Ng mRNA Research Articles

The Alpha Subunit of Hemoglobin Interacts with Endothelial Nitric Oxide Synthase to Regulate Feedback Vasodilation in Human Resistance Arteries

Alpha globin, but not beta globin, is expressed in mouse resistance arteries where it binds endothelial nitric oxide (NO) synthase (eNOS) and limits NO signaling. To understand this signaling pathway in humans, we studied omental resistance arteries. Omental tissue was collected during clinically indicated surgery on protocol 13-C-0176 and transferred to the lab on ice. Human resistance arteries 80 to 200 um in diameter were microdissected, cannulated, and perfused with cold PBS to expel blood. We measured gene expression of HBA1 (2658 ± 1190 gene transcripts per 1 ng mRNA ± SEM), HBA2 (3319 ± 1667), HBB (1831 ± 725), and NOS3 (408 ± 49) using RT-ddPCR in human arteries obtained from 7 different subjects. Expression of SLC4A1, a control for blood contamination, was nearly undetectable at (4.2 ± 2.3). To determine the binding partners of vascular alpha globin, protein lysates from omental arteries from three donors were subjected to co-immunoprecipitation with an anti-alpha globin antibody. Western blot revealed both eNOS and beta globin to co-immunoprecipitate (co-IP) with alpha globin. Multiphoton imaging of perfused omental arteries revealed co-localization of alpha globin, beta globin and eNOS to distinct punctates within the plane of the internal elastic lamina (IEL). Given the broadly autofluorescent nature of these complexes (similar to hemoglobin in red blood cells), we used fluorescence lifetime imaging microscopy (FLIM) to identify distinct peak lifetimes of fluorophores labeling antibodies against each of alpha globin, beta globin, and eNOS in the complex. To interrogate the function of endothelial hemoglobin, we performed ex vivo pressure myography on omental arteries obtained from five donors. Maximal vasoconstriction of omental arteries to the alpha-1-adrenergic agonist phenylephrine (PE) was 40.8 ± 3.1% of baseline diameter. Incubation with an alpha globin mimetic peptide that disrupts binding of alpha globin to eNOS, reduced maximal constriction in response to PE to 65.9 ± 2.7% of baseline (p < 0.0001 compared to PE alone), whereas a control peptide had little effect (46.5 ± 7.6% of baseline). Inhibition of eNOS by L-NAME in mimetic-peptide treated arteries restored PE-induced vasoconstriction (44.2 ± 2.3% of baseline). We identified expression of not only alpha but also beta globin in perfused human omental arteries. Co-IP and multiphoton microscopy identified alpha globin, beta globin, and eNOS as components of a complex that localizes near the IEL, likely in myoendothelial junctions. Disruption of alpha globin-eNOS binding enhanced feedback vasodilation to phenylephrine in an NO synthase-dependent fashion. In conclusion, we report that hemoglobin is expressed in human omental arteries where it binds eNOS and regulates alpha-1-adrenergic feedback vasodilation by limiting nitric oxide signaling.

Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion.

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

Rapid, experience-dependent translation of neurogranin enables memory encoding

Experience induces de novo protein synthesis in the brain and protein synthesis is required for long-term memory. It is important to define the critical temporal window of protein synthesis and identify newly synthesized proteins required for memory formation. Using a behavioral paradigm that temporally separates the contextual exposure from the association with fear, we found that protein synthesis during the transient window of context exposure is required for contextual memory formation. Among an array of putative activity-dependent translational neuronal targets tested, we identified one candidate, a schizophrenia-associated candidate mRNA, neurogranin (Ng, encoded by the Nrgn gene) responding to novel-context exposure. The Ng mRNA was recruited to the actively translating mRNA pool upon novel-context exposure, and its protein levels were rapidly increased in the hippocampus. By specifically blocking activity-dependent translation of Ng using virus-mediated molecular perturbation, we show that experience-dependent translation of Ng in the hippocampus is required for contextual memory formation. We further interrogated the molecular mechanism underlying the experience-dependent translation of Ng, and found that fragile-X mental retardation protein (FMRP) interacts with the 3'UTR of the Nrgn mRNA and is required for activity-dependent translation of Ng in the synaptic compartment and contextual memory formation. Our results reveal that FMRP-mediated, experience-dependent, rapid enhancement of Ng translation in the hippocampus during the memory acquisition enables durable context memory encoding.

