Unlock instant, AI-driven research and patent intelligence for your innovation.

Human liver chimeric non-human animal with deficient p450 oxidoreductase and methods of using same

A non-human animal, non-human technology, applied in oxidoreductase, compound screening/testing, biochemical equipment and methods, etc., can solve problems such as endangering human life and driving the cost of drug development

Active Publication Date: 2019-04-26
BAYLOR COLLEGE OF MEDICINE
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lack of experimental animal models that accurately predict human xenobiotic metabolism is a major limitation that endangers human life and drives up drug development costs

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Human liver chimeric non-human animal with deficient p450 oxidoreductase and methods of using same
  • Human liver chimeric non-human animal with deficient p450 oxidoreductase and methods of using same
  • Human liver chimeric non-human animal with deficient p450 oxidoreductase and methods of using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1: Por-add on both sides of the sequence loxP loci for the generation of mouse strains

[0124] Por First knockout targeting vector was purchased from National Institutes of Health (NIH) Knock-OutMouse Program (KOMP) ( Figure 4A ). The vector was linearized with the AsisI restriction enzyme and the DNA was electroporated into Jm8A3 mouse embryonic stem cells (ESCs) by Baylor College of Medicine's Mouse Embryonic Stem Cell Core (Pettitt, S.J. et al. "Agouti C57BL / 6N embryonic stem cells for mouse genetic resources.” Nat Methods 6, 493-495 (2009)). Integrating clones were selected using neomycin resistance. DNA from ESC clones was digested with NSiI restriction enzyme following the manufacturer's instructions and screened for site-specific integration by Southern blotting using the DIG non-isotopic detection system (Roche Applied Biosciences) ( Figure 17 All blots in ). Use the following primer sets to synthesize 5' and 3' probes of 500 bp size that ...

Embodiment 2

[0127] Example 2: X-Gal staining

[0128] Embryos and fresh liver sections were fixed in 4% PFA at 4°C for 1 h and washed 2x for 30 min in X-Gal wash buffer (PBS 1x, containing 0.02% Igepal and 0.01% deoxycholate), followed by washing with X-Gal. -Gal staining solution (PBS 1x, containing 5mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , 0.02% Igepal, 0.01% deoxycholate, 2 mM MgCl 2 , 5 mMEGTA and 1 mg / ml fresh X-Gal) were incubated overnight. Samples were post-fixed overnight at 4°C in 4% PFA.

Embodiment 3

[0129] Embodiment 3: PIRF ( Por c- / c - / Il2rg- / - / Rag2- / - / Fah- / - ) generation of mouse strains

[0130] Targeting was selected using two different online tools (crispr.mit.edu and CORMID) Rag2 , Il2-rg or Fah 6 gRNA sequences of key exons of the gene ( Figure 1A, Figures 6 and 7) (Cradick, T.J., Qiu, P., Lee, C.M., Fine, E.J. & Bao, G. “COSMID: A Web-based Tool for Identifying and Validating CRISPR / Cas Off-target Sites.” Molecular therapy. Nucleic acids 3, e214(2014)). Complementary oligonucleotides were annealed and ligated into DR274 vector (Addgene plasmid #42250) with the restriction enzymes BsaI (NEB) and T4 DNA ligase (NEB) using standard molecular cloning techniques (Hwang, W.Y. et al. "Efficient genome editing in Zebrafish using a CRISPR-Cas system.” Nat Biotechnol 31, 227-229 (2013)). Using standard molecular cloning techniques, the T7 bacterial promoter sequence was inserted into the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (Addgene plasmid #42230) u...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present disclosure provides a chimeric non-human animal comprising human hepatocytes, methods for preparing the chimeric non-human animal comprising human hepatocytes and methods of utilizing thechimeric non-human animal comprising human hepatocytes to screening and identifying metabolites for any type of drugs, typically small molecule drugs, which might affect human liver functions and anyother bodily function.

Description

[0001] Cross references to related applications [0002] This application claims the benefit of and the benefit of U.S. Provisional Application No. 62 / 355,102, filed June 27, 2016, and U.S. Provisional Application No. 62 / 509,942, filed May 23, 2017. The entire content of each of these applications is incorporated herein by reference in its entirety. [0003] Incorporation of the Sequence Listing as a reference [0004] The contents of the text file named "KARL-001-WO_ST25" created on June 20, 2017 and having a size of 67 KB are hereby incorporated by reference in its entirety. Background of the invention [0005] Only one in ten drugs in development are approved for clinical use. Most fail during clinical trials due to ineffectiveness or toxicity in humans. The lack of experimental animal models that accurately predict xenobiotic metabolism in humans is a major limitation that endangers human lives and drives up drug development costs. Therefore, there is an urgent need to...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027A01K67/02
CPCC12N15/8509A01K67/0271A01K67/0276C12Y106/02004C12N9/0042A01K2207/15A01K2217/075A01K2217/206A01K2227/105C12N2800/107C12N2800/30C12N9/0008C12N9/93G01N33/5088C12N2015/8527C12Y101/01022C12Y603/02003A61K49/0008
Inventor K-D.比西F.P.潘科维奇M.d.l.M.B.迪盖斯
Owner BAYLOR COLLEGE OF MEDICINE