Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA) / Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Dicing to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80o C. Alternatively, chemically synthesiz

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (1

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos / In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then

Características anatômicas e teciduais em cabras alimentadas por longos períodos com resíduo da indústria do biodiesel da mamona / Anatomic and tissue characteristics in goats fed for extended periods with residue of castor biodiesel production

Vinte e cinco cabras adultas mestiças, divididas em dois grupos, foram alimentadas por um período de 16 meses com dietas a base de feno de tifton e concentrado sem e com farelo de mamona destoxificado em substituição ao farelo de soja. Aos 480 dias, foram coletadas amostras de sangue para dosagem de lactato desidrogenase, aspartato aminotransferase, alanina aminotransferase, uréia, albumina e creatinina. Em seguida, os animais foram sacrificados e foi realizada a pesagem dos componentes anatômicos (pulmões, coração, baço, fígado, rins, língua, estomago vazio, intestino vazio, omento, tecido adiposo cardíaco e renal), da carcaça e dos cortes comerciais (paleta, pernil, lombo, costelas e pescoço). Posteriormente, foi realizada a dissecção anatômica do lombo, separando tecidos muscular, adiposo e ósseo. Na porção muscular do lombo, Longissimus dorsii, foram analisadas a composição centesimal, o perfil de ácidos graxos e a expressão dos genes SEW-1, IGF-I e IGF-II. Verificou-se uma incidência superior de tecido ósseo na dissecção anatômica do lombo e uma incidência inferior de gordura na composição centesimal do Longissimus dorsii do grupo FMD em relação ao grupo SFMD (P 0,05). A expressão do gene IGFII resultou superior (P 0,001) no tecido muscular dos animais FMD, bem como para o gene SEW-1 (P 0,001). Diante do exposto, podemos concluir que a utilização do farelo de

Twenty-five adult crossbred goats, divided in two groups, were fed over a period of 16 months with diets based on Tifton hay and concentrate feed with (DCO) or without (WDCO) detoxified castor bean meal as a substitute for soybean meal. Throughout 480 days, blood samples were taken to measure lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, urea, albumin and creatinine. The animals were euthanized, and the anatomical components (lungs, heart, spleen, liver, kidneys, tongue, empty stomach, empty intestines, omentum, cardiac and renal adipose tissue), carcass and commercial cuts (shoulder, ham, loin, ribs and neck) were weighed. Thereafter, an anatomic dissection of the loin was performed, separating the muscle, adipose and bone tissues. On the muscular part of the loin, longissimus dorsi, the proximate composition, fatty acid profile and the expression of SEW-1, IGF-I and IGF-II were analyzed. A higher incidence of bone tissue was observed in the anatomical dissections of the loin and a lower incidence of fat in the proximate composition of the longissimus dorsi of the DCO group compared to the WDCO group (p 0.05). The expression of the IGF-II and SEW-1 genes was higher (p 0.001 for each) in the muscle tissue of the DCO animals. Thus, using detoxified castor bean meal for long periods does not produce significant changes in the anatomical compos

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA) / Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Dicing to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80o C. Alternatively, chemically synthesiz

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (1

Características anatômicas e teciduais em cabras alimentadas por longos períodos com resíduo da indústria do biodiesel da mamona / Anatomic and tissue characteristics in goats fed for extended periods with residue of castor biodiesel production

Vinte e cinco cabras adultas mestiças, divididas em dois grupos, foram alimentadas por um período de 16 meses com dietas a base de feno de tifton e concentrado sem e com farelo de mamona destoxificado em substituição ao farelo de soja. Aos 480 dias, foram coletadas amostras de sangue para dosagem de lactato desidrogenase, aspartato aminotransferase, alanina aminotransferase, uréia, albumina e creatinina. Em seguida, os animais foram sacrificados e foi realizada a pesagem dos componentes anatômicos (pulmões, coração, baço, fígado, rins, língua, estomago vazio, intestino vazio, omento, tecido adiposo cardíaco e renal), da carcaça e dos cortes comerciais (paleta, pernil, lombo, costelas e pescoço). Posteriormente, foi realizada a dissecção anatômica do lombo, separando tecidos muscular, adiposo e ósseo. Na porção muscular do lombo, Longissimus dorsii, foram analisadas a composição centesimal, o perfil de ácidos graxos e a expressão dos genes SEW-1, IGF-I e IGF-II. Verificou-se uma incidência superior de tecido ósseo na dissecção anatômica do lombo e uma incidência inferior de gordura na composição centesimal do Longissimus dorsii do grupo FMD em relação ao grupo SFMD (P 0,05). A expressão do gene IGFII resultou superior (P 0,001) no tecido muscular dos animais FMD, bem como para o gene SEW-1 (P 0,001). Diante do exposto, podemos concluir que a utilização do farelo de

Twenty-five adult crossbred goats, divided in two groups, were fed over a period of 16 months with diets based on Tifton hay and concentrate feed with (DCO) or without (WDCO) detoxified castor bean meal as a substitute for soybean meal. Throughout 480 days, blood samples were taken to measure lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, urea, albumin and creatinine. The animals were euthanized, and the anatomical components (lungs, heart, spleen, liver, kidneys, tongue, empty stomach, empty intestines, omentum, cardiac and renal adipose tissue), carcass and commercial cuts (shoulder, ham, loin, ribs and neck) were weighed. Thereafter, an anatomic dissection of the loin was performed, separating the muscle, adipose and bone tissues. On the muscular part of the loin, longissimus dorsi, the proximate composition, fatty acid profile and the expression of SEW-1, IGF-I and IGF-II were analyzed. A higher incidence of bone tissue was observed in the anatomical dissections of the loin and a lower incidence of fat in the proximate composition of the longissimus dorsi of the DCO group compared to the WDCO group (p 0.05). The expression of the IGF-II and SEW-1 genes was higher (p 0.001 for each) in the muscle tissue of the DCO animals. Thus, using detoxified castor bean meal for long periods does not produce significant changes in the anatomical compos

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos / In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then