scholarly journals Successful i-GONAD in Brown Norway Rats by Modification of in vivo Electroporation Conditions

2020 ◽  
Vol 4 (4) ◽  
Author(s):  
Shuji Takabayashi ◽  
◽  
Takuya Aoshima ◽  
Yukari Kobayashi ◽  
Hisayoshi Takagi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Preimplantation Embryos ◽  
Brown Norway ◽  
Sprague Dawley ◽  
In Vivo Electroporation ◽  
Nucleic Acids Delivery ◽  
A Current

Improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) was developed for in situ genome editing of the preimplantation embryos present within the oviductal lumen of mice. This method is based on intra-oviductal instillation of genome editing components and subsequent in vivo electroporation (EP) in the entire oviduct. Therefore, i-GONAD differs from the previous methods (i.e., zygote microinjection and in vitro EP) in producing genome-edited mice, which relied on ex vivo handling of preimplantation embryos and egg transfer to the recipient females. We have previously demonstrated that i-GONAD can be successfully applied to produce genome-edited rats, including albino Sprague-Dawley and albino Lewis rats (however, not pigmented Brown Norway [BN] rats). We observed that the successful i-GONAD was dependent on the mouse strain used; for example, in random-bred mice, such as ICR and C3H/He × C57BL/6, it was successful under relatively stringent electrical conditions but not in the C57BL/6 strain. Under less stringent conditions, i-GONAD was successful in the C57BL/6 strain. We speculated that this would also be true for i-GONAD using BN rats. On applying a current of >500 mA, we failed to obtain rat offspring (fetuses/newborns); however, i-GONAD under a current of 100-300 mA using NEPA21 (NEPA GENE) led to the production of genome-edited BN rats with efficiencies of 75%-100%. Similarly, i-GONAD, under a current of 150-200 mA using CUY21EDIT II (BEX Co.) led to the production of genome-edited BN rats with efficiencies of 24%-55%. These experiments suggest the importance of selecting the appropriate current value, depending on the rat strain used, when performing i-GONAD.

scholarly journals Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 546 ◽  
Author(s):  
Masahiro Sato ◽  
Rico Miyagasako ◽  
Shuji Takabayashi ◽  
Masato Ohtsuka ◽  
Izuho Hatada ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Preimplantation Embryos ◽  
Nucleic Acid Delivery ◽  
Cell Stage ◽  
Initial Development ◽  
Genetic Manipulations ◽  
Target Sites

Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4–1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4–1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called “sequential i-GONAD (si-GONAD).”

scholarly journals Studies to Elucidate the Mechanism of Cardio Protective and Hypotensive Activities of Anogeissus acuminata (Roxb. ex DC.) in Rodents

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3471
Author(s):  
Fatima Saqib ◽  
Muhammad Arif Aslam ◽  
Khizra Mujahid ◽  
Luigi Marceanu ◽  
Marius Moga ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Ex Vivo ◽  
Inflammatory Cells ◽  
Left Ventricular ◽  
Guanosine Monophosphate ◽  
Sprague Dawley ◽  
Positive Effects ◽  
Creatine Kinase Mb

Anogeissus acuminata (Roxb. ex DC.) is a folkloric medicinal plant in Asia; including Pakistan; used as a traditional remedy for cardiovascular disorders. This study was planned to establish a pharmacological basis for the trivial uses of Anogeissus acuminata in certain medical conditions related to cardiovascular systems and to explore the underlying mechanisms. Mechanistic studies suggested that crude extract of Anogeissus acuminata (Aa.Cr) produced in vitro cardio-relaxant and vasorelaxant effects in isolated paired atria and aorta coupled with in vivo decrease in blood pressure by invasive method; using pressure and force transducers connected to Power Lab Data Acquisition System. Moreover; Aa.Cr showed positive effects in left ventricular hypertrophy in Sprague Dawley rats observed hemodynamically by a decrease in cardiac cell size and fibrosis; along with absence of inflammatory cells; coupled with reduced levels of angiotensin converting enzyme (ACE) and renin concentration along with increased concentrations of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP). In Acute Myocardial Infarction (AMI) model; creatine kinase (CK), creatine kinase-MB (CK-MB) and lactic acid dehydrogenase (LDH levels) were found to be decreased; along with decreased necrosis; edema and recruitment of inflammatory cells histologically. In vivo and ex vivo studies of Anogeissus acuminata provided evidence of vasorelaxant; hypotensive and cardioprotective properties facilitated through blockage of voltage-gated Ca++ ion channel; validating its use in cardiovascular diseases

Elevated IL-1β contributes to antibody suppression produced by stress

2002 ◽  
Vol 93 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Albert Moraska ◽  
Jay Campisi ◽  
Kien T. Nguyen ◽  
Steven F. Maier ◽  
Linda R. Watkins ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Acute Stress ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Keyhole Limpet Hemocyanin ◽  
Acquired Immunity ◽  
Sprague Dawley

