified from 2-cell embryos plus [32P]dCTP. Lane 3: Q7/Q9 RT-PCR products amplified from 8-cell embryos plus [32P]dCTP. Lane 4: Q7/Q9 RT-PCR products amplified from blastocyst embryos plus [32P]dCTP. Lane 5: Q7/Q9 RT-PCR products amplified from 1-cell embryos plus [32P]dGTP. Lane 6: Q7/Q9 RT-PCR products amplified from 2-cell embryos plus [32P]dGTP. Lane 7: Q7/Q9 RT-PCR products amplified from 8-cell embryos plus [32P]dGTP. Lane 8: Q7/Q9 RT-PCR products amplified from blastocyst embryos plus [32P]dGTP. RI, Relative intensity. The error lines represent the standard error of the mean.

Effect of -IFN on Q6, Q7, Q8, and Q9 Gene Expression in Preimplantation Embryos

Since the Q6, Q7, Q8, and Q9 genes all possess interferon-responsive elements, the last set of experiments was designed to determine the effect of -IFN on the expression of these four genes. In order to confirm that mouse recombinant -IFN is active and has the ability to up-regulate expression of MHC class I antigens, we treated WEHI-3 cells with various concentrations of -IFN for 48 h. We then monitored the expression level of the MHC class I molecule, H-2 D^b, by FACScan analysis. The results showed that H-2 D^b expression was enhanced by -IFN in a dose-dependent manner (Fig. 11). The concentration of murine -IFN that resulted in the maximal enhancement of H-2D^b expression on WEHI-3 cells was 10³ U/ml.

View larger version (16K): [in this window] [in a new window]   FIG. 11. Up-regulation of H-2 Db expression on WEHI-3 cells by -IFN. WEHI-3 cells were treated with various concentrations of -IFN for 48 h; then the expression of H-2 Db on the cell surface was analyzed by FACScan analysis. The error lines represent the standard error of the mean.

Next, the effect of -IFN on the expression of Q6, Q7, Q8, and Q9 genes in preimplantation development was examined. Two-cell embryos from C57BL/6, B6.K2, and CBA/Ca mice were collected and cultured in WB medium in the presence of 10³, 10⁴, and 5 x 10⁴ U/ml of -IFN for 48 h; and then Q6, Q7, Q8, and Q9 expression was analyzed. The RT-PCR results showed that the overall qualitative expression pattern of the Q6, Q7, Q8, and Q9 genes was not altered upon -IFN treatment of either C57BL/6 or B6.K2 embryos (Fig. 12). The SNuPE results showed that the expression of both Q7 and Q9 was up-regulated approximately 2-fold in preimplantation embryos from both C57BL/6 and B6.K2 mice (Fig. 13).

View larger version (72K): [in this window] [in a new window]   FIG. 12. RT-RCR detection of expression of both the Q7/Q9 gene pair and the Q6/Q8 gene pair before (lanes 1-6) and after (lanes 7-12) -IFN treatment. Lane MW: a 100-bp DNA ladder.

View larger version (33K): [in this window] [in a new window]   FIG. 13. The effect of -IFN treatment on the expression of the Q7 and Q9 genes as determined by the SNuPE assay. In A, 2-cell embryos from C57BL/6 mice were used. In B, 2-cell embryos from B6.K2 were used. Lane 1: embryos with -IFN treatment plus [32P]dCTP. Lane 2: embryos with -IFN treatment plus [32P]dGTP. Lane 3: embryos without -IFN treatment plus [32P]dCTP. Lane 4: embryos without -IFN treatment plus [32P]dGTP. RI, Relative intensity. Student's t-test analysis of the data shows that both -IFN treatment groups are significantly higher than the control groups (p < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Ped gene is one of the genes that influences the rate of cleavage division of preimplantation mouse embryos. In this study, we continued our experiments to identify the Ped gene at the molecular level. We assessed the contribution of four Ped gene candidates—Q6, Q7, Q8, and Q9—each of which encodes the Qa-2 antigen, to total embryonic mRNA during preimplantation development. We showed that only the Q7/Q9 gene pair, not the Q6/Q8 gene pair, is detectable during preimplantation mouse development. Since only actively transcribed genes can express functional proteins and play a role in the regulation of preimplantation development, we could eliminate the Q6 and Q8 genes as possible Ped genes. Therefore, the Ped gene candidates could be narrowed to the Q7 and Q9 genes.

