scholarly journals DNase I activity in pig MII oocytes: implications in transgenesis

Reproduction ◽  
2006 ◽  
Vol 131 (3) ◽  
pp. 461-468 ◽  
Author(s):  
Augusta Zannoni ◽  
Marcella Spinaci ◽  
Chiara Bernardini ◽  
Maria Laura Bacci ◽  
Eraldo Seren ◽  
...  
Keyword(s):  
Conformational Changes ◽  
High Efficiency ◽  
Transgenic Animals ◽  
Nuclease Activity ◽  
Dnase I ◽  
Great Opportunity ◽  
Exogenous Dna ◽  
Male Genome ◽  
Sperm Dna

Several reliable methods to produce transgenic animals utilize the male genome. After penetration into oocyte, sperm DNA undergoes dramatic conformational changes that could represent a great opportunity for exogenous DNA to be integrated in the zygote genome. Among the enzymes responsible for sperm remodeling, a nuclease could be involved. The presence of a DNase I in oocytes has not been much investigated. To date, an immunolocalization of DNase I has been reported only in rat immature oocytes and the presence of nuclease activities has been shown in avian oocytes. The present study was conducted to verify whether a DNase-I like activity is present in MII mature pig oocytes. To do this, oocyte extracts were assessed for nuclease activity by a plasmid degradation assay and by zymography; these analyses evidenced a 33 kDa, Ca2+/Mg2+ dependent DNase I-like activity that was inhibited by Zn2+. A further identification of DNase I was achieved by Western blot, immunofluorescence and RT-PCR experiments. Moreover, the presence of the enzyme activity was confirmed by the rapid degradation of exogenous DNA microinjected into the ooplasm. Finally, the exogenous DNA transferred to oocyte by spermatozoa during sperm mediated gene transfer in vitro fertilisation protocol seemed to be protected from DNase I degradation and to persist in the ooplasm till 6 h. These results, together with the high efficiency of sperm based transgenesis methods, suggest that the association with spermatozoa protects exogenous DNA from nuclease activities.

scholarly journals Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 259-272 ◽  
Author(s):  
Francisco A García-Vázquez ◽  
Salvador Ruiz ◽  
Carmen Matás ◽  
M José Izquierdo-Rico ◽  
Luis A Grullón ◽  
...  
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Reactive Oxygen Species Generation ◽  
Reca Protein ◽  
Transgenic Animals ◽  
Membrane Lipid ◽  
Transgenic Pigs ◽  
Exogenous Dna

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA–DNA complexes at 5 μg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT–ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

240 QUALITY AND VIABILITY OF IVF BOVINE EMBRYOS AFTER INTRACYTOPLASMIC INJECTION OF DNA–LIPOSOME COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 232
Author(s):  
L. N. Moro ◽  
G. Vichera ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Dna Fragmentation ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Egfp Expression ◽  
Bovine Embryos ◽  
Exogenous Dna ◽  
Intracytoplasmic Injection

Transgenic animals have important applications in agriculture and human medicine; nevertheless the available techniques still remain inefficient and technically difficult. We have recently developed a novel method to transfect bovine embryos that consists of intracytoplasmic injection of exogenous DNA–liposome complexes (eDNA-LC) in IVF zygotes. This study was designed to evaluate the quality and viability of IVF bovine embryos, after intracytoplasmic injection of pCX-EGFP–liposome complexes (EGFP-LC) or pBCKIP2.8-liposome complexes (plasmid that codifies the human insulin gene, HI-LC). First, we evaluated embryo development and enhanced green fluorescent protein (EGFP) expression of IVF embryos injected with both plasmids separately. This treatment was analysed by Fisher's Exact test (P ≤ 0.05). Cleavage rates for EGFP-LC, HI-LC and IVF embryos injected with liposomes alone (IVF-L) and IVF control (IVF-C) were 62% (63/102), 67% (67/100), 66% (67/101) and 79% (98/124); blastocysts rates were 17% (17/102), 21% (21/100), 21% (21/101) and 23% (28/124), respectively. No statistical differences were seen among groups. The percentage of EGFP-positive embryos (EGFP+) after EGFP-LC injection was 42.9% after 3 days of culture and 41.8% at the blastocyst stage. In the second experiment, the blastocysts obtained, EGFP+ or EGFP-negative (EGFP–), were analysed by TUNEL assay at Day 6 (Bd6), 7 (Bd7) and 8 (Bd8) of in vitro culture, in order to evaluate the effect of the transgene and culture length, on DNA fragmentation. This treatment was analysed by the difference of proportions test (P ≤ 0.05) using statistical INFOSTAT software. All EGFP+ blastocysts showed TUNEL positive cells (T+). The percentage of T+ in Bd6, Bd7 and Bd8 were 91, 73.7 and 99.5%, respectively (P ≤ 0.05). EGFP– blastocysts showed lower fragmented nuclei (0, 44.6 and 85%, respectively; P ≤ 0.05). Groups IVF-L and IVF-C were also evaluated. In both groups, there was no evidence of DNA fragmentation in Bd6 and Bd7, but T+ were detected in Bd8 (66.4 and 85.8%, respectively; P ≤ 0.05). In the third experiment, bovine blastocysts obtained from the HI-LC group were individually transferred to recipient cows after 6 (n = 11), 7 (n = 5) and 8 (n = 5) days of culture post-IVF and HI-LC injection. The pregnancies obtained were from Bd6 [18.2% (2/11)] and Bd7 [40% (2/5)], although none of the recipients receiving Bd8 were diagnosed pregnant. Two pregnancies developed to term, one derived from Bd6 and the other from Bd7. Analysis by PCR determined that none of the born cows were transgenic. In summary, IVF bovine embryos could be easily transfected after the injection of eDNA-LC and the technique did not affect offspring viability. The results indicate that extended time in in vitro culture increases the percentage of fragmented nuclei in blastocysts. Moreover, this parameter increases in blastocysts with transgene expression compared with those without expression. Finally, more transfers are required in order to obtain the real efficiency of this new technique and to overcome the drawbacks generated by in vitro culture length and transgene expression.

