Ce travail a été soutenu par l&#39;Université de Western Ontario, le Département d&#39;obstétrique et de gynécologie, et une subvention ER06-02-188 à partir de la recherche et l&#39;innovation Ministryof, bourse de nouveau chercheur. MMD a été soutenue par un programme de formation des IRSC sur la reproduction, le développement des jeunes et l&#39;impact sur les bourses d&#39;études supérieures de la santé (REDIH).

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bisulfite+method+of+5-methylcytosine+mapping+that+minimizes+template+degradation.">A bisulfite method of 5-methylcytosine mapping that minimizes template degradation.</a> Anal. Biochem. 226, 161-166 (1995).</li><li>Patterson, K., Molloy, L., Qu, W., Clark, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Methylation:+Bisulphite+Modification+and+Analysis.">DNA Methylation: Bisulphite Modification and Analysis.</a> J. Vis. Exp. (56), e3170 (2011).</li><li>Olek, A., Oswald, J., Walter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+and+improved+method+for+bisulphite+based+cytosine+methylation+analysis.">A modified and improved method for bisulphite based cytosine methylation analysis.</a> Nucleic Acids Res. 24, 5064-5066 (1996).</li><li>Mann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+loss+of+imprinting+in+the+placenta+following+preimplantation+development+in+culture.">Selective loss of imprinting in the placenta following preimplantation development in culture.</a> Development. 131, 3727-3735 (2004).</li><li>Market-Velker, B. A., Zhang, L., Magri, L. S., Bonvissuto, A. C., Mann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+effects+of+superovulation:+loss+of+maternal+and+paternal+imprinted+methylation+in+a+dose-dependent+manner.">Dual effects of superovulation: loss of maternal and paternal imprinted methylation in a dose-dependent manner.</a> Hum. Mol. Genet. 19, 36-51 (2010).</li><li>Meng, L. H., Xiao, S. Q., Huang, X. F., Zhou, Y., Xu, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+on+bisulfite+sequencing+method+for+methylation+status+of+imprinted+genes+in+single+human+oocytes.">A study on bisulfite sequencing method for methylation status of imprinted genes in single human oocytes.</a> Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 25, 289-292 (2008).</li><li>Denomme, M. M., Zhang, L., Mann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+imprinting+perturbations+do+not+originate+from+superovulation-induced+defects+in+DNA+methylation+acquisition.">Embryonic imprinting perturbations do not originate from superovulation-induced defects in DNA methylation acquisition.</a> Fertil. Steril. 96, 734-738 (2011).</li><li>Tahiliani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1.">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324, 930-935 (2009).</li><li>Hajj, N. 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Les auteurs n&#39;ont rien à révéler.

Ce test seul ovocyte contient de nombreuses étapes avec un certain nombre qui sont essentiels et nécessitent des soins spéciaux. La première est le lavage des ovocytes. Il est particulièrement important de se laver chaque ovocyte à plusieurs reprises dans un milieu frais tombe suite à un traitement hyaluronidase pour enlever le plus les cellules du cumulus plus grand nombre possible. En outre, lors du transfert des ovocytes à la solution acide de Tyrode pour zone pellucide retrait assurez-vous du milieu environn...