Next, we compared the individual expression of the Q7 and Q9 genes in two Ped fast mouse strains, C57BL/6 and B6.K2. We found that both the Q7 and Q9 genes were expressed in all stages of preimplantation development in C57BL/6 mice, but only Q9 was expressed in B6.K2 embryos. B6.K2 mice have the same background genes as C57BL/6 mice, and they differ only in the TL region (Table 1). It has been suggested that the differential expression of the Q7 and Q9 genes in both strains may be due to a regulatory effect that resides in the TL region or a mutation that has inactivated the Q7 gene [32]. On the basis of the quantitative SNuPE assay, we were able to show that the levels of Q7 and Q9 transcripts in preimplantation embryos from C57BL/6 mice are equivalent to each other and that the level of the Q9 transcripts in B6.K2 mice is the same as in C57BL/6 (Table 4). If one calculates the amount of mRNA for Q7 and Q9 on a per cell basis, the amount of mRNA per cell remains constant throughout development. The Ped fast phenotype is conferred by 30-40 mRNA molecules/embryonic cell, with no major difference in rate of embryonic development with twice the number of mRNA molecules/embryonic cell (compare the C57BL/6 and B6.K2 results in Tables 4 and 6). In addition, our results indicate that the Q7/Q9 transcript level is similar to H-2K mRNA levels at different stages of preimplantation embryo development [33]. A summary of the expression of the Q6, Q7, Q8, and Q9 genes in splenic cells and embryos from C57BL/6 and B6.K2 mice is shown in Table 5.

In order to investigate further the relationship between the differential expression patterns of the Q7 and Q9 genes and the Ped gene phenotype, we compared the expression pattern of the Q7 and Q9 genes, the Qa-2 levels in splenic cells, and the Ped phenotype in 14 mouse strains (Table 6) [2, 17, 32, 34]. The double-positive strains expressing both the Q7 and Q9 genes and the single-positive strains expressing either the Q7 or the Q9 gene are Ped fast, whereas double-negative strains, which do not express either of the Q7 or Q9 genes, are Ped slow. However, the double-positive strains do not have a faster Ped phenotype than the single-positive strains. It seems that cell surface expression of Qa-2 antigen may be similar whether one or both genes are transcribed (compare Qa-2 sites/cell for B6.K3 mice that express Q7 and Q9 with those for B10.A[2R] mice that express only Q7). Thus, one can speculate that strains expressing only the Q7 or Q9 gene might have a level of Qa-2 antigen in preimplantation mouse embryos similar to the double-positive strains due to yet-to-be-defined compensation effects. It seems likely that similar Qa-2 levels in embryos will account for the similar rates of embryonic development in all Ped fast strains.

The Q7 and Q9 genes both encode the Ped fast phenotype in the mouse. However, we still do not know whether both the Q7 and Q9 genes contribute equally to the Ped fast phenotype. This awaits a functional study of the Qa-2 proteins encoded by the Q7 and Q9 genes. These Qa-2 proteins have only one amino acid difference in the 2 domain: glutamine from the Q7 gene and glutamic acid from the Q9 gene [15]. This difference may not be of biological significance since it is outside the peptide binding site.