Pparγ Expression in T Cells as a Prognostic Marker of Sepsis.

Translating murine data to the human situation, we proposed that the level of peroxisome proliferator-activated receptor γ (PPARγ) expression in T cells from septic patients correlates with clinical outcome. In this preliminary report, we analyzed PPARγ mRNA expression in CD3 T cells derived from blood of a very small number of septic patients (n = 18) on various days up to 2 weeks after the initial diagnosis. CD3 T cell count was determined by flow cytometry. T cells from n = 11 healthy donors were included as controls. Maximal PPARγ mRNA expression was observed on the day of sepsis diagnosis (day 0; 5,896 ± 1,523 copies PPARγ mRNA/25 ng mRNA, P < 0.05 vs. controls). In contrast, the number of CD3 T cells was significantly decreased in septic patients compared with healthy controls (296 ± 31 vs. 1,803 ± 134 T cells/μL blood, P < 0.001). Setting two arbitrary limits: patients with a PPARγ expression in T cells higher than 7,000 copies/25 ng mRNA, of whom five of six patients died during the ICU stay, and patients with a T cell count below 100 T cells/μL blood, of whom five of eight patients died, we identified a correlation between sepsis survival and low T cell number, paired with high T cell-specific PPARγ expression. Among all 18 sepsis patients, four fulfilled the criteria for both arbitrary settings and all four of these patients subsequently died. We suggest that both high PPARγ expression in T cells and low absolute T cell number in blood of septic patients may have the potential as a new prognostic marker for a poor sepsis outcome.

Increased Erythropoiesis in Mice Injected With Submicrogram Quantities of Pseudouridine-containing mRNA Encoding Erythropoietin

Advances in the optimization of in vitro-transcribed mRNA are bringing mRNA-mediated therapy closer to reality. In cultured cells, we recently achieved high levels of translation with high-performance liquid chromatography (HPLC)-purified, in vitro-transcribed mRNAs containing the modified nucleoside pseudouridine. Importantly, pseudouridine rendered the mRNA non-immunogenic. Here, using erythropoietin (EPO)-encoding mRNA complexed with TransIT-mRNA, we evaluated this new generation of mRNA in vivo. A single injection of 100 ng (0.005 mg/kg) mRNA elevated serum EPO levels in mice significantly by 6 hours and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10–100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma. Using macaques, we could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. These results demonstrate that HPLC-purified, pseudouridine-containing mRNAs encoding therapeutic proteins have great potential for clinical applications.

Effects of sleep deprivation for 24 hours on long-term potentiation and neurogranin mRNA expression in hippocampus of rats

Objective To study 24 hours of sleep deprivation(SD)on long-term potentiation(LTP)and neurogranin(Ng)mRNA expression of hippocampus in rats.Methods Male Wistar rats were divided into two groups:sleep deprivation group(SD group)and control group(C group).After 24 hours of SD by sleep deprivation box,LTP was induced in hippocampal dentate gyrus(DG)by high-frequency stimulation(HFS).One step methods by Trizol was used to extract hippocampus neuronal total RNA.The changes of Ng mRNA expression were detected by SYBRA green ⅠRT-PCR.Results Compared with the C group,the changes of amplitude of population spike (PS)were lower in SD group,especially at 25min,30min,35min,40min,45min and 50rain after HFS(P＜0.05).The expression of Ng mRNA of SD group(0.48±0.38)Was significantly lower than that of C group(1.40±1.07,P＜0.05).Conclusion The expression of Ng mRNA was down regulated by 24 hours of SD,which might be relevant to the impairment of LTP in hippocampus of rats. Key words: Sleep deprivation; Hippocampus; LTP; Neurogranin

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: Difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP

