Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation.

The functionality of Moloney murine leukemia virus reverse transcriptase (MMLV RT) will increase with the improvement of its solubility and thermal stability. Introduce directed mutation at specific positions of the MMLV RT sequence and codon optimization is needed to achieve these properties. The two RT coding sequences with (rRT-K) and without directed mutations (rRT-L) were versatility optimized and expressed to analyze the ribonuclease H (RNase H) inactivity and thermostable polymerase activity. For this purpose, the five-point mutations (438-442aa) and three-point mutations (530, 568, and 659 aa) were done at the RT connection domain and RNase H active site, respectively. High expression levels of rRT-L and rRT-K were obtained in E. coli BL21(DE3) and BL21(shuffle) strains, 0.5 mM IPTG concentration at 37 °C, and 8 hours' post-induction condition. Then, recombinant enzymes were purified and verified by Ni-NTA resin and western blotting. Insilico analysis (IUpred 3.0) showed that the directed mutation in the RNase H domain caused the formation of disorder regions or instability in the RNase H domain of rRT-K compared to rRT-L. The modified RT-PCR and the RT-LAMP reactions proved the RNase H inactivity of rRT-K. In addition, increasing of thermostability of rRT-K compared to rRT-L and commercial RT was evaluated by the RT-PCR and RT-LAMP reactions. The results showed that rRT-K could successfully tolerate 60 ºC in the two methods. This study revealed that the directed mutations and the versatile sequence optimization can promise to produce thermostable commercial enzymes to decrease non-specific one-step RT-PCR and RT-LAMP products.

Expression of soluble moloney murine leukemia virus-reverse transcriptase in Escherichia coli BL21 star (DE3) using autoinduction system.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

Development of Moloney Murine Leukemia Virus Reverse Transcriptase Fused with Archaeal DNA-binding Protein Sis7a.

INTRODUCTION: Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is a common enzyme used to convert RNA sequences into cDNA. However, it still has its shortcomings, especially in terms of processivity and thermostability. According to a previous patent, the fusion of polymerase enzyme to an archaeal DNA-binding protein has been proven to enhance its performance. Furthermore, recent studies have also stated that the fusion of a polymerase enzyme to an archaeal DNA-binding protein is predicted to improve its thermostability and processivity. AIM: As an early stage of enzyme development, this study aimed to design, express, and purify enzymatically active MMLV RT fused with archaeal DNA-binding protein. METHODS: RT fusion proteins were designed and evaluated using in silico methods. The RT fusion enzyme was then expressed in Escherichia coli BL21(DE3) and purified. Its reverse transcriptional activity was proved using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: This study showed that MMLV RT fusion with Sis7a protein at its C-terminal end using commercial linker (GGVDMI) produced the best in silico evaluation results. The RT fusion was successfully expressed and purified. It was also known that the optimal condition for expression of the RT fusion was using 0.5 mM IPTG with post-induction incubation at room temperature (± 26°C) for 16 hours. In addition, the activity assay proved that the RT fusion has the reverse transcriptional activity. CONCLUSION: This study shows that the designed MMLV RT Sis7a fusion can be expressed and purified, is enzymatically active, and has the potential to be developed as an improved RT enzyme. Further study is still needed to prove its thermostability and processivity, and further characterize, and plan production scale-up of the MMLV RT Sis7a fusion for commercial use.

Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase.

Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, in vivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

Phosphoryl guanidine oligonucleotides as primers for RNA-dependent DNA synthesis using murine leukemia virus reverse transcriptase.

