भ्रूण चूहे की आंत अंग से प्रतिदीप्ति सक्रिय सेल छँटाई (FACS) स्वायत तंत्रिका progenitors के अलगाव के लिए एक अनुकूलित प्रक्रिया

Summary

एक अनुकूलित भ्रूण माउस ऊतकों से तंत्रिका शिखा व्युत्पन्न neuronal progenitors शुद्ध करने की प्रक्रिया में वर्णित है. इस विधि के फ्लोरोसेंट संवाददाता alleles से अभिव्यक्ति का लाभ लेता है प्रतिदीप्ति सक्रिय सेल छँटाई (FACS) असतत आबादी अलग. तकनीक के विकास भर में या वयस्क ऊतकों से neuronal subpopulations अलग लागू किया जा सकता है.

Abstract

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS).

The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine¹ and TH is also present in a subset of adult pelvic ganglia neurons^2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc¹. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP.

To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine^2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (>70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

Protocol

1. मीडिया की तैयारी (सभी कदम ऊतक संस्कृति हुड में किया) निम्नलिखित हारवेस्टर: 44 मिलीलीटर एल -15 मध्यम, 0.5 मिलीग्राम 100X पेनिसिलीन / स्ट्रेप्टोमाइसिन (पी / एस), 0.5 मिलीग्राम 100 मिलीग्राम / एमएल गोजातीय सीरम albumin (BS…

Discussion

माउस संवाददाता फ्लोरोसेंट संवाददाताओं से व्यक्त लाइनों व्यापक रूप से उपलब्ध से murine आनुवंशिकी ^1,8,9 समुदाय में कई प्रयासों के माध्यम से होते जा रहे हैं. एक परिणाम के रूप में पृथक्करण विधि यहाँ सचित्र ?…

Materials

Reagent Name	Vendor	Catalog number	Comments
Accumax	Sigma(mfr: Innovative Cell Technologies)	A7089-100ML	Store frozen in 1 ml aliquots
DNase I	Sigma	D-4527	Stored frozen at -20 °C 5 mg/ml in 1xHBSS, (Used in Quench, Quench 1:5)
10X PBS pH 7.4	Gibco	70011-044	Make up to 1x with tissue culture grade water then sterile filter
10X HBSS w/o Ca or Mg	Gibco	14185-052	Make up to 1x with tissue culture grade water then sterile filter
Leibovitz’s L-15 medium	Gibco	21083027
Penicillin /Streptomycin 100X	Gibco	15140-133	Store aliquoted at -20 °C
BSA	Sigma	A3912-100G	Store aliquoted at -20 °C, 100 mg/ml in water
Biowhittaker 1M HEPES in 0.85% NaCl	Lonza	17-737E
38 μm NITEX Nylon Mesh Membrane	Sefar America	3-38/22	Cut into ~3 cm squares. UV treat overnight to sterilize in the tissue culture hood.
7-AAD	Invitrogen	A1310	1 mg/ml
TRIzol LS	Invitrogen	10296-028
5 ml polystyrene tubes	Falcon	352058
15 ml conical tubes	Corning	430790
Fine Dissecting Forceps	Fine Science Instruments	11251-30	Dumont#5 forcep, Dumoxel, standard tip 0.1×0.06mm
Dissecting Spoon	Fine Science Instruments	10370-18