Acute stressor exposure can facilitate innate immunity and suppress acquired immunity. The present study further characterized the potentiating effect of stress on innate immunity, interleukin-1β (IL-1β), and demonstrated that stress-induced potentiation of innate immunity may contribute to the stress-induced suppression of acquired immunity. The long-term effect of stress on IL-1β was measured by using an ex vivo approach. Sprague-Dawley rats were challenged with lipopolysaccharide (LPS) in vivo, and the IL-1β response was measured in vitro. Splenocytes, mesenteric lymphocytes, and peritoneal cavity cells had a dose- and time-dependent ex vivo IL-1β response to LPS. Rats that were exposed to inescapable shock (IS, 100 1.6 mA, 5-s tail shocks, 60-s intertrial interval) and challenged with a submaximal dose of LPS 4 days later had elevated IL-1β measured ex vivo. To test whether the acute stress-induced elevation in IL-1β contributes to the long-term suppression in acquired immunity, IL-1β receptors were blocked for 24 h after stress. Serum anti-keyhole limpet hemocyanin (KLH) immunoglobulin (Ig) was measured. In addition, the acute elevation (2 h post-IS) of splenic IL-1β in the absence of antigen was verified. Interleukin-1 receptor antagonist prevented IS-induced suppression in anti-KLH Ig. These data support the hypothesis that stress-induced increases in innate immunity (i.e., IL-1β) may contribute to stress-induced suppression in acquired immunity (i.e., anti-KLH Ig).

scholarly journals Sympathetic cardiac hyperinnervation and atrial autonomic imbalance in diet-induced obesity promote cardiac arrhythmias

2013 ◽  
Vol 305 (10) ◽  
pp. H1530-H1537 ◽  
Author(s):  
Belinda H. McCully ◽  
Wohaib Hasan ◽  
Cole T. Streiff ◽  
Jennifer C. Houle ◽  
William R. Woodward ◽  
...  
Keyword(s):  
High Fat Diet ◽  
Parasympathetic Nervous System ◽  
Ex Vivo ◽  
System Control ◽  
Sprague Dawley ◽  
High Fat ◽  
Sprague Dawley Rats ◽  
Normal Chow

Obesity increases the risk of arrhythmias and sudden cardiac death, but the mechanisms are unknown. This study tested the hypothesis that obesity-induced cardiac sympathetic outgrowth and hyperinnervation promotes the development of arrhythmic events. Male Sprague-Dawley rats (250–275 g), fed a high-fat diet (33% kcal/fat), diverged into obesity-resistant (OR) and obesity-prone (OP) groups and were compared with rats fed normal chow (13% kcal/fat; CON). In vitro experiments showed that both OR and OP rats exhibited hyperinnervation of the heart and high sympathetic outgrowth compared with CON rats, even though OR rats are not obese. Despite the hyperinnervation and outgrowth, we showed that, in vivo, OR rats were less susceptible to arrhythmic events after an intravenous epinephrine challenge compared with OP rats. On examining total and stimulus-evoked neurotransmitter levels in an ex vivo system, we demonstrate that atrial acetylcholine content and release were attenuated in OP compared with OR and CON groups. OP rats also expressed elevated atrial norepinephrine content, while norepinephrine release was suppressed. These findings suggest that the consumption of a high-fat diet, even in the absence of overt obesity, stimulates sympathetic outgrowth and hyperinnervation of the heart. However, normalized cardiac parasympathetic nervous system control may protect the heart from arrhythmic events.

Abstract 247: Gender-specific Angiogenesis Suppression By Btg2 May Be Mediated Through Suppressed Renin Expression

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Timothy J Stodola ◽  
Daniela Didier ◽  
Michael Flister ◽  
Jozef Lazar ◽  
Andrew Greene
Keyword(s):  
Luciferase Activity ◽  
Proximal Promoter ◽  
Brown Norway ◽  
Sprague Dawley ◽  
Hek 293 ◽  
Congenic Lines ◽  
Sprague Dawley Rats ◽  
Males And Females