Modern approaches to the detection and analysis of low-copy-number RNAs are often based on the use of RNA-dependent DNA polymerases, for example, in reverse-transcription PCR. The accuracy and eff iciency of cDNA synthesis in the reverse-transcription reaction catalyzed by reverse transcriptase (RNA-dependent DNA polymerase) signif icantly affect the correctness of the results of PCR diagnostic assays and/or RNA sequencing. In this regard, many studies are focused on the optimization of the reverse-transcription reaction, including the search for more perfect primers necessary to obtain a full-length DNA copy of RNA under study. The best-known completely uncharged analogs of oligonucleotides - morpholine oligonucleotides and peptide nucleic acids - cannot be substrates for enzymes that process nucleic acids. The aim of this work was to conduct a pilot study of uncharged phosphoryl guanidine oligodeoxyribonucleotides (PGOs) as primers for mouse leukemia virus reverse transcriptase (MMLV H-). Specif ic features of elongation of partially and completely uncharged PGO primers were investigated. It was demonstrated that PGOs can be elongated eff iciently, e. g., in the presence of a fragment of human ribosomal RNA having complex spatial structure. It was shown that the proportion (%) of abortive elongation products of a PGO primer depends on buffer ionic strength, nucleotide sequence of the primer, and the presence and location of phosphoryl guanidine groups in the primer. The results indicate the suitability of PGOs, including completely electroneutral ones, as primers for reverse-transcription PCR, thereby opening up new prospects for the creation of experimental models for the analysis of highly structured RNA.

Culturing Chinese hamster ovary cells on cyclo olefin polymer triggers epithelial-mesenchymal transition and spheroid formation, which increases the foreign gene expression driven by the Moloney murine leukemia virus long terminal repeat promoter.

Chinese hamster ovary (CHO) cells are frequently used for recombinant protein production (RPP) as a host. While the RPP has been proven successful, there is still a compelling need for further improvement. Cyclo olefin polymer (COP) is a plastic material widely utilized due to its properties including its low protein absorption. We applied this as a raw material for RPP cell culture to see if the COP is suitable. A recombinant CHO cell line expressing the human erythropoietin (hEPO) gene under the control of the Moloney murine leukemia virus-long terminal repeat (MMLV-LTR) was established. When the cells were cultured in a dish made from COP, the cells attached to the bottom, and then started to float and form spheroids. RNASeq data analysis suggested the epithelial-mesenchymal transition (EMT) was triggered with receptor tyrosine kinase activation shortly after cultivation. It coincided with the hEPO transcription increase. After the cell floating, though EMT marker gene expression subsided, a hEPO expression increase sustained. When fibronectin was applied to COP dish surface, the cell floating was suppressed and hEPO expression decreased. We then treated cells with MßCD, a drug that destroys the lipid raft, eliminating molecules in the raft. This facilitated cell floating and spheroid formation coincided with hEPO expression enhancement. These results suggest interactions between a cell and COP surface might trigger the EMT and the subsequent event, both of which activated the MMLV-LTR promoter. Thus, employing COP for culturing cells, a potent RPP system could be established with its advantage for efficient protein purification.

Simple, Inexpensive RNA Isolation and One-Step RT-qPCR Methods for SARS-CoV-2 Detection and General Use.

The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis. Commercial one-step master mixes-which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well-are key reagents for SARS-CoV-2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M-MLV reverse transcriptase and assemble a homemade one-step RT-qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS-CoV-2 RNA detection by RT-qPCR: heat-inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT-qPCR using the homemade master mix, how to prepare in vitro-transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT-PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one-step RT-qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT-PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One-step RT-qPCR of RNA samples using a real-time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One-step RT-PCR using endpoint fluorescence detection.

Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering.

Cloning and expressing a highly functional and substrate specific farnesoic acid o-methyltransferase from the Asian citrus psyllid (Diaphorina citri Kuwayama).

