Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Summary

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Abstract

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

Introduction

The ability to culture mouse or human crypts cells as three dimensional (3D) organoids from the small intestines or colon over prolonged time periods provided a major breakthrough because these cultures display defining features of intestinal epithelium in vivo^1,2,3. Organoids derived from primary crypts are capable of self-renewal and self-organization, exhibiting cellular functions similar to their tissues of origin. Indeed, organoids recapitulate not only the structural organization of crypts in vivo, but also many molecular features, thus providing useful tools to study n....

Protocol

This protocol was approved by the Johns Hopkins Medical Institutions Animal Care and Use Committee (IACUC). This protocol is modified from a previously published methods^10,11,12,13.

1. Preparation of Reagents

1. Prepare fresh 293T medium several hours in advance and warm to 37 °C in a water bath for at least 10 min before use (Table 1).
2. Prepare plasmid DNA^2,13,14 for viral packagin....

Results

Here, we describe a rapid and highly efficient transduction technique which harnesses magnetic nanoparticles exposed to a magnetic field to deliver lentivirus to cells of interest. With readily available tools, we have applied this approach not only to transduce freshly isolated crypt cells (Figure 1A), but also for organoids (Figure 2) and other cells that are refractory to more routine transduction approaches. Lentiviral partic.......

Discussion

Primary culture of adult intestinal epithelium as organoids provides a powerful tool to study molecular mechanisms involved in stem cell function, intestinal epithelial homeostasis, and pathology^1,2,3,4. Although CRISPR/Cas9 technology can be used to genetically engineer organoids⁹, it is limited by the need for extensive screening and selection based on sequence analysi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Thermo Fisher Scientific	11965092	Base medium for 293T cells
DMEM/F12+	Thermo Fisher Scientific	12634010	Base medium for organoid culture medium and organoid digestion buffer
OPTI-MEM	Thermo Fisher Scientific	11058021	Virus plasmids transfection medium
Fetal Bovine Serum	Corning	35-011-CV	Component of virus collection medium and 293T medium
Pen/Strep	Thermo Fisher Scientific	15140122	Component of organoid culture medium and crypt dissociation buffer
PBS (without Ca2+, Mg2+)	Thermo Fisher Scientific	10010049	A wash buffer and component of crypt dissociation buffer
Mem-NEAA	Thermo Fisher Scientific	11140050	Component of organoid culture medium
GlutamaxII	Thermo Fisher Scientific	35050061	Component of organoid culture medium
HEPES	Thermo Fisher Scientific	15630080	Component of organoid culture medium
EGF	Millipore Sigma	E9644	Component of organoid culture medium
Noggin	Peprotech	250-38 B	Component of organoid culture medium
R-spondin	R&D	7150-RS-025/CF	Component of organoid culture medium
Human recombinant insulin	Millipore Sigma	I9278-5ml	Component of organoid culture medium
Nicotinamide	Millipore Sigma	N3376-100G	Component of Transduction medium
Wnt3A	R&D	5036-WN-010	Component of Transduction medium
Y27632	Millipore Sigma	Y0503-1MG	Component of Transduction medium
0.5 M EDTA	Thermo Fisher Scientific	15575020	Component of Crypts dissociation buffer
DTT (dithiothreitol)	Thermo Fisher Scientific	R0861	Component of Crypts dissociation buffer
Dispase I	Millipore Sigma	D4818-2MG	Component of organoid digestion buffer
DNase I	Millipore Sigma	11284932001	Component of organoid digestion buffer
matrigel(Growth factor reduced)	Corning	356231	Used as a matrix to embed organoids
Opti-MEM	Thermo Fisher Scientific	31985070	Medium for transfection in viral production
ViralMag R/L	Oz Biosiences	RL40200	Magnetic particles of viral transduction
Magnetic plate	Oz Biosiences	MF10000	Magnetic plate to facilitate viral transduction
Lipofectamine 2000	Thermo Fisher Scientific	11668019	Transfection agent in viral production
Poly-D-Lysine	Millipore Sigma	A-003-E	Coating for plates before seeding 293T cells
4% Formaldehyde Solution	Boster	AR1068	Solution to fix organoids
O.C.T embedding compound	Thermo Fisher Scientific	4583S	For embedding of the the organoids
5 mL Falcon polystyrene tubes	Corning	352054
50 mL Falcon Tubes	Sarstedt	62.547.100
Orbitron rotator II Rocker Shaker	Boekel Scientific	260250
Olympus Inverted microscop CK30	Olympus	CK30	for scanning and counting crypts
Zeiss Axiovert 200 inverted fluorescence	Nikon	Axiovert 200	for viewing fluorescence in the crypts
Amicon Ultra-15 Centrifugal Filter unit with Ultracel-100 membrane	Milipore Sigma	UFC910024	For concentrating viruses
pluriStrainer 20 µm (Cell Strainer)	pluriSelect	SKU 43-50020	For preparing organoid fragments
Falcon Cell Strainer	Fisher Scientific	352340	For preparing cyrpts of similar size after crypt isolation
Greiner CELLSTAR multiwell culture plates 48 wells (TC treated with lid)	Millipore Sigma	M8937-100EA	ForD2:D37+D16:D37g organoid fragments
Animal strain: C57BL/6J	Jackson Lab	#000664	For organoid culture