218 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS (TM-INTRACYTOPLASMIC SPERM INJECTION) USING BULL SPERM PRETREATED WITH HEPARIN AND GLUTATHIONE

2014 ◽  
Vol 26 (1) ◽  
pp. 223
Author(s):  
N. C. Canel ◽  
R. J. Bevacqua ◽  
M. I. Hiriart ◽  
D. F. Salamone
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Transgenic Animals ◽  
Blastocyst Stage ◽  
Control Group ◽  
Sham Control ◽  
Exogenous Dna ◽  
Developmental Rates ◽  
Bull Sperm ◽  
Sperm Decondensation

TM-intracytoplasmic sperm injection (ICSI) was demonstrated to be an effective technique for the production of transgenic animals. However, this method has not been widely applied for transgenesis in cattle, because of the low embryo developmental rates. This problem may be related to the incomplete sperm decondensation and subsequent pronuclei formation that occurs in cattle after ICSI (Malcuit et al. 2006 Reprod. Fertil. Dev. 18, 39–51). Delgado et al. showed that pretreatment with heparin-sodium salt combined with reduced glutathione (Hep-GSH) could improve bull sperm decondensation (2001 Archives of Andrology 47, 47–58). The objective of this work was to test the use of pretreated sperm with Hep-GSH for TM-ICSI, because an improvement of male pronucleus formation could cause an increase on the frequency of exogenous DNA integration. To this aim, cumulus-oocyte complexes were collected from slaughtered cow ovaries and in vitro matured for 21 h. Frozen sperm from a bull that was previously determined to produce low developmental rates post ICSI and IVF was used. It was thawed and washed twice by centrifuging at 390 × g for 10 min. After that, sperm were incubated with Tris medium supplemented with 80 μM Hep and 15 mM GSH for 20 h. After washing, semen was co-incubated with 50 ng μL–1 of pCX-EGFP plasmid for 5 min on ice and used for ICSI (Hep-GSH ICSI group). An ICSI control group was injected with semen not treated with Hep-GSH. Sham controls were injected with 50 ng μL–1 of pCX-EGFP. Haploid and diploid parthenogenetic controls were also included (Haplo PA and Diplo PA groups). Oocytes were activated by a 4 min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h, and treated with 1.9 mM DMAP for 3 h; Diplo PA were immediately exposed to DMAP after ionomycin treatment. Embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post ICSI, respectively. Expression of egfp was assayed at Day 4 and at the blastocyst stage. Results: Hep-GSH ICSI group showed higher cleavage rates than ICSI control (68.5%, n = 89 v. 35%, n = 60), and lower than Sham, Diplo PA, and Haplo Pa groups (94% n = 50, 95.1% n = 61, and 85.1% n = 47, respectively; Fisher's exact test, P ≤ 0.05). Although blastocyst rates from ICSI groups did not differ from Haplo PA (21.2%) and Sham groups (8%), Hep-GSH ICSI produced higher rates than ICSI control (19.1 v. 5%). The higher blastocyst rates were observed for Diplo PA (47.5%; P ≤ 0.05). Transgene expression levels at Day 4 were higher for both Hep-GSH ICSI and ICSI control than for Sham control (24.7 and 11.7% v. 0%, respectively; P ≤ 0.05). Rates of egfp expressing blastocysts/injected oocytes were significantly higher for Hep-GSH ICSI than for ICSI and Sham control groups (13.5 v. 1.7 and 0%, respectively; P ≤ 0.05). Conclusions: Pretreatment of bull sperm with Hep-GSH can increase blastocyst rates after ICSI, even when low quality semen is used. Additionally, the employment of Hep-GSH treatment increased rates of transgene expressing blastocysts. It could be a useful strategy for massively implementing TM-ICSI in bovine, for the production of transgenic animals.