The Asian citrus psyllid, Diaphorina citri, transmits a phloem-limited bacterium, Candidatus 'Liberibacter' asiaticus that causes citrus greening disease. Because juvenile hormone (JH) plays an important role in adult and nymphal development, we studied the final steps in JH biosynthesis in D. citri. A putative JH acid methyltransferase ortholog gene (jmtD) and its cognate cDNA were identified by searching D. citri genome database. Expression analysis shows expression in all life stages. In adults, it is expressed in the head-thorax, (containing the corpora allata), and the abdomen (containing ovaries and male accessory glands). A 3D protein model identified the catalytic groove with catalytically active amino acids and the S-adenosyl methionine (SAM)-binding loop. The cDNA was expressed in Escherichia coli cells and the purified enzyme showed high preference for farnesoic acid (FA) and homoFA (kcat of 0.752 × 10(-3) and 0.217 × 10(-3) s(-1), respectively) as compared to JH acid I (JHA I) (cis/trans/cis; 2Z, 6E, 10cis), JHA III (2E, 6E, 10cis), and JHA I (trans/cis/cis; 2E, 2Z, 10cis) (kcat of 0.081 × 10(-3), 0.013 × 10(-3), and 0.003 × 10(-3) s(-1), respectively). This suggests that this ortholog is a DcFA-o-methyl transferase gene (fmtD), not a jmtD, and that JH biosynthesis in D. citri proceeds from FA to JH III through methyl farnesoate (MF). DcFA-o-MT does not require Ca(2+), Mg(2+) or Zn(2+), however, Zn(2+) (1 mM) completely inhibits the enzyme probably by binding H115 at the active groove. This represents the first purified FA-o-MT from Hemiptera with preferred biological activity for FA and not JHA.

GALNT11 as a new molecular marker in chronic lymphocytic leukemia.

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B-CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [א(2)(1)=18.26; P<0.0001], lipoprotein lipase expression [א(2)(1)=13.72; P=0.0002] and disease prognosis [א(2)(1)=15.49; P<0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.

Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection).

Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400mg/kg) and then challenged 48h later with 600mg APAP/kg. Livers were obtained 4 or 24h later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430_2 GeneChip. Statistically significant genes were determined and gene expression changes were also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection.

BDNF signaling is necessary for the antidepressant-like effect of naringenin.

Previous studies in our laboratory have demonstrated that naringenin produced antidepressant-like action in tail suspension test (TST). However, the underlying mechanisms involved in neurotrophin system by which naringenin works have not been investigated. The present study extends earlier works on the role of brain-derived neurotrophic factor (BDNF) in regulating the antidepressant-like actions of naringenin in chronic unpredictable mild stress (CUMS). We showed that a 21-day regimen with naringenin reversed the decreased sucrose preference in sucrose preference test (SPT) and the prolonged first feeding latency in novelty-suppressed feeding test (NSFT), without affecting home-cage feeding consumption. In addition, we also found that naringenin promoted BDNF expression in the hippocampus but not in the frontal cortex in both non-stressed and CUMS mice. Moreover, the antidepressant-like effect of naringenin in SPT and NSFT induced by naringenin administration were totally abolished by K252a, an inhibitor of BDNF receptor tropomyosin-related kinase receptor B (TrkB). In conclusion, our findings suggest that the antidepressant-like effect of naringenin may be mediated, at least in part, by the activation of BDNF signaling in the hippocampus.

Genetic manipulation of B cells for the isolation of rare therapeutic antibodies from the human repertoire.

Antibody based therapies are increasingly applied to prevent and treat human disease. While the majority of antibodies currently on the market are chimeric or humanized antibodies from rodents, the focus has now shifted to the isolation and development of fully human antibodies. By retroviral transduction of B cell lymphoma-6 (BCL-6), which prevents terminal differentiation of B cells and, the anti-apoptotic gene B-cell lymphoma-extra large (Bcl-xL) into primary human B cells we efficiently immortalize antibody-producing B cells allowing the isolation of therapeutic antibodies. Selection of antigen-specific B cell clones was greatly facilitated because the transduced B cells retain surface immunoglobulin expression and secrete immunoglobulin into the culture supernatant. Surface immunoglobulin expression can be utilized to stain and isolate antigen specific B cell clones with labeled antigen. Immunoglobulins secreted in culture supernatant can directly be tested in functional assays to identify unique B cell clones. Here we describe the key features of our Bcl-6/Bcl-xL culture platform (AIMSelect).

The effect of syndecan-4 and glypican-1 expression on age-related changes in myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness.

