A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Summary

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

Abstract

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans.  There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.

Introduction

Humanized mice (hu-mice) have transformed the study of many aspects of human health and disease including hematopoiesis, immunity, cancer, autoimmune disease, and infectious disease^{1,2,3,4,5,6,7,8,9}. These hu-mice have the distinct advantage over other mouse models in that they have a functional human immune system and can be infected with human specific pathogens. Neve....

Protocol

All methods described here were conducted in accordance with Institutional Animal Care and Research Committee (IACUC)-approved protocols at the University of Nebraska-Lincoln (UNL). The IACUC at UNL has approved two protocols related to generating and using hu-BLT mice, including double hu-mice. Additionally, the Scientific Research Oversight Committee (SROC) at UNL has also approved the use of human embryonic stem cells and fetal tissues, which are procured from the Advanced Bioscience Resources for humanized mice studi.......

Discussion

The protocol described here is for the creation of double hu-BLT mice that feature both a functional human immune system and a stable human-like gut microbiome. This protocol can be adapted to other humanized or non-humanized mice models without the need for GF animals and gnotobiotic facilities. While the methods described here are relatively simple, there are several critical details that are important for the successful creation of double hu-BLT mice. NSG mice are extremely immunodeficient and preventing infections is.......

Materials

Name	Company	Catalog Number	Comments
Animal Feeding Needles 18G	Cadence Science	9928B
Clidox-s Activator	Pharmacal Research Laboratories	95120F
Clidox-s Base	Pharmacal Research Laboratories	96125F
DGM 108 cage rack	Techniplast
Flat Brown Grocery Bag 3-5/8"D x 6"W x 11-1/16"L	Grainger	12R063
FMT Upper Delivery Microbiota Preparations	OpenBiome	FMP30
Grape Kool-Aid	Kraft Foods Inc.
hCD19-PE/Cy5	Biolegend	302209
hCD3-PE	Biolegend	300408
hCD4-Alexa 700	Biolegend	300526
hCD45-FITC	Biolegend	304006
hCD8-APC/Cy7	Biolegend	301016
Lactate Buffered Ringer's Solution	Boston BioProducts Inc	PY-906-500
mCD45-APC	Biolegend	103111
Microvette 100 K3E	Microvette	20.1278.100
Neosporin First Aid Antibiotic/Pain Relieving Ointment	Neosporin
NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ)	The Jackson Laboratory	005557
PrecisionGlide 25 G Needle	BD	305127
RS200 X-ray irradiator	RAD Source Technologies
Sealsafe Plus GM500 microisolator cages	Techniplast
Sterile Non-woven Gauze	Fisherbrand	22-028-558
Teklad global 16% protein irradiated mouse chow	Teklad	2916