Suppression Subtractive Hybridization Technology Research Articles

Comparative transcript profiling of gene expression of fresh and frozen–thawed bull sperm

Although frozen semen is widely used commercially in the cattle breeding industry, the resultant pregnancy rate is lower than that produced using fresh semen. Cryodamage is a major problem in semen cryopreservation; it causes changes to sperm transcripts that may influence sperm function and motility. We used suppression subtractive hybridization technology to establish a complementary DNA subtractive library, and combined microarray technology and sequence homology analysis to screen and analyze differentially expressed genes in the library, comparing fresh sperm with the frozen–thawed sperm of nine bulls. Overall, 19 positive differentially expressed unigenes were identified using microarray data and Significance Analysis of Microarrays software (|score (d)| ≥ 2, fold change > 1, and false discovery rate < 0.05). Of 15 differentially expressed unigenes exhibited high sequence homology (E-value ≤ 1 × 10−3), 12 were upregulated in frozen–thawed sperm, the remaining 3 were upregulated in fresh sperm, and 4 other clones were identified as unknown because of incomplete sequences or because there was no significant sequence homology (E-value > 1E−03) and were considered novel genes. The expression of five of these genes—RPL31, PRKCE, PAPSS2, PLP1, and R1G7—was verified by quantitative real-time reverse transcription–polymerase chain reaction. There was a significant differential expression of the RPL31 gene (P < 0.05). Our preliminary results provide an overview of differentially expressed transcripts between fresh and frozen–thawed sperm of Holstein bulls.

Identification of differentially expressed genes related to metabolic syndrome induced with high-fat diet in E3 rats.

Understanding the genes differentially expressing in aberrant organs of metabolic syndrome (MetS) facilitates the uncovering of molecular mechanisms and the identification of novel therapeutic targets for the disease. This study aimed to identify differentially expressed genes related to MetS in livers of E3 rats with high-fat-diet-induced metabolic syndrome (HFD-MetS). E3 rats were fed with high-fat diet for 24 weeks to induce MetS. Then, suppression subtractive hybridization (SSH) technology was used to identify the genes differentially expressed between HFD-MetS and control E3 rat livers. Twenty positive recombinant clones were chosen randomly from forward subtractive library and sent to sequence. BLAST analysis in GenBank database was used to determine the property of each cDNA fragment. In total, 11 annotated genes, 3 ESTs, and 2 novel gene fragments were identified by SSH technology. The expression of four genes (Alb, Pip4k2a, Scd1, and Tf) known to be associated with MetS and other five genes (Eif1, Rnase4, Rps12, Rup2, and Tmsb4) unknown to be relevant to MetS was significantly up-regulated in the livers of HFD-MetS E3 rats compared with control rats using real-time quantitative PCR (RT-qPCR). By analyzing the correlations between the expression of these nine genes and serum concentrations of TG, Tch, HDL-C, and LDL-C, we found that there were significant positive correlations between TG and the expression of five genes (Alb, Eif1, Pip4k2a, Rps12, and Tmsb4x), Tch and three genes (Rnase4, Scd1, and Tmsb4x), and LDL-C and two genes (Rnase4 and Scd1), as well there were significant negative correlations between HDL-C and the expression of three genes (Rup2, Scd1, and Tf). This study provides important clues for unraveling the molecular mechanisms of MetS.

Gene expression profiles of the heterotrophic microalga Chlorella pyrenoidosa F-9.