Satellite cells are multipotential stem cells responsible for muscle growth and regeneration. Satellite cell proliferation, differentiation, and responsiveness to fibroblast growth factor 2 (FGF2) is, in part, regulated by the heparan sulfate proteoglycans syndecan-4 and glypican-1. Syndecan-4 and glypican-1 expression declines with satellite cell age and may be associated with decreased satellite cell activity. The objective of the current study was to determine if overexpression of syndecan-4 and glypican-1 would increase proliferation, differentiation and FGF2 responsiveness in satellite cells isolated from pectoralis major muscle from 16-wk-old turkeys. Overexpression of syndecan-4 and glypican-1 did not have a significant effect on proliferation and differentiation in 1d, 7 wk, and 16 wk satellite cells, and did not affect FGF2 responsiveness during proliferation. Expression of syndecan-4 and glypican-1 increased differentiation at 48 h in 1d, 7 wk, and 16 wk cells treated with FGF2. Expression of myogenic regulatory factors MyoD, myogenin, and MRF4 was affected by the overexpression of syndecan-4 and glypican-1. However, changes in myogenic regulatory factor expression did not have a significant effect on proliferation or differentiation. These data demonstrate that syndecan-4 and glypican-1 are likely not directly associated with the age related decrease in satellite cell activity.

Knockdown of NOB1 expression by RNAi inhibits cellular proliferation and migration in human gliomas.

NOB1 (NIN1/RPN12 binding protein 1 homolog), a ribosome assembly factor, is thought to be essential for the processing of the 20S pre-rRNA into the mature 18S rRNA. It is also reported to participate in proteasome biogenesis. However, the contribution of NOB1 gene dysfunction to the pathology of human diseases, such as gliomas, has not been addressed. Here, we detected expression levels of NOB1 mRNA in U251, U87, U373, and A172 cells by quantitative real-time PCR. To analyze the expression levels of NOB1 protein in glioma tissues, we performed immunohistochemistry on 56 pathologically confirmed glioma samples (7 Grade I cases, 19 Grade II cases, 16 Grade III cases, and 14 Grade IV cases). A recombinant lentivirus expressing NOB1 short hairpin RNA (shNOB1) was constructed and infected into U251 and U87-MG human glioma cells. We found that NOB1 mRNA was expressed in all four cell lines. The expression level of the NOB1 protein was significantly higher in high-grade gliomas than in low-grade gliomas. Knockdown of the NOB1 gene resulted in suppression of the proliferation and the colony-forming abilities of U251 and U87-MG cells, cell cycle arrest during the G0/G1 phase, and a significant enhancement of cell apoptosis. In addition, cell migration was significantly suppressed in U251 and U87-MG cells that were infected with the shNOB1-expressing lentivirus. These results suggest that NOB1 promotes glioma cell growth and migration and could be a candidate for molecular targeting during gene therapy treatments of glioma.

First functional analysis of a novel splicing mutation in the B3GALTL gene by an ex vivo approach in Tunisian patients with typical Peters plus syndrome.

Peters plus syndrome is a rare recessive autosomal disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities and distinctive facial features. It was related only to mutations in the B3GALTL gene in the 13q12.3 region. In this study, we undertook the first functional analysis of a novel c.597-2 A>G splicing mutation within the B3GALTL gene using an ex-vivo approach. The results showed a complete skipping of exon 8 in the B3GALTL cDNA, which altered the open reading frame of the mutant transcript and generated a PTC within exon 9. This finding potentially elicits the nonsense mRNA to degradation by NMD (nonsense-mediated mRNA decay). The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to ex-vivo results. The findings confirmed the key role played by the B3GALTL gene in typical Peters-plus syndromes and the utility of mRNA analysis to understand the primary impacts of this mutation and the phenotype of the disease.

A M-MLV reverse transcriptase with reduced RNaseH activity allows greater sensitivity of gene expression detection in formalin fixed and paraffin embedded prostate cancer samples.