Here we report a method for efficient transfection of in vitro-transcribed mRNA into two different types of human adherent cells, the neuroblastoma cell line SK-N-AS, and the transformed kidney cell line HEK293. By using newly trypsinized adherent cells in suspension and Lipofectaminetrade mark 2000, we detected a transfection efficiency of 80-90% in both cell lines and a cell viability of 90% in SK-N-AS and 60% in HEK293, 24 h after transfection when using cytoplasmic enhanced green fluorescent protein (EGFP)-mRNA. We have evaluated the different effects of the generally used EGFP that mainly localizes to the cytoplasm and nuclear EGFP, where the nuclear EGFP are more toxic to the cells than the cytoplasmic EGFP. In order to develop a null experiment, we constructed a short non-functional mRNA including a nuclear localization signal and evaluated the concentrations at which mRNA encoding nuclear proteins can be added without a general toxicity, depending on the fact that the proteins are localized to the nucleus. For both SK-N-AS and HEK293 cells, a concentration of up to 100 ng mRNA in 10(5) cells, encoding a nuclear protein with no other function, did not affect the cells. For evaluation of the method, we screened four different human mRNAs, PDG, DFFA, CORT and PEX14, for their ability to affect cell proliferation in these cells. PEX14 was the only gene that significantly (p=0.03) reduced cell proliferation for both cell types, DFFA significantly (p=0.04) reduced cell proliferation in SK-N-AS but not in HEK293 cells. PGD and CORT did not have any effect on cell proliferation. We have developed an easy method for efficient delivery of in vitro-transcribed mRNA into the adherent cell lines, SK-N-AS and HEK293. This method is useful for a quick screening of how different genes affect cell proliferation.

Combined laser capture microdissection and serial analysis of gene expression from human tissue samples

Cell-specific gene expression profiling from heterogeneous human tissues is confounded by cell purification limitations. Here, we describe a technique to generate gene expression profiles of pure populations of prostate cancer cells obtained from fresh-frozen prostatectomy specimens and small initial quantities of RNA by combining laser capture microdissection and microserial analysis of gene expression (LCM–microSAGE). Two microSAGE libraries were obtained from approximately 100 000 laser pulses, estimated to contain fewer than 3 × 105 cells and 20–30 ng mRNA. Two libraries were sequenced to a depth of 10 111 and 10 463 unique tags from normal and cancer cells, representing 6453 and 6923 genes, respectively. Most transcripts were expressed at similar levels, but cancer cells compared with normal cells had increased expression of 385 tags and decreased expression of 389 tags. A total of 20 genes were differentially expressed (P<0.05); five of these genes were upregulated and 15 were downregulated in cancer cells. Quantitative reverse transcriptase-polymerase chain reaction results from three selected genes corroborated the existence of cell-specific gene expression in LCM–microSAGE-derived libraries. In conclusion, the LCM–microSAGE approach demonstrates that large-scale expression profiles of known and unknown transcripts can be generated from pure populations of target cells obtained from human tissue samples comprised of heterogeneous mixtures of cell types.

Optimized transfection of mRNA transcribed from a d(A/T) 100 tail-containing vector

Studies employing mRNA transfection are currently limited by a lack of transcription vectors for generating a long poly(A) tail-containing mRNA and published methods for efficient mRNA transfection. We have constructed a transcription vector containing firefly luciferase gene (pBS-FLuc-A100) to generate luciferase mRNA with A 100 tail followed by no heterologous sequence. The pBS-FLuc-A100 was propagated in XL1-Blue, in which the plasmid was more stable than in other bacterial strains. Optimal mRNA transfection conditions were determined using TransMessenger Transfection Reagent (Qiagen) and yeast tRNA as a carrier. Firefly luciferase expression, which peaked at about 12 h post-transfection, was detected with as little as 5 ng mRNA and was linear with mRNA amount up to 100 ng. When cells were transfected with luciferase mRNA containing different lengths of poly(A) tail, luciferase expression increased proportionally with poly(A) tail length up to 60A residues and then declined. Cell lines from monkey, mouse, and rat were transfected efficiently by this method. Like cellular ferritin heavy chain mRNA, which contains an iron response element in its 5′UTR, translation of transfected luciferase mRNA containing the 5′UTR of ferritin mRNA was iron-dependent. Our results demonstrate that the poly(A) vector and the transcription method described will be useful to study the regulation of gene expression at the mRNA level by UTRs.