Table des réactifs spécifiques et des équipements. <table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td colspan="4"> Collection d&#39;ovocytes </td></tr><tr><td> Hyaluronidase </td><td> Sigma </td><td> H4272 </td><td></td></tr><tr><td> Acide de Tyrode </td><td> Sigma </td><td> T1788 </td><td></td></tr><tr><td> Protéinase K </td><td> Sigma </td><td> P5568 </td><td></td></tr><tr><td> IGEPAL 10% </td><td> Bioshop </td><td> NON999.500 </td><td></td></tr><tr><td colspan="4"> Solution de lyse </td></tr><tr><td> Tris, pH 7,5 </td><td> Bioshop </td><td> TRS001.5 </td><td></td></tr><tr><td> LiCl </td><td> Sigma </td><td> L9650 </td><td> &amp; Nbsp; </td></tr><tr><td> EDTA pH 8,0 </td><td> Sigma </td><td> E5134 </td><td></td></tr><tr><td> LiDS </td><td> Bioshop </td><td> LDS701.10 </td><td></td></tr><tr><td> TNT </td><td> Invitrogen </td><td> P2325 </td><td></td></tr><tr><td colspan="4"> Tampon de lyse SDS </td></tr><tr><td> PH 7,5 TE </td><td> Bioshop (Tris) Sigma (EDTA) </td><td> TRS001.5 E5134 </td><td></td></tr><tr><td> SDS à 10% </td><td> Bioshop </td><td> SDS001.500 </td><td></td></tr><tr><td colspan="4"> Conversion au bisulfite </td></tr><tr><td> Hydroxyde de sodium </td><td> Sigma </td><td> S8045 </td><td></td></tr><tr><td> Hydrogénosulfite de sodium (bisulfite de sodium) </td><td> Sigma </td><td> 243973 </td><td>; </td></tr><tr><td> Hydroquinone </td><td> Sigma </td><td> H9003 </td><td></td></tr><tr><td> Bas point de fusion (LMP) Agarose </td><td> Sigma </td><td> A9414 </td><td></td></tr><tr><td> Huile minérale </td><td> Sigma </td><td> M8410 </td><td></td></tr><tr><td> Milieu M2 </td><td> Sigma </td><td> M7167 </td><td></td></tr><tr><td> GIBCO eau distillée </td><td> Invitrogen </td><td> 15230-196 </td><td></td></tr><tr><td> Autoclavée bidistillée (jj) de l&#39;eau </td><td></td><td></td><td></td></tr><tr><td colspan="4"> PCR </td></tr><tr><td> Illustra Hot Mix Démarrer RTG </td><td> GE Healthcare </td><td> 28-9006-54 </td><td></td></tr><tr><td> 240 ng / ml ARNt de levure </td><td> Invitrogen </td><td> 15401-011 </td><td></td></tr><tr><td> 5x tampon vert réaction GoTaq </td><td> Proméga </td><td> M7911 </td><td></td></tr><tr><td> Intérieure et extérieure amorces imbriquées </td><td> Sigma </td><td></td><td></td></tr><tr><td colspan="4"> La ligature </td></tr><tr><td> Promega vecteur pGEM-T Facile </td><td> Fisher Scientific </td><td> A1360 </td><td></td></tr><tr><td colspan="4"> TA Cloning </td></tr><tr><td> Compétentes cellules de E. coli </td><td> Zymo Research Corp </td><td> T3009 </td><td></td></tr><tr><td colspan="4"> Équipement </td></tr><tr><td> Microscope à dissection </td><td></td><td></td><td></td></tr><tr><td> 70 ° C et 90 ° C à chaleur blocs </td><td></td><td></td><td></td></tr><tr><td> 37 ° C et 50 ° C Bain-marie (42 ° C pour les transformations) </td><td></td><td></td><td></td></tr><tr><td>Bascule </td><td></td><td></td><td></td></tr><tr><td> Machine à PCR </td><td></td><td></td><td></td></tr></tbody></table>

