High efficiency, high throughput generation of genetically modified mammals by electroporation

A mammalian and genetic modification technology, applied in the field of high-efficiency and high-throughput generation of genetically modified mammals by electroporation, which can solve problems such as unsuccessful germline transmission

Inactive Publication Date: 2017-08-01
JACKSON LAB THE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In a follow-up experiment using a high-salt medium designed to increase the total energy delivered during the electroporation pulse, the authors again report preliminary data suggesting that germline transmission was unsuccessful

Method used

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  • High efficiency, high throughput generation of genetically modified mammals by electroporation
  • High efficiency, high throughput generation of genetically modified mammals by electroporation
  • High efficiency, high throughput generation of genetically modified mammals by electroporation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0218] Example 1 Electroporation of mouse zygotes and putative zygotes with reporter-encoding polynucleotides

[0219] This experiment describes the results of electroporation of mouse zygotes or putative zygotes ("zygotes" for short) with reporter-gene encoding plasmids.

[0220]The pMAXGFP vector (Lonza, USA) carrying the CMV promoter and SV40 polyadenylation signal supporting ubiquitous expression was chosen for this experiment. B6D2F2 mouse embryos were collected and treated in acidic Tyrode solution (AT) (P / N T1788, SigmaAldrich) for 5 s, washed twice in KOSMaa / BSA (P / N Zeks-050, Zenith Biotech), and placed in 25 μL of Opti-MEM (P / N 31985, Life Technologies). Embryos in 25 μL were then mixed with an equal volume (25 μL) of 80 ng / μL of pMAXGFP in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5) to achieve a final DNA concentration of approximately 40 ng / μL, and the mixture was Load into a 1-mm shock cuvette and perform electroporation using the following settings: 30 V, pulse...

Embodiment 2

[0224] Example 2 Electroporation of mouse zygotes and putative zygotes with CRISPR / Cas system

[0225] This experiment demonstrates the use of the methods of the present invention to deliver the CRISPR / Cas system to mouse zygotes or putative zygotes ("zygotes" for short) for CRISPR / Cas-mediated targeted gene disruption.

[0226] To this end, Tet1 exon 4 and Tet2 exon 3 were selected as targets using the aforementioned guide RNAs (Wang et al., 2013, incorporated herein by reference). A convenience of the Tet1 and Tet2 systems is that the Sac1 (Tet1) or EcoRV (Tet2) restriction sites overlap with the PAM (protospacer adjacent motif) proximal sequence. Therefore, restriction fragment length polymorphism (RFLP) analysis can be used to detect mutant alleles using PCR products amplified from embryos containing the target site (Wang et al., 2013).

[0227] In Experiment 6, mouse B6D2F2 zygotes were first treated with AT for 10 s, mixed with 40 / 20 ng / μL or 100 / 50 ng / μL of Cas9 mRNA / T...

Embodiment 3

[0235] The development of the mouse zygote of embodiment 3 electroporation

[0236] The in vitro culture and analysis of zygotes is an important method based on which a large number of parameters related to gene editing technologies can be tested and analyzed with a fast turnaround time and preferably low operating costs. However, when electroporated zygotes were cultured using conventional in vitro culture systems, subsequent embryonic development appeared to be arrested or aborted. Typically, after electroporation of Cas9 mRNA / sgRNA at different concentration ranges (e.g., 50 / 25 ng / μL to 1000 / 500 ng / μL), at the end of the 3.5-day culture period, embryos progressed to different developmental stages, including 1-cell, Those at the 2-cell or 4-cell stage, the morula stage and occasionally the blastocyst stage (Experiment 8). In contrast, control embryos that were not electroporated reached the blastocyst stage at the end of the same time period, whether or not the control embr...

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Abstract

The invention described herein provides high throughput methods and reagents for generating transgenic animals (e.g., mammals) through introducing materials into gametes or preimplantation stage (e.g., one-cell embryo or zygotes) via electroporation, leading to genetically inheritable modification to the genome of the animal.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of the filing date of US Provisional Application No. 62 / 056,687, filed September 29, 2014, which is hereby incorporated by reference in its entirety, under 35 U.S.C. § 119(e). Background technique [0003] Delivery of biological or genetic material into mammalian zygotes is often the first step in modifying mammalian genomes, such as the creation of genetically modified animal models. This is traditionally and currently accomplished by microinjecting the desired biological / genetic material into the zygote using a glass needle. [0004] Direct microinjection of DNA has been used as early as the late 1980s to introduce the herpes simplex virus (HSV) TK gene into cultured mammalian cells (Capecchi, Cell, 22:479-488, November 1980). However, even when small pBR 322 / TK DNA was co-injected with SV40 DNA, ie, 15 transformants per 1,000 cells, the transformation rate was extremely low. Furt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/873C12N15/90C12N15/877
CPCC12N15/8509C12N15/902C12N15/873A01K67/00C12N15/00A01K67/027A61K35/12C12N5/0603C12N5/10C12N15/09C12N2510/00
Inventor 覃文宁S·L·迪昂P·M·库特奈王皓毅
Owner JACKSON LAB THE
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