Detection of deoxyribonuclease I and II activities in Japanese quail oocytes

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Urszula Stepińska ◽  
Bozenna Olszańska
Keyword(s):  
Japanese Quail ◽  
Dna Degradation ◽  
Dnase I ◽  
Early Cleavage ◽  
Experimental Conditions ◽  
Dnase Ii ◽  
Exogenous Dna ◽  
Deoxyribonuclease I ◽  
Female Pronucleus

Birds exhibit physiological polyspermy, i.e. numerous spermatozoa enter the germinal disc of an oocyte and form pronuclei during fertilisation. However, only one of them unites with the female pronucleus to form a zygote nucleus; the supernumerary spermatozoal nuclei degenerate at the early cleavage stages. To establish a factor responsible for spermatozoal degeneration, the presence of DNase activity was studied in vitro in extracts of Japanese quail oocytes using λ DNA/HindIII as a substrate. The experimental conditions were designed to reveal the presence of either DNase I or DNase II activities, separately. Degradation of the substrate DNA was evaluated by electrophoresis on agarose gels stained with ethidium bromide. High activities of DNase I and DNase II were found in the germinal discs of the largest vitellogenic oocytes. DNase I activity was estimated to be about 3 × 10−3 Kunitz units and DNase II about 4 × 10−2 Kunitz units per germinal disc. DNase I activity in an oocyte seems to increase during oogenesis since DNA degradation by the extracts from the germinal discs of the largest vitellogenic oocytes was much higher than by those from previtellogenic and small vitellogenic oocytes. The presence of high DNase I and II activities in the largest vitellogenic oocytes would point to their role in degradation of DNA from supernumerary spermatozoa entering the ovum during polyspermic fertilisation in birds. The enzymes could be a factor, or one of the factors, in the late block to polyspermy in the cytoplasm of avian eggs. It is suggested here that the DNase activities might also be responsible for poor efficiency in obtaining transgenic birds by microinjection of exogenous DNA into the fertilised chick ovum.

In vitro Assay Revealed Mismatches between Guide RNA and Target DNA can Enhance Cas9 Nuclease Activity

2020 ◽  
Vol 1 (1) ◽  
pp. 69-72
Author(s):  
Ji Luan ◽  
Zhen Li ◽  
Hailong Wang ◽  
Jun Fu ◽  
Youming Zhang
Keyword(s):  
Dna Sequences ◽  
High Efficiency ◽  
Nuclease Activity ◽  
Ease Of Use ◽  
Unexpected Finding ◽  
Vitro Assay ◽  
Guide Rna ◽  
Cleavage Activity ◽  
Target Dna

Background: CRISPR-Cas9 is a powerful technology that allows us to modify DNA sequences in a specific manner across a variety of organisms. Due to its high efficiency and specificity, and ease of use, it becomes a commonly used method for gene editing. Although many structural and biochemical studies have been carried out to understand the fundamental mechanism of CRISPR/Cas9, our understanding of CRISPR/Cas9 caused off-target effects is still lacking. Methods: The enhanced in vitro cleavage activity of Cas9 protein from Streptococcus pyogenes (SpCas9) was evaluated by both synthetic crRNA-tracrRNA duplexes and in vitro transcribed single guide RNAs. Results: Here, we report an unexpected finding that mismatches between the guide RNA and target DNA significantly enhanced the in vitro cleavage activity of SpCas9 by more than 2 folds. Conclusion: Our observation that mismatches between the guide RNA and target DNA can dramatically increase the in vitro cleavage of Cas9 suggests the potential sequence preference for the CRSIPR/Cas9 system.

scholarly journals Sperm DNA fragmentation induced by DNAse I and hydrogen peroxide: an in vitro comparative study among different mammalian species

Reproduction ◽  
2010 ◽  
Vol 140 (3) ◽  
pp. 445-452 ◽  
Author(s):  
Paola Villani ◽  
Patrizia Eleuteri ◽  
Maria Giuseppa Grollino ◽  
Michele Rescia ◽  
Pierluigi Altavista ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Comet Assay ◽  
Chromatin Structure ◽  
Dnase I ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Dna Breaks ◽  
Sperm Dna