A strain of the microalga Chlorella pyrenoidosa F-9 in our laboratory showed special characteristics when transferred from autotrophic to heterotrophic culture. In order to elucidate the possible metabolic mechanism, the gene expression profiles of the autonomous organelles in the green alga C. pyrenoidosa under autotrophic and heterotrophic cultivation were compared by suppression subtractive hybridization technology. Two subtracted libraries of autotrophic and heterotrophic C. pyrenoidosa F-9 were constructed, and 160 clones from the heterotrophic library were randomly selected for DNA sequencing. Dot blot hybridization showed that the ratio of positivity was 70.31% from the 768 clones. Five chloroplast genes (ftsH, psbB, rbcL, atpB, and infA) and two mitochondrial genes (cox2 and nad6) were selected to verify their expression levels by real-time quantitative polymerase chain reaction. Results showed that the seven genes were abundantly expressed in the heterotrophic culture. Among the seven genes, the least increment of gene expression was ftsH, which was expressed 1.31-1.85-fold higher under heterotrophy culture than under autotrophy culture, and the highest increment was psbB, which increased 28.07-39.36 times compared with that under autotrophy conditions. The expression levels of the other five genes were about 10 times higher in heterotrophic algae than in autotrophic algae. In inclusion, the chloroplast and mitochondrial genes in C. pyrenoidosa F-9 might be actively involved in heterotrophic metabolism.

Effects of SIPL1 screened by suppression subtractive hybridization (SSH) on biological function and drug resistance of renal cell carcinoma cells

To screen the differentially expressed genes in human renal clear-cell carcinoma (RCC) cells using suppression subtractive hybridization (SSH), and to explore their biological function and underlying mechanism in RCC cells. Total RNAs were extracted from human renal clear-cell carcinoma cell line RLC-310 and human normal renal cell line HK-2 cells, and SSH technology was used to construct a RCC cell library of differential expression genes and to screen the most differentially expressed genes. RNA interference vector was constructed to silence the expression of the differentially expressed gene SIPL1 in human renal cell lines RLC-310 and GRC-1. Proliferation index was estimated by cell counting, MTT and tumor xenograft assay. Cell cycle analysis was performed using fluorescence activated cell sorting. Drug resistance potential to adriamycin was assessed by MTT. A subtractive cDNA library of highly expressed genes in the RCC cells was constructed and 12 differentially expressed genes were screened from the subtractive library, in which SIPL1 was the most differently expressed gene in the RCC cell line. SIPL1 overexpression in the RCC cells and clinical samples was confirmed by RT-PCR and Western blot analyses. The shRNA expression plasmid targeting to SIPL1 gene was constructed and transfected into RLC-310 and GRC-1 cells, resulting in downregulation of SIPL1. SIPL1 knockdown inhibited the cell proliferation (P < 0.05) and tumorgenesis. The tumor weights formed by RLC-310 cells transfected with SIPL1 shRNA was (0.22 ± 0.07)g and that of negative control vector was (0.85 ± 0.06)g. The tumor weight formed by GRC-1 cells was (0.32 ± 0.07)g and that of control vectors was (1.21 ± 0.11)g (P < 0.05). SIPL1 shRNA-transfected RLC-310 cells showed that more cells were arrested at G0/G1 phase [(71.13 ± 4.58)%] than that in the negative control RLC-310 cells [(53.27 ± 3.34)%, P < 0.05]. The proportion of G0/G1 phase in the SIPL1 shRNA transfected GRC-1 cells was (73.83 ± 3.97)%, significantly higher than that of (59.33 ± 3.03)% in the negative control GRC-1 cells (P < 0.05), and enhanced their sensitivity to adriamycin (P < 0.05). Silence of SIPL1 caused inactivation of AKT signaling and up-regulated expression of P27(Kip1) and P21(Cip1) proteins. A differentially expressed gene SIPL1 in the renal clear-cell carcinoma is successfully screened using SSH technology. SIPL1 functions as an oncogene in RCC, and may become a novel molecular target for RCC diagnosis and therapy.

