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Humanized M-CSF mice

A humanized and mouse technology, applied in the field of humanized M-CSF mice, can solve problems such as poor myeloid differentiation

Active Publication Date: 2014-01-29
REGENERON PHARM INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although significant progress has been made in generating immunocompromised mice with human T lymphocytes, B lymphocytes, NK cells, and dendritic cells (DCs), several challenges remain in the field, one of which is Poor myeloid differentiation in humanized mice

Method used

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  • Humanized M-CSF mice
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  • Humanized M-CSF mice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143] Example 1: Cell Preparations, Analytical Methods, and Assays

[0144] CD34 +Cell isolation and transplantation. Human fetal liver samples were obtained from the Human Fetal Liver Tissue Collection at Albert Einstein College of Medicine, Bronx, NY and from Advance Biosciences Resources, Inc., Alameda, CA. All experiments involving human tissue were performed under the approval of the Yale Human Investigations Committee.

[0145] To isolate human CD34 + For cells, the fetal liver tissue was rinsed once with PBS, cut into small pieces, and treated with collagenase D (100 ng / mL) at 37° C. for 45 minutes. Single cell suspensions were prepared and mononuclear cells were isolated using density gradient centrifugation (lymphocyte separation medium, MP biomedicals). After treatment of cells with anti-human CD34 beads, followed by MACS TM technology (Miltenyi Biotech) to isolate CD34 + cell.

[0146] For transplantation, newborn puppies (day 1 of birth) were sublethally ir...

Embodiment 2

[0160] Example 2: Genetically Modified Mice for Grafting

[0161] Human M-CSF knock-in strategy. use The technical construction is used to use human M-CSF nucleic acid sequence in a single targeting step ( Allele identification number 5093) to replace the targeting construct of the mouse M-CSF nucleic acid sequence, as previously described (Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nat.Biotechnol .21:652-659). Mouse and human M-CSF DNA were obtained from bacterial artificial chromosome (BAC) RPCI-23, clone 373B18 and from BAC RPCI-11, clone 101M23, respectively. Briefly, linearized targeting constructs generated by gap repair cloning were electroporated into RAG2 + / - gamma c - / -In mouse embryonic stem (ES) cells generated from a commercial V17ES cell line (BALB / c x129F1), the linearized targeting construct contained 633nt downstream of non-coding exon 9 extending from exon 2 The mouse M-CS...

Embodiment 3

[0168] Example 3: Differentiation of Human Monocytes / Macrophages in Humanized M-CSF Mice

[0169] To assess the impact of M-CSF humanization, sublethally irradiated neonatal Rag2 - / - gamma c - / - M-CSF m / m ,Rag2 - / - gamma c - / - M-CSF h / m and Rag2 - / - gamma c - / - M-CSF h / h Intrahepatic (i.h) transplantation of pups approximately 2x10 5 purified human fetal liver CD34 + cell. Recipients were then bled at 8 weeks post-transplantation to confirm donor-derived cells (based on human CD45 expression). Twelve weeks after transplantation, recipients were sacrificed and their BM, SP and PB were harvested. Analysis revealed with M-CSF m / m Mice compared to M-CSF h / m and M-CSF h / h CD14 in BM, SP and PB of both mice + CD33 + Increased relative and absolute frequencies of monocyte / macrophage lineage cells ( Figure 3A -C). Although M-CSF h / m Mice exhibit elevated CD14 + CD33 + cell frequency, but CD14 + CD33 + The greatest frequency of cells exists in M-CSF h / h in mice...

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Abstract

Genetically modified mice comprising a nucleic acid sequence encoding a human M-CSF protein are provided. Also provided are genetically modified mice comprising a nucleic acid sequence encoding a human M-CSF protein that have been engrafted with human cells such as human hematopoietic cells, and methods for making such engrafted mice. These mice find use in a number of applications, such as in modeling human immune disease and pathogen infection; in in vivo screens for agents that modulate hematopoietic cell development and / or activity, e.g. in a healthy or a diseased state; in in vivo screens for agents that are toxic to hematopoietic cells; in in vivo screens for agents that prevent against, mitigate, or reverse the toxic effects of toxic agents on hematopoietic cells; in in vivo screens of human hematopoietic cells from an individual to predict the responsiveness of an individual to a disease therapy, etc.

Description

field of invention [0001] The present invention relates to genetically modified mice comprising the gene encoding human M-CSF protein, and mice comprising additional modifications that support engraftment of human hematopoietic cells. Background of the invention [0002] The development of animal models to study human disease has significantly advanced the understanding of the mechanisms underlying several diseases, including cancer. To date, animal models, especially mice, have proven to be excellent candidates for evaluating the efficiency and efficacy of drugs and therapy options. While the use of these alternative models to study human biology and disease can be largely justified (due to ethical and technical constraints on conducting experimental therapies in humans), studies have highlighted the limitations of extrapolating data from mice to humans. Potential limitations (Mestas J, Hughes CC. (2004) Of mice and not men: differences between mouse and human immunology. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12Q1/68C12Q1/6897
CPCA01K2227/105A01K67/027A01K67/0271A01K67/0278C07K14/53C12N15/8509C12N2800/107A01K2267/0337A01K2217/072A01K2217/15A01K2207/15A01K2207/12A01K2267/03A61K49/0008G01N33/5088G01N2500/10C12N2015/8536
Inventor A·J·墨菲S·史蒂文斯C·雷西南E·艾农M·曼茨R·弗拉维尔G·D·雅克波罗斯
Owner REGENERON PHARM INC
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