Angiogenesis is the formation of new microvessels from existing vascular beds. AngII, a downstream product of renin, has been shown to be essential mediator of skeletal muscle angiogenesis in Dahl Salt Sensitive (SS) and Sprague Dawley rats, C57BL/6 mice, and human endothelial cells in vitro. Using partial chromosome introgression from the Brown Norway (BN) rat into the SS rat we have created two congenic lines with small (<300 Kbp) BN substitutions that differ by 23 Kbp. The congenic regions define an angiogenesis locus that contains one gene, Btg2 . Sanger sequencing revealed no sequence variants in exons of Btg2 between the BN and SS strains. Angiogenesis was measured using an in vivo electrical stimulation model of one hindlimb, with the contralateral leg acting as the control, in males and females of both new congenic strains. Males from Btg2 BN have angiogenesis (TA=20.0±4.5% increase in vessel density in stimulated leg relative to unstimulated leg, EDL=15.8±5.8%,) while Btg2 SS males did not have angiogenesis (TA=5.1±2.2%, EDL=4.4±2.7%). Females of both strains had angiogenesis: Btg2 BN (TA=15.8±4.3%, EDL=3.4%), Btg2 SS (TA=14.5±2.0%, EDL=14.6±2.5%). Stimulation significantly increased Btg2 expression in both males and females. Btg2 SS males had a significantly greater increase in Btg2 mRNA in the stimulated muscle relative to unstimulated than Btg2 BN males (5.0±0.9 versus 2.1± 0.3), but there was no difference in stimulated females (Btg2 BN 2.5±0.2, Btg2 SS 3.2±1.5). To test the hypothesis that Btg2 impacted renin expression, we cloned the renin proximal promoter into a vector to drive luciferase, and co-transfected HEK-293 cells with this and vector(s) expressing Btg2 or Hoxb9 and Btg2 . Normalized luciferase activity was 4.52±0.30 (arbitrary units) with an empty vector control and suppressed with the Btg2 vector to 1.45±0.07. Co-expression of Btg2 and Hoxb9 lead to a further reduction in luciferase activity to 0.40±0.04. These data suggest the elevated Btg2 expression observed in Btg2 SS strain may be acting to suppress renin expression through renin proximal promoter transcription factor Hoxb9 , leading to an inhibited angiogenic response.

scholarly journals Antifungal Activity of Oleylphosphocholine on In Vitro and In Vivo Candida albicans Biofilms

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Michelle Holtappels ◽  
Erwin Swinnen ◽  
Lies De Groef ◽  
Jurgen Wuyts ◽  
Lieve Moons ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Biofilm Formation ◽  
Ex Vivo ◽  
Oral Candidiasis ◽  
Sprague Dawley ◽  
Content Type ◽  
Polyurethane Catheters

ABSTRACT In this study, we investigated the potential antifungal activity of the alkylphospholipid oleylphosphocholine (OlPC), a structural analogue of miltefosine, on in vitro and in vivo Candida albicans biofilm formation. The effect of OlPC on in vitro and in vivo C. albicans biofilms inside triple-lumen polyurethane catheters was studied. In vivo biofilms were developed subcutaneously after catheter implantation on the lower back of Sprague-Dawley rats. Animals were treated orally with OlPC (20 mg/kg of body weight/day) for 7 days. The effect of OlPC on biofilms that developed on the mucosal surface was studied in an ex vivo model of oral candidiasis. The role of OlPC in C. albicans morphogenesis was investigated by using hypha-inducing media, namely, Lee, Spider, and RPMI 1640 media. OlPC displayed activity against both planktonic cells and in vitro C. albicans biofilms. To completely abolish preformed, 24-h-old biofilms, higher concentrations (8, 10, and 13 mg/liter) were needed. Moreover, OlPC was able to reduce C. albicans biofilms formed by caspofungin-resistant clinical isolates and acted synergistically when combined with caspofungin. The daily oral administration of OlPC significantly reduced in vivo C. albicans biofilms that developed subcutaneously. In addition, OlPC decreased biofilm formation on mucosal surfaces. Interestingly, the application of subinhibitory concentrations of OlPC already inhibited the yeast-to-hypha transition, a crucial virulence factor of C. albicans. We document, for the first time, the effects of OlPC on C. albicans cells and suggest the potential use of OlPC for the treatment of C. albicans biofilm-associated infections.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Significance of Platelet β-Adrenoceptors for Platelet Responses In Vivo and In Vitro

1992 ◽  
Vol 68 (06) ◽  
pp. 687-693 ◽  
Author(s):  
P T Larsson ◽  
N H Wallén ◽  
A Martinsson ◽  
N Egberg ◽  
P Hjemdahl
Keyword(s):  
Ex Vivo ◽  
High Dose ◽  
Platelet Aggregability ◽  
Adrenoceptor Agonist ◽  
Dose Infusion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen

SummaryThe significance of platelet β-adrenoceptors for platelet responses to adrenergic stimuli in vivo and in vitro was studied in healthy volunteers. Low dose infusion of the β-adrenoceptor agonist isoprenaline decreased platelet aggregability in vivo as measured by ex vivo filtragometry. Infusion of adrenaline, a mixed α- and β-adrenoceptor agonist, increased platelet aggregability in vivo markedly, as measured by ex vivo filtragometry and plasma β-thromboglobulin levels. Adrenaline levels were 3–4 nM in venous plasma during infusion. Both adrenaline and high dose isoprenaline elevated plasma von Willebrand factor antigen levels β-Blockade by propranolol did not alter our measures of platelet aggregability at rest or during adrenaline infusions, but inhibited adrenaline-induced increases in vWf:ag. In a model using filtragometry to assess platelet aggregability in whole blood in vitro, propranolol enhanced the proaggregatory actions of 5 nM, but not of 10 nM adrenaline. The present data suggest that β-adrenoceptor stimulation can inhibit platelet function in vivo but that effects of adrenaline at high physiological concentrations are dominated by an α-adrenoceptor mediated proaggregatory action.

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