Construction and analysis of subtractive cDNA library of head kidney in humphead snapper, Lutjanus sanguineus

A subtracted cDNA library of humphead snapper (Lutjanus sanguineus) was constructed by suppression subtractive hybridization technology (SSH) to screen immune-related EST.The cDNA library has been constructed by the mRNA of the test group and driven by SSH.Differential ESTs from the subtracted cDNA library have been identified by both PCR technology and dot blot hybridization.Six hundred and eighty positive clones were sequenced by Sangon Biological Engineering Technology Services Co.,Ltd.The homology of the sequences was analyzed by BLASTx tool and BLASTn tool in GenBank database.Functional distribution was performed based on the features of ESTs by gene ontology annotation (GO) and 30 immune-related ESTs of L.sanguineus,such as major histocompatibility complex gene (MHC Ⅰ and MHC Ⅱ),immunoglobulin gene (IgH and IgL),heat shock protein gene (HSP10,HSP70 and HSP90) and so on,were found.In this study,the subtracted cDNA library was successfully constructed by SSH and a number of immune-related ESTs of L.sanguineus were identified,which laid a foundation for the immune-related genes research of L.sanguineus.

Expression Analysis of Restorer Alleles-lnduced Genes in Pepper

Fertility restoration of cytoplasmic male-sterility in pepper ( Capsicum annuum L.) is useful for commercial production of hybrid seeds. However, the mechanism of fertility restoration has not been determined. We previously constructed a cDNA library and identified some genes related to fertility restoration in pepper using suppression subtractive hybridization technology. In this study, the expression patterns of 20 genes were investigated using semi-quantitative RT-PCR. Three genes expressed only in restorer lines, but not in sterility lines. Four genes expressed only in anther, but not in other organs. Among these 7 genes, the clone TG31 was observed to specifically express in anther of restorer lines. The work described here provides a comprehensive overview on the expression pattern of the genes that are induced by restorer alleles in pepper. It will also contribute to the current understanding of molecular networks for the regulation of fertility restoration.

Isolation and expression analysis of tuberous root development related genes in Rehmannia glutinosa

As one kind of important modified storage organs, tuberous roots are attractive for their economic and biological values. Although considerable progresses have been made in the past, molecular information regarding the tuberous root development is still limited. In this study, we focused on the molecular profiling of the tuberous root development of Rehmannia glutinosa. Suppression subtractive hybridization technology was employed to compare gene expression between adventitious root and developing tuberous root. As a result, a tuberous root subtractive library was constructed and 199 development-related unique expressed sequence tags were identified, which represent different groups of genes involved in metabolism, protein synthesis, protein fate, cell fate, signaling, transcription and development, etc. In order to further validate the obtained result, 18 genes were selected for expression analysis and the genes most likely being involved in tuberous root development were discussed. Our present study provided the first molecular profiling of tuberous root development-related genes in Rehmannia glutinosa, which will establish the basis for future deciphering the tuberous root development mechanism.

Identification of differentially expressed genes in the growth plate of broiler chickens with thiram-induced tibial dyschondroplasia

Tibial dyschondroplasia (TD) is characterized by expansion of the proximal growth plates of the tibiotarsus that fail to form bone, lack blood vessels, and contain non-viable cells. Thiram (a carbamate pesticide), when fed to young broiler chicks, induces TD with high regularity and precision. We used this experimental model to understand the cause of the defects associated with TD by selecting and identifying the genes differentially expressed in the TD growth plate of broiler chickens. Broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 96 h to induce TD (thiram-fed). mRNA was purified from the growth plates of control and thiram-fed broilers. Forward and reverse-subtracted cDNA libraries were generated by suppression subtractive hybridization technology. Ten selected genes from cDNA libraries were identified by real-time quantitative polymerase chain reaction. All were differentially expressed in TD growth plates (P<0.05 or P<0.01). The levels of collagen type X (Col X), pro-alpha-1 collagen type I (Col I α1), collagen type IX (Col IX), NADH dehydrogenase (NADH DH), cytochrome C oxidase subunit III (COX III), enolase 1, alpha (ENO1), carbonic anhydrase II (CA2) and heat shock protein 90 (Hsp90) mRNA transcripts were up-regulated, while the expression levels of Matrilin 3 (MATN3) and chondromodulin-I (ChM-I) were down-regulated. Col I and Hsp90 were detected by immunohistochemistry at different stages. Given that these genes are involved in matrix formation, endochondral ossification, developmental regulation, electron transport in the mitochondrial respiratory chain and vascularization, our findings may provide new insights into understanding the pathogenesis of TD.