In C57BL/6 and B6.K2 mice the expression of the Q6/Q8 genes is detectable in splenic lymphocytes but not in preimplantation mouse embryos. The mechanisms of this differential gene expression are still unknown. It has been shown that the 5' upstream regulatory sequence is important in the control of MHC class I expression [35]. A recent report has shown that a 150-bp 5' region of a human MHC class I gene is sufficient to direct a tissue-specific expression pattern [36]. A DNA sequence comparison among the promoters of Q6, Q7, Q8, and Q9 [15] shows that the four Qa-2-encoding genes have the same promoter structure as the classical MHC class I genes [37, 38], and also reveals 27 bp differences out of 800 in the 5' regulatory region. Of special interest is that 4 of the 27 differences are in a 150-bp 5' region containing the three regulatory regions, Class I regulatory element (CRE)/enhancer A, IRS, and enhancer B, and may account for the differential expression of the Q6/Q8 gene pair versus the Q7/Q9 gene pair in embryos compared to splenic lymphocytes. It is possible that a single cis-element, the CRE, is responsible for directing appropriate expression of Q6/Q8 genes in both embryos and lymphocytes, since it has been shown that the CRE acts as a negative regulator in undifferentiated cells and a positive regulator in differentiated cells [39]. There are two differences in the CRE sequences between the Q7/Q9 and Q6/Q8 genes. Therefore, one possibility that may account for the expression of the Q7/Q9 genes, but not the Q6/Q8 genes, in preimplantation embryos is that the CRE of the Q6/Q8 genes may suppress promoter activity in preimplantation embryos while the CRE sequences of the Q7/Q9 genes may have lost this negative regulatory effect due to the two mutations. However, we cannot rule out the possibility that the regulation of tissue-specific expression of the Q7/Q9 and Q6/Q8 genes is exerted by regulatory sequences further 5' upstream and/or by the 1 kilobase of extra sequence in the 3' untranslated region in the Q6/Q8 genes compared to the Q7/Q9 genes [8].

Both DNA sequence (cis-acting factors) and regulatory proteins (trans-acting factors) play important roles in the regulation of gene expression. For the Q6/Q8 genes, the cis-acting elements are the same in embryos and splenic lymphocytes from the same mouse strain, so the observed tissue-specific differences might be related to trans-acting factors. It may be possible, in the future, to identify trans-acting factors that regulate Qa-2 expression.

Finally, the response of the four genes—Q6, Q7, Q8, and Q9—to -IFN was investigated. -IFN can up-regulate the expression of class I MHC genes in cells that express -IFN receptors. It has been shown that -IFN can induce MHC antigen expression in cells that do not normally express these antigens [40]. Therefore, we tested whether -IFN could induce Q6/Q8 expression in preimplantation embryos. We found that -IFN could not induce the expression of the Q6/Q8 genes in C57BL/6 and B6.K2 preimplantation embryos. However, -IFN enhanced the expression of the Q7/Q9 genes. This confirms a previous study that showed that -IFN can up-regulate the expression of the Ped gene product, Qa-2, and speed up the rate of preimplantation development [19]. The fact that -IFN cannot turn on transcription of the Q6/Q8 genes in embryos, even though the cis-regulatory IRS sequence is present, argues in favor of control by trans-regulatory factors of the suppression of Q6/Q8 transcription in preimplantation embryos. It has been shown that -IFN and -IFN receptor genes are transcribed during preimplantation mouse development [41]. However, it is still unknown whether the embryos utilize similar JAK-STAT signaling pathways [42–44] that transmit signals from -IFN receptors to modulate gene expression. The analysis of the expression of the components involved in -IFN signaling pathways will be the first step towards the understanding of the function of -IFN in early embryonic development.

In conclusion, the results reported in this paper show that both the Q7 and the Q9 genes can confer the Ped fast phenotype to preimplantation embryos. There seems to be a threshold level of both mRNA and protein for Qa-2 antigen, the product of both the Q7 and Q9 genes, that is sufficient for embryos to cleave at a fast rate. Although the Q6 and Q8 genes are present in Ped fast embryos, they are not transcribed, in constrast to lymphocytes, which transcribe all four Qa-2-encoding genes—Q6, Q7, Q8, and Q9. The molecular mechanisms of the differential transcription pattern of the Qa-2 antigen-encoding genes in embryos compared to lymphocytes remains to be determined.

	ACKNOWLEDGMENTS

 
We thank Abby McElhinny and Miriam Paschetto for their help with embryo collection and careful reading of the manuscript. We also thank Melissa Amendola for taking care of the mouse colony.

	FOOTNOTES

 
¹ Supported by NIH grant HD 31505.

² Correspondence: Carol M. Warner, Department of Biology, 414 Mugar Hall, Northeastern University, Boston, MA 02115. FAX: 617 373 3724; cmw{at}neu.edu

Accepted: June 3, 1998.

Received: February 18, 1997.

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