A NOVEL METHOD TO INTERFERE WITH GENE EXPRESSION IN MICE

Transgenic animals are generated by injection of recombinant DNA sequences into fertilized oocytes. Here I applied a new methodology for the generation of transgenic knockdown mice using the LentiLox 3.7 lentivirus as a transfer vehicle bearing an U6-promoter dependent shRNA expression cassette. Lentiviruses are a sub-class of retroviruses that have the capability to infect non-dividing post-mitotic cells. Recently, in addition to their use to transform primary cells and established cell lines, lentiviruses have also been used to generate transgenic mice, pigs, cattle, rats and chickens. Thus, we hoped that lentiviral vectors containing U6/RNAi expression cassettes could serve as a fast and attractive alternative for the generation of mice with reduced expression of specific genes. As a model for this in vivo knockdown approach I chose the hepatocyte nuclear receptor 4gamma for which a knock out model was not available. Expression analyses of the HNF4gamma gene demonstrated synthesis of the protein in the embryonic gut at day E16.5. In adult animals its expression is restricted predominantly to the differentiated, absorptive brush border cells of the small intestine (enterocytes) and to the cells of pancreatic islets (islets of Langerhans). In order to knockdown the HNF4gamma gene, a panel of five shRNA hairpin sequences was selected by the public Sirna software and their activity was validated by transfection experiments in cell culture. After re-cloning of the U6/shRNA cassettes into the pLL 3.7 vector, infectious virus particles were generated and injected within the perivitelline space of one cell stage mouse embryos. 56% of the LentiLox 3.7 lentivirus founder mice were PCR-positive, however expression from the transgene was highly mosaic. The high mosaicism of F0 mice precluded their use for immediate expression analysis as it was hoped when the project was started. The high degree of mosaicism is also reflected by a low rate of germ line transmission. Only 6% of F1 mice expressed the indicator gene for EGFP as well as the shRNA transgene. Often expression was not ubiquitous probably reflecting the dependence of expression on the chromosomal integration site. A good correlation between EGFP activity and siRNA accumulation in organs of F1 mice was found as evidenced by Northern blot hybridisation. Despite the general low efficiency of transgenesis the down regulation of HNF4gamma gene in one F1 line (A-I) reached 50% in the gut and 80% in pancreas proving that this targeted knockdown approach is working in living animals

Effects of riboflavin on hyperalgesia and serum glutamine-to-glutamate ratio in rats with painful diabetic neuropathy

Previous studies have explored the antinociceptive effects of riboflavin (vitamin B2) across various experimental models. However, there remains a gap in the literature regarding its potential to alleviate neuropathic pain in diabetes. This study aims to investigate the effects of riboflavin on hyperalgesia and serum glutamine-to-glutamate ratio in rats with painful diabetic neuropathy. In fasted rats, a model of painful diabetic neuropathy was induced through intraperitoneal injection of streptozotocin. In the fifth week post-injection, diabetic rats experiencing neuropathic pain were administered daily doses of riboflavin (25 or 50 mg), dissolved in their drinking water, for a duration of two weeks. Results demonstrate that riboflavin significantly reduced mechanical and cold-induced hyperalgesia in diabetic rats compared to controls. Formalin-induced hyperalgesia was alleviated by riboflavin in the second phase. Additionally, riboflavin supplementation increased the serum glutamine-to-glutamate ratio in these animals. These findings highlight the therapeutic potential of riboflavin in managing neuropathic pain associated with diabetes

Effects of riboflavin on hyperalgesia and serum glutamine-to-glutamate ratio in rats with painful diabetic neuropathy

Previous studies have explored the antinociceptive effects of riboflavin (vitamin B2) across various experimental models. However, there remains a gap in the literature regarding its potential to alleviate neuropathic pain in diabetes. This study aims to investigate the effects of riboflavin on hyperalgesia and serum glutamine-to-glutamate ratio in rats with painful diabetic neuropathy. In fasted rats, a model of painful diabetic neuropathy was induced through intraperitoneal injection of streptozotocin. In the fifth week post-injection, diabetic rats experiencing neuropathic pain were administered daily doses of riboflavin (25 or 50 mg), dissolved in their drinking water, for a duration of two weeks. Results demonstrate that riboflavin significantly reduced mechanical and cold-induced hyperalgesia in diabetic rats compared to controls. Formalin-induced hyperalgesia was alleviated by riboflavin in the second phase. Additionally, riboflavin supplementation increased the serum glutamine-to-glutamate ratio in these animals. These findings highlight the therapeutic potential of riboflavin in managing neuropathic pain associated with diabetes

Reduced Expression of the Retinoblastoma Protein Shows That the Related Signaling Pathway Is Essential for Mediating the Antineoplastic Activity of Erufosine

<div><p>Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27^Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G₂ phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by eru-fosine in the expression of these proteins and abrogated the induction of G₂ arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status.</p></div

Cytotoxicity and clonogenic activity of erufosine in Rb-deficient cells.