Identification of Botrytis cinerea genes up-regulated during infection and controlled by the Galpha subunit BCG1 using suppression subtractive hybridization (SSH).

The Galpha subunit BCG1 plays an important role during the infection of host plants by Botrytis cinerea. Delta bcg1 mutants are able to conidiate, penetrate host leaves, and produce small primary lesions. However, in contrast to the wild type, the mutants completely stop invasion of plant tissue at this stage; secondary lesions have never been observed. Suppression subtractive hybridization (SSH) was used to identify fungal genes whose expression on the host plant is specifically affected in bcg1 mutants. Among the 22 differentially expressed genes, we found those which were predicted to encode proteases, enzymes involved in secondary metabolism, and others encoding cell wall-degrading enzymes. All these genes are highly expressed during infection in the wild type but not in the mutant. However, the genes are expressed in both the wild type and the mutant under certain conditions in vitro. Most of the BCG1-controlled genes are still expressed in adenylate cyclase (bac) mutants in planta, suggesting that BCG1 is involved in at least one additional signaling cascade in addition to the cAMP-depending pathway. In a second SSH approach, 1,500 clones were screened for those that are specifically induced by the wild type during the infection of bean leaves. Of the 22 BCG1-controlled genes, 11 also were found in the in planta SSH library. Therefore, SSH technology can be successfully applied to identify target genes of signaling pathways and differentially expressed genes in planta.

Chicken interleukin-6. cDNA structure and biological properties.

Suppression subtractive hybridization technology was used to identify differentially expressed genes in spleens of chickens that had been treated with the synthetic immune modifier S-28463. One induced chicken gene encoded a protein with about 35% sequence identity to human interleukin-6 (IL-6). It consists of 241 amino acids including a putative N-terminal signal peptide of 47 residues. Bacterially expressed chicken IL-6 (ChIL-6) carrying a histidine tag in place of the signal peptide was biologically active: it induced proliferation of the IL-6-dependent murine hybridoma cell line 7TD1. The concentration of ChIL-6 required for half-maximal proliferative response was approximately 60 pg.mL-1. When injected intravenously into adult chickens, purified recombinant ChIL-6 induced an increase in serum corticosterone levels. Supernatants of chicken LMH and monkey COS-7 cells transiently transfected with a ChIL-6 expression construct induced proliferation of 7TD1 cells, demonstrating that recombinant ChIL-6 from eukaryotic cells is also active.

Analysis of altered gene expression in rat soleus muscle atrophied by disuse.

The present study involved a global analysis of genes whose expression was modified in rat soleus muscle atrophied after hindlimb suspension (HS). HS muscle unloading is a common model for muscle disuse that especially affects antigravity slow-twitch muscles such as the soleus muscle. A cDNA cloning strategy, based on suppression subtractive hybridization technology, led to the construction of two normalized soleus muscle cDNA libraries that were subtracted in opposite directions, i.e., atrophied soleus muscle cDNAs subtracted by control cDNAs and vice versa. Differential screening of the two libraries revealed 34 genes with altered expression in HS soleus muscle, including 11 novel cDNAs, in addition to the 2X and 2B myosin heavy chain genes expressed only in soleus muscles after HS. Gene up- and down-regulations were quantified by reverse Northern blot and classical Northern blot analysis. The 25 genes with known functions fell into seven important functional categories. The homogeneity of gene alterations within each category gave several clues for unraveling the interplay of cellular events implied in the muscle atrophy phenotype. In particular, our results indicate that modulations in slow- and fast-twitch-muscle component balance, the protein synthesis/secretion pathway, and the extracellular matrix/cytoskeleton axis are likely to be key molecular mechanisms of muscle atrophy. In addition, the cloning of novel cDNAs underlined the efficiency of the chosen technical approach and gave novel possibilities to further decipher the molecular mechanisms of muscle atrophy.