Validating and Optimizing A Combination of INTACT and FACS Techniques for the Isolation of Mouse Astrocyte Nuclei Upon Ablation of the <i>Atrx</i> Intellectual Disability Gene
Notice bibliographique
Résumé
The ATRX gene codes for the ATRX chromatin remodelling protein, which interacts with DAXX protein to deposit histone variant H3.3 at telomeres and pericentromeric heterochromatin. Mutations of the ATRX gene cause intellectual disability. One noticeable example is the ATRX syndrome, which is characterized by distinctive craniofacial features, severe developmental delays, intellectual disability, and mild‐to‐moderate anemia. It is therefore apparent that the ATRX gene likely plays an important role in the central nervous system (CNS). An astrocyte is a cell type that, by the most conservative survey, has the same abundance as neurons in the human brain. However, unlike neurons, astrocyte function is far less understood, and its role other than a supporting function was not known until a decade ago, when astrocytes were discovered to actively participate in higher neuronal processing through the tripartite synapse. The specific role of ATRX in astrocytes has never been reported before. A challenge of elucidating ATRX function in astrocyte is a lack of an optimized technique to isolate a reasonable number of highly enriched Atrx ‐ablated astrocyte nuclei for various sequencing purposes, including ChiP‐seq and RNA‐seq. This project aims to fulfill such research demand by validating and optimizing the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) technique with mouse brain tissue at postnatal day 30. By adjusting various experimental conditions such as tissue homogenization time, the concentration of octylphenoxy poly(ethyleneoxy)ethanol (IGEPAL) in buffer solutions, the dilution ratio of the tissue homogenate and the centrifugation time, a total nuclei yield of 90% was obtained. The structural integrity of nuclei was verified by laser microscopy with nuclei sample stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). However, subsequent enrichment of GFP‐tagged Atrx F/y ; Cre +/− ;Sun1‐GFP +/− nuclei using anti‐G‐protein coated magnetic bead, as per the original INTACT protocol, is plagued with non‐specific binding of less than 32% specificity. To improve the enrichment factor, a preliminary investigation was performed to validate the use of Florescent‐Assisted Cell Sorting (FACS) to enrich Sun1GFP‐tagged nuclei from the previously‐produced total nuclei preparation. A 92% enrichment of GFP‐tagged nuclei was obtained, demonstrating the promising potential of a combined INTACT‐FACS technique to effectively isolate structurally‐sound nuclei from transcriptionally‐sensitive cell types, within a complex and intermingled tissue environment. Support or Funding Information This work was supported by a Dean's Undergraduate Research Opportunity Program award to Yuxuan Jiang and the Canadian Institutes for Health Research MOP#142369. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,001 | 0,001 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,001 | 0,000 |
| Études des sciences et des technologies | 0,001 | 0,000 |
| Communication savante | 0,001 | 0,000 |
| Science ouverte | 0,001 | 0,001 |
| Intégrité de la recherche | 0,001 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,002 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».