Chronic ethanol consumption restores the age-related decrease in neurogranin mRNA level in the hippocampus of mice

Neurogranin (Ng) is a Ca 2+-sensitive calmodulin-binding neuron-specific protein that has been implicated in the regulation of numerous post-synaptic signalling pathways. Here, we investigate the effects of 5 months low level ethanol consumption (∼20% of total calories intake) on Ng mRNA expression in the brain of adult (∼7–8 months) and aged (∼21–22 months) mice using in situ hybridization histochemistry. Results showed that ageing was accompanied by a decrease in amounts of mRNA coding for Ng, especially in the hippocampus (∼25% of adults) known to play a critical role in higher cognitive functions. Chronic ethanol consumption restored this decline up to pre-senescent (adult) levels without altering Ng mRNA levels in adult mice. On the basis of recent data indicating a central role for Ng in the regulation of hippocampal synaptic plasticity and spatial learning, our results suggest that moderate ethanol consumption might have a beneficial influence on cognitive deterioration during senescence. Such a possibility is in fact congruent with recent follow-up studies conducted in elderly people.

A novel sensitive microarray approach for differential screening using probes labelled with two different radioelements.

We have developed a novel microarray approach for differential screening using probes labelled with two different radioelements. The complementary DNAs from the reverse transcription of mRNAs from two different biological samples were labelled with radioelements of significantly different energies (3H and 35S or 33P). Radioactive images corresponding to the expressed genes were acquired with a MicroImager, a real time, high resolution digital autoradiography system. An algorithm was used to process the data such that the initially acquired radioactive image was filtered into two subimages, each representative of the hybridisation result specific for one probe. The simultaneous screening of gene expression in two different biological samples requires <100 ng mRNA without any amplification. In such conditions, the technique is sensitive enough to directly quantify the amount of mRNA even when present in small amounts: 10(7) molecules in the probe as assessed with an added control sequence and 2 x 10(5) molecules with an endogenous tyrosine hydroxylase mRNA. This novel technique of double radioactive labelling on a microarray is thus suitable for the comparison of gene expression in two different biological samples available in only small quantities. Consequently, it has great potential for various biological fields, such as neuroscience.

Selective age-related changes in the PKC-sensitive, calmodulin-binding protein, neurogranin, in the mouse brain.

Brain ageing is associated with a dysregulation of intracellular calcium (Ca(2+)) homeostasis which leads to deficits in Ca(2+)-dependent signalling pathways and altered neuronal functions. Given the crucial role of neurogranin/RC3 (Ng) in the post-synaptic regulation of Ca(2+) and calmodulin levels, age-dependent changes in the levels of Ng mRNA and protein expression were analysed in 3, 12, 24 and 31-month-old mouse brains. Ageing produced significant decreases in Ng mRNA expression in the dorsal hippocampal subfields, retrosplenial and primary motor cortices, whereas no reliable changes were seen in any other cortical regions examined. Western blot indicated that Ng protein expression was also down-regulated in the ageing mouse brain. Analysis of Ng immunoreactivity in both hippocampal CA1 and retrosplenial areas indicated that Ng protein in aged mice decreased predominantly in the dendritic segments of pyramidal neurones. These data suggest that age-related changes of post-synaptic Ng in selected brain areas, and particularly in hippocampus, may contribute to altered Ca(2+)/calmodulin-signalling pathways and to region-specific impairments of synaptic plasticity and cognitive decline.

Differential expression of thyrotropin receptor mRNA in the porcine heart.

Thyrotropin receptor (TSHR) mRNA expression has previously been detected in human heart, suggesting a possible role for the receptor in cardiac function and pathophysiology. In the present study we examined the regional distribution of TSHR mRNA in pig heart to map potential cardiac sites of TSH action. Polyadenylated mRNA extracted from thyroid, atria, ventricles, aorta, coronary arteries, epicardial fat, and purified preparations of atrial and ventricular cardiomyocytes was subjected to reverse-transcriptase polymerase chain reaction (RT-PCR) using primers designed to amplify a 311 base pair (bp) DNA segment of the human TSHR. After reverse transcription of 100 ng mRNA, cDNA was amplified by PCR using TSHR primers and compared by electrophoresis on 2% agarose gels. Relative levels of TSHR cDNA (normalized to glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) were as follows: Coronary arteries, epicardial fat > right atrium > left atrium > right ventricle, aorta > left ventricle, ventricular cardiocytes. In contrast to ventricular cardiocytes, purified atrial cardiocytes expressed levels of TSHR mRNA readily detectable with RT-PCR. These findings demonstrate that TSHR mRNA expression in porcine heart varies regionally, and furthermore suggest that areas of highest expression (coronary arteries, adipose tissue, right atrium) are potential sites for a functional or pathologic role of the TSHR.