single oocyte bisulfite mutagenesis

L&#39;épigénétique englobe toutes héréditaires et des modifications réversibles de la chromatine que l&#39;accessibilité des gènes alter, et sont donc les principaux mécanismes de régulation de la transcription du gène 1. Méthylation de l&#39;ADN est une modification épigénétique qui agit principalement comme une marque de répression. Grâce à l&#39;addition covalente d&#39;un groupement méthyle sur les cytosines dans les dinucléotides CpG, on peut recruter d&#39;autres protéines de répression et les modifications des histones pour lancer le processus impliqués dans la condensation de la chromatine et faire taire des gènes 2. Méthylation de l&#39;ADN est essentielle pour le développement normal car elle joue un rôle essentiel dans la programmation du développement, la différenciation cellulaire, la répression des éléments rétroviraux, inactivation du chromosome X et l&#39;empreinte génomique. Une des méthodes les plus puissants pour l&#39;analyse méthylation de l&#39;ADN est la mutagenèse bisulfite. Bisulfite de sodium est un agent mutagène ADN qui désamine cytosines en uraciles. Après amplification par PCR et seque<html> ncing, ces phénomènes de conversion sont détectés comme thymines. Cytosines méthylées sont protégés de la désamination et restent donc en cytosines, permettant l&#39;identification de la méthylation de l&#39;ADN au niveau individuel nucléotide 3. Développement de l&#39;essai de mutagenèse bisulfite a progressé de ceux initialement déclarés 4-6 vers ceux qui sont plus sensible et reproductible 7. Un progrès important a été l&#39;incorporation de petites quantités d&#39;ADN dans un bourrelet d&#39;agarose, protégeant ainsi l&#39;ADN provenant du traitement bisulfite dure 8. Cette analyse de la méthylation permis à être exécuté sur des pools d&#39;ovocytes et embryons au stade blastocyste-9. Le protocole le plus sophistiqué du bisulfite de mutagenèse à ce jour est pour individuels blastocyste-embryons au stade 10. Toutefois, étant donné que les blastocystes ont en moyenne 64 cellules (contenant 120-720 pg d&#39;ADN génomique), cette méthode n&#39;est pas efficace pour les études de méthylation sur les ovocytes individuels ou clivage au stade d&#39;embryons. tente &quot;&gt; Prendre des indices de plongement d&#39;agarose des montants d&#39;ADN minute, y compris les ovocytes 11, nous présentons ici une méthode par laquelle les ovocytes sont directement intégrés dans une solution de lyse agarose et perles de récupération qui suit immédiatement et le retrait de la zone pellucide de l&#39;ovocyte. Cela nous permet de contourner les deux principaux défis de la mutagenèse ovocyte unique: bisulfite. protégeant une infime quantité d&#39;ADN de la dégradation et la perte subséquente au cours des étapes du protocole de nombreuses important, car les données sont obtenues à partir d&#39;ovocytes simples, la question de la partialité PCR au sein des pools est éliminée plus. , par inadvertance la contamination de cellules du cumulus est détectable par cette méthode depuis n&#39;importe quel échantillon avec plus d&#39;un modèle de méthylation peuvent être exclus de l&#39;analyse 12. Ce protocole fournit une méthode améliorée pour les analyses réussies et reproductibles de méthylation de l&#39;ADN au niveau unicellulaire et est parfaitement adapté des ovocytes individuels ainsi que le clivage au stade d&#39;embryons.

Watch this Scientific Journal Video about Single Oocyte Bisulfite Mutagenesis at JoVE.com

Single Oocyte Bisulfite Mutagenesis

Mutagenèse bisulfite est l&#39;étalon-or pour l&#39;analyse de méthylation de l&#39;ADN. Notre protocole modifié permet une analyse méthylation de l&#39;ADN au niveau unicellulaire et a été spécialement conçu pour les ovocytes individuels. Il peut également être utilisé pour le clivage-embryons au stade.

Simple mutagenèse bisulfite d&#39;ovocytes

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

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Biology

14.0K vues.  Schulich School of Medicine and Dentistry, University of Western Ontario. Mutagenèse bisulfite est l'étalon-or pour l'analyse de méthylation de l'ADN. Notre protocole modifié permet une analyse méthylation de l'ADN au niveau unicellulaire et a été spécialement conçu pour les ovocytes individuels. Il peut également être utilisé pour le clivage-embryons au stade.

Vidéo: Single Oocyte Bisulfite Mutagenesis

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Mutagenèse dirigée maison des plasmides entiers

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

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Biologie

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Une introduction au laboratoire Worm: La culture de Worms à la mutagenèse

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Protocoles de sélection mutagenèse et fonctionnelles pour l&#39;évolution dirigée de protéines dans les E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Évaluation microinjection des ovocytes de souris, la maturation et de ploïdie

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

La<em&gt; Xenopus</em&gt; Ovocytes Cut-ouvert vaseline Gap voltage-clamp Technique Avec Fluorimétrie

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Génération de<em&gt; Enterobacter</em&gt; Sp. UEE auxotrophes utilisant mutagénèse par transposon

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Un protocole pour l&#39;évaluation fonctionnelle de Whole-Protein Saturation mutagenèse Bibliothèques Utilisant à haut débit de séquençage

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

Hydrogels d’Alginate et microbioréacteurs hydrophobes protéolytically dégradés pour l’encapsulation des oocytes porcins

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

Protocoles pour la mutagenèse CRISPR/Cas9 de la mouche orientale des fruits Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

Isolement et analyse transcriptome de types de cellules végétales

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...