<p>The figure depicts the survival (A) or clonogenicity (B) of SKW-3 cell clones with 99% (shRNA1) or 83% (shRNA 2) stable Rb-knockdown after treatment with 2, 4, 8, 16 or 32 µM erufosine for 48 h. A significant difference versus the respective nonsense control is marked by an asterisk (Student’s t-test; p<0.05). Bars denote standard deviation. The table under the graphs gives the IC₅₀ values of erufosine after 48 h of treatment as well as the respective 95% confidence intervals.</p

Erufosine-induced apoptosis is inhibited by Rb-loss.

<p>The expression of proteins is shown, which are involved in induction of the intrinsic apoptotic pathway, respectively, for nonsense or antisense-transduced cell clones with 99% or 83% Rb-knockdown after exposure to erufosine (16 µM, 48 h). The values under the protein bands denote their intensity compared to the untreated control and are calculated after densitometric analysis with the Quantity One 4.6.6 Program (Bio-Rad).</p

Rb-loss inhibits the erufosine-induced G2 cell cycle arrest.

<p>The flow cytometry histograms present the distribution of nonsense- or antisense transduced SKW-3 cell clones with 99% (shRNA 1) and 83% (shRNA 2) Rb-knockdown in G1-, S- and G2-phases of the cell cycle before and after exposure to 16 µM erufosine for 24 and 48 h. The percentage of the cell fractions is calculated with the ModFit LT software and given in the table below the graphs.</p

Survival of cells with stable Rb-knockdown after 48

<p> <b>h treatment with 5-floururacil, cytosine arabinoside, doxorubicin and cisplatin.</b> SKW-3 cell clones (nonsense control – NSO, cells with 99% Rb-knockdown – shRNA 1 and cells with 83% Rb-knockdown – shRNA 2) were treated with five different concentrations of the four cytostatics. A significant difference versus the respective nonsense control is marked by an asterisk (Student’s t-test; p<0.05). Bars denote standard deviation. The table under the graphs gives the IC₅₀ values of the drugs after 48 h of treatment with the respective 95% confidence intervals.</p

Efficacy of the Rb-knockdown.

<p>(A) Fluorescence imaging of SKW-3 cells at 72 h after viral transduction with three different pLL 3.7-constructs (nonsense shRNA – NSO, antisense shRNAs–21 bp, 1 or 27 bp, 2) and Cell Sorting using the eGFP signal. (B) The efficacy of the Rb-knockdown estimated by Western blot before and after selection. The Rb-knockdown on protein level was calculated as percentage of the respective nonsense-control cells after densitometric analysis of the protein bands using the Quantity One 4.6.6 Program (Bio-Rad Laboratories).</p

Deletion of Prostaglandin E-2 Synthesizing Enzymes in Brain Endothelial Cells Attenuates Inflammatory Fever

Fever is a hallmark of inflammatory and infectious diseases. The febrile response is triggered by prostaglandin E-2 synthesis mediated by induced expression of the enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1). The cellular source for pyrogenic PGE(2) remains a subject of debate; several hypotheses have been forwarded, including immune cells in the periphery and in the brain, as well as the brain endothelium. Here we generated mice with selective deletion of COX-2 and mPGES1 in brain endothelial cells. These mice displayed strongly attenuated febrile responses to peripheral immune challenge. In contrast, inflammation-induced hypoactivity was unaffected, demonstrating the physiological selectivity of the response to the targeted gene deletions. These findings demonstrate that PGE(2) synthesis in brain endothelial cells is critical for inflammation-induced fever.Funding Agencies|Swedish Medical Research Council; Swedish Cancer Foundation; European Research Council; Knut and Alice Wallenberg Foundation; Swedish Brain foundation; County Council of stergotland; Wenner-Gren Fellowship</p