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Embryo lysate and application thereof

A lysate and embryo technology, applied in the field of cell lysis, can solve the problems of high cost, time-consuming cutting activity detection procedure, etc., and achieve the effect of improving controllability

Pending Publication Date: 2022-03-01
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on this, it is necessary to provide an embryo lysate capable of lysing fertilized eggs of rodents and embryos developed in vitro for several days and its application in view of the time-consuming and high-cost problems of the traditional sgRNA cleavage activity detection procedure

Method used

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  • Embryo lysate and application thereof
  • Embryo lysate and application thereof
  • Embryo lysate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The formula and preparation method of embodiment 1 embryo lysate

[0059] Embryo lysate is composed of two kinds of proportion A, B solution components. Based on a volume of 100ml, solution A contains 5mmol potassium chloride, 1mmol ethylenediaminetetraacetic acid-hydrogen chloride (pH 8.5), 0.25mmol magnesium chloride, 10mg gelatin, 450μl ethylphenyl polyethylene glycol and 450μl Tween 20, and the remaining The amount is enzyme-free water. Under the condition that the temperature is 20-25°C and the humidity is less than 60%, the corresponding solid raw materials or liquid raw materials are accurately weighed with a precision balance of 1 / 100,000 or 1 / 100,000. In a 200ml Erlenmeyer flask, and then in a 100ml constant volume bottle, the embryo lysate A solution was obtained after aliquoting. Solution B was proteinase K at 20 mg / ml and pronase at 1 mg / ml. Weigh the powder corresponding to solution B, add water to volume, and obtain solution B. Solution A and solution ...

Embodiment 2

[0060] Embodiment 2 uses the effect comparison of the embryo lysate of embodiment 1 and other cell lysates

[0061]Embryo samples were taken from equally microinjected embryos with normal development (mouse embryos, taken out at 0.5 days after fertilization, microinjected Cas9 protein and sgRNA in vitro, and then lysed at 1.5 days after development), with commercially purchased cell lysis reagents The box is used as an experimental control group to illustrate the effect of using the embryo lysate in Example 1. The final embryo lysate was prepared according to the embryo lysate formula in Example 1, and added to a PCR tube containing 20 embryos, which was sample E0. Control group 1 used commercially purchased cell lysate (GBdirect cell direct PCR kit, Suzhou Shenzhou Gene Co., Ltd., involving commercial secrets, and the specific components were not reflected in the instructions), and according to the instructions of the kit, A 100 microliters of reagents, 10 microliters of rea...

Embodiment 3

[0073] In this example, the lysate was configured according to the preparation methods of the four samples in Example 2, with the difference that 20 embryos in each group were changed to 10 embryos, and samples E0', E1', E2', and E3' were prepared. Perform PCR according to the same conditions and primers. figure 2 It is the graph of the genomic DNA electrophoresis results cleaved by different lysates in Example 3. The order of loading is 2000bp makerDL, the PCR product of the lysed sample E0', the PCR product of the lysed sample E1', the PCR product of the lysed sample E2', and the lysed sample E3 'PCR product, no-template control. The results showed that when the content of the target gene was very small, only the formula lysate in Example 1 had higher sensitivity and could detect the microgenome produced after the embryo was lysed, and the rest could not be detected. It shows that the lysing efficiency of the embryo lysate of the present invention is higher, and the lysing...

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Abstract

The invention discloses