Expression and characterization of a new species of 11 beta-hydroxysteroid dehydrogenase in Xenopus oocytes

11 beta-Hydroxysteroid dehydrogenase (11-OHSD) plays a critical role in conferring aldosterone specificity to mineralocorticoid target cells. We have recently described a novel isoform of 11-OHSD in the collecting duct (11-OHSD/CD), which differs from the previously characterized isoform (11-OHSD-1) in kinetic parameters, cofactor dependency, and reversibility of the reaction. Unlike 11-OHSD-1, the collecting duct enzyme catalyzes irreversible dehydrogenation of endogenous glucocorticoids, has a very high affinity for its substrate, and is located in mineralocorticoid target cells; it thus appears well suited to "protect" the mineralocorticoid receptors from occupancy by glucocorticoids. As a first step in attempting to isolate the cDNA for the 11-OHSD/CD isoform, we isolated mRNA from immunodissected cortical collecting duct (CCD) cells and characterized the 11-OHSD in oocytes injected with this mRNA. Water-injected oocytes had no measurable 11-OHSD activity. In contrast, oocytes injected with as little as 1 ng CCD mRNA expressed detectable 11-OHSD activity. Expression of 11-OHSD activity was dependent on the amount of mRNA injected and was maximal with 30 ng mRNA. Similar to the findings in CCD cells, the expressed enzyme preferred NAD over NADP (activity was 0.46 +/- 0.04 and 0.011 +/- 0.01 fmol.min-1.oocyte-1 with 0.1 mM NAD and NADP, respectively). The Michaelis constant (Km) for corticosterone was 11.5 +/- 3.7 nM. Similar to the findings in CCD cells, the expressed enzyme worked predominantly in the oxidative direction, as back-conversion of [3H]dehydrocorticosterone to corticosterone was negligible. (ABSTRACT TRUNCATED AT 250 WORDS)

Ribulose bisphosphate carboxylase gene expression in subtropical marine phytoplankton populations

Oceanic phytoplankton are known to fix CO2 primarily through the action of the enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase). The amino acid and nucleotide sequence of the large subunit of this enzyme have been conserved across the evolution of the chlorophytic plants (from cyanobacteria to higher plants via green algae) with approximately 80 and 70 % homology at the amino acid and nucleotide levels respectively. To understand the molecular regulation of this enzyme in phytoplankton, we have measured levels of the RuBPCase large subunit (rbcL) mRNA and DNA, in combination with rates of photosynthetic CO2 fixation, autofluorescent cell counts, and chlorophyll a in natural phytoplankton communities of Tampa Bay (Flonda, USA) and the southeastern Gulf of Mexico. We measured rbcL mRNA, rbcL DNA, and the ratio of rbcL mRNA to rbcL DNA by extracting RNA and DNA and probing the extracts with the Synechococcus PCC 6301 rbcL gene as a probe. Additionally, rbcL mRNA was amplified from certain samples using a reverse transcriptase-linked polymerase chain reaction procedure. In a transect from Tampa Bay seaward, levels of rbcL mRNA decreased 3to 8-fold from the estuarine environment to the offshore environment, and followed similar trends as photosynthetic CO2 fixation, picocyanobacterial counts, and chlorophyll a. In an offshore vertical profile, the subsurface maximum in the rbcL mRNA/DNA ratio coincided with the 60 m maximum in photosynthetic assimilation rates. In a die1 study using 150 1 of offshore water in a deck-top incubator, the rbcL mRNA.DNA ratio was nearly an order of magnitude greater (76 ng mRNA ng-' DNA) during the light period than in the dark (17 ng mRNA ng-' DNA). Likewise, the photosynthetic rate (PB) at constant illun~ination was highest during daylight hours (6.2 pg C pg-' chl a h-') and lowest at night (1.9 pg C pg-' chl a h-'). These results indicate that (1) carbon fixation in oceanic phytoplankton may be transcriptionally regulated at the level of the RuBPCase gene and (2) that the quantitatlon of particular target mRNAs and DNAs is an effective means to study the regulation of conserved gene functions in natural water column microbial populations.

35] Construction of primary and subtracted cDNA libraries from early embryos

By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification and study of novel genes or gene families, and it is an important new tool for understanding the molecular control of mammalian development.