Sperm DNA damage may have adverse effects on reproductive outcome. Sperm DNA breaks can be detected by several tests, which evaluate DNA integrity from different and complementary perspectives and offer a new class of biomarkers of the male reproductive function and of its possible impairment after environmental exposure. The remodeling of sperm chromatin produces an extremely condensed nuclear structure protecting the nuclear genome from adverse environments. This nuclear remodeling is species specific, and differences in chromatin structure may lead to a dissimilar DNA susceptibility to mutagens among species. In this study, the capacity of the comet assay in its two variants (alkaline and neutral) to detect DNA/chromatin integrity has been evaluated in human, mouse, and bull sperm. The hypothesis that chromatin packaging might influence the amount of induced and detectable DNA damage was tested by treating sperm in vitro with DNAse I, whose activity is strictly dependent upon its DNA accessibility. Furthermore, hydrogen peroxide (H2O2) was used to assess whether spermatozoa of the three species showed a different sensitivity to oxidative stress. DNAse I-induced damage was also assessed by the sperm chromatin structure assay and the TUNEL assay, and the performances of these two assays were compared and correlated with the comet assay results. Results showed a different sensitivity to DNAse I treatment among the species with human sperm resulting the most susceptible. On the contrary, no major differences among species were observed after H2O2 treatment. Furthermore, the three tests show a good correlation in revealing sperm with DNA strand breaks.

Dimethyl sulphoxide and electrolyte-free medium improve exogenous DNA uptake in mouse sperm and subsequently gene expression in the embryo

Zygote ◽  
2018 ◽  
Vol 26 (5) ◽  
pp. 403-407
Author(s):  
Soleiman Kurd ◽  
Sara Hosseini ◽  
Fardin Fathi ◽  
Vahid Jajarmi ◽  
Mohammad Salehi
Keyword(s):  
Sperm Motility ◽  
Transgenic Animals ◽  
Blastocyst Stage ◽  
Control Group ◽  
Dna Uptake ◽  
Free Medium ◽  
Exogenous Dna ◽  
Mouse Sperm ◽  
Better Than

SummaryOne of the methods to generate transgenic animals is called sperm-mediated gene transfer (SMGT). Mature sperm cells can take up exogenous DNA molecules intrinsically and transfer them into the oocyte during fertilization. This study assessed the effect of dimethyl sulfoxide (DMSO) and electrolyte-free medium (EFM) on DNA uptake (EGFP–N1plasmid) in mouse sperm. Sperms cells cultured in human tubular fluid (HTF) without any treatment were considered as the control group. Sperms cells that were incubated in EFM and HTF with DNA/DMSO at 4°C were classified into EFM and HTF groups. Sperm motility and viability were assessed following treatment. In vitro fertilization (IVF) with sperm in all groups was performed. Fertilization, embryo development and GFP-positive blastocyst rates were analyzed and compared. The result showed that sperm motility and viability in EFM were better than those in the HTF group. The rate of development to reach the blastocyst stage and GFP-positive blastocysts was significantly higher in the EFM group compared with the HTF group (P<0.05). Our data demonstrate that sperm stored in the EFM group can improve the efficiency of SMGT for the generation of GFP-positive blastocysts.

scholarly journals Phage Display of Adenovirus Type 5 Fiber Knob as a Tool for Specific Ligand Selection and Validation

2001 ◽  
Vol 75 (15) ◽  
pp. 7107-7113 ◽  
Author(s):  
Alexander Pereboev ◽  
Larisa Pereboeva ◽  
David T. Curiel
Keyword(s):  
Conformational Changes ◽  
High Efficiency ◽  
A Priori ◽  
Adenovirus Type ◽  
Target Cells ◽  
Genetic Modifications ◽  
Filamentous Bacteriophages ◽  
Serotype 5

ABSTRACT Adenovirus (Ad) vectors are most potent for use as gene delivery vehicles to infect human cells in vitro and in vivo with high efficiency. The main limitation in utilization of Ad as a gene transfer vector is the lack of specificity. Genetic modifications of Ad capsid proteins resulting in incorporation of foreign polypeptide ligand sequences can redirect the vector towards target cells. However, in many cases the incorporated ligands lose specificity or lead to conformational changes influencing virion integrity. In order to select target-specific ligands a priori structurally compatible with Ad, we propose a system for displaying polypeptide sequences in the context of the Ad fiber knob on the surfaces of filamentous bacteriophages. To establish this concept, we displayed the wild-type Ad serotype 5 knob and knobs containing c-Myc epitopes and six-histidine sequences in the pJuFo phage system. The knobs remained trimeric and bound the coxsackievirus-Ad receptor, and the phage knob-displayed ligands recognized and bound their cognates in the phage-displayed knob context. Further development of this system may be useful for candidate ligand fidelity and Ad structural compatibility validation prior to Ad modification.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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