Formalin fixed and paraffin embedded (FFPE) human tissue collections are an invaluable resource for retrospective gene expression studies. However formalin fixation results in chemical modification of RNA and increased RNA degradation. This can affect RNA yield and quality. A critical step when analysing gene expression is the conversion of RNA to complementary DNA (cDNA) using a reverse transcriptase (RT) enzyme. FFPE derived RNA may affect the performance and efficiency of the RT enzyme and cDNA synthesis. We directly compared three commonly used FFPE RNA isolation methods and measured RNA yield, purity and integrity. We also assessed the effectiveness of three commercially available Moloney Murine Leukemia Virus (M-MLV) RTs on cDNA synthesis and gene expression sensitivity when using FFPE RNA as a template. Our results show that gene detection sensitivity is dependent on the isolation method, RT and length of the PCR amplicon (<200bp) when using FFPE RNA. The use of an M-MLV RT enzyme with reduced RNaseH activity gave significantly increased qRT-PCR sensitivity when using FFPE RNA derived from prostate tissue. The choice of RT can also affect perceived changes in target gene expression and thus the same RT should be used when attempting to reproduce results from different studies. This study highlights the need to optimise and evaluate RNA isolation methods and RTs when using FFPE RNA as a template in order to maximise a successful outcome in PCR applications.

Neonatal expression of amh, sox9 and sf-1 mRNA in Caiman latirostris and effects of in ovo exposure to endocrine disrupting chemicals.

Caiman latirostris is a reptilian species that exhibits temperature-dependent sex determination (TSD). Male-to-female sex reversal can be achieved after in ovo estrogen/xenoestrogen exposure. This is known as hormone-dependent sex determination (HSD). The amh, sox9 and sf-1 genes are involved in sex determination, sex differentiation, and steroidogenesis. The aims of this study were: (a) to establish the expression patterns of amh, sox9 and sf-1 mRNA in the gonad-adrenal-mesonephros (GAM) complexes of neonatal TSD-male and TSD-female caimans, (b) to compare the expression of these genes between TSD-females and HSD-females (born from E2-exposed eggs incubated at the male-producing temperature) and (c) to evaluate whether in ovo exposure to a low dose of E2 or bisphenol A (BPA) or to a high dose of endosulfan (END) modifies amh, sox9 or sf-1 mRNA expressions in neonatal males. The mRNA expressions of amh, sox9 and sf-1 in GAM complexes from TSD-males and TSD-females and from HSD-females were quantitatively compared by RT-PCR. A sexually dimorphic pattern of amh and sox9 mRNA expression was found, with a higher expression in TSD-males than in TSD-females. sf-1 mRNA did not differ between TSD-males and TSD-females. HSD-females exhibited a higher expression of sox9 than TSD-females. In males, increased mRNA expression of sex-determining genes was observed after in ovo exposure to END. E2 decreased sox9 but increased sf-1 mRNA expression. Changes induced by BPA were evident although not significant. These results provide new insights into the potential mechanisms that lead to the gonadal histo-functional alterations observed in caimans exposed to contaminated environments.

Human T47D-ERß breast cancer cells with tetracycline-dependent ERß expression reflect ERα/ERß ratios in rat and human breast tissue.

T47D-ERß breast cancer cells with tetracycline-dependent ERß expression and constant ERα expression can be used to investigate effects of varying ERα/ERß ratios on estrogen-induced cellular responses. This study defines conditions at which ERα/ERß ratios in T47D-ERß cells best mimic ERα/ERß ratios in breast and other estrogen-sensitive tissues in vivo in rat as well as in human. Protein and mRNA levels of ERα and ERß were analyzed in T47D-ERß cells exposed to a range of tetracycline concentrations and compared to ERα and ERß levels found in breast, prostate, and uterus from rat and human origin. The ERα/ERß ratio in T47D-ERß cells exposed to >150ng/ml tetracycline is comparable to the ratio found in rat mammary gland and in human breast tissue. The ERα/ERß ratio of other estrogen-sensitive rat and human tissues can also be mimicked in T47D-ERß cells. The ERα/ERß ratio found in MCF-7 and native T47D breast cancer cell lines did not reflect ratios in analyzed rat and human tissues, which further supports the use of T47D-ERß cells as model for estrogen-responsive tissues. Using 17ß-estradiol and the T47D-ERß cells under the conditions defined to mimic various tissues it could be demonstrated how these different tissues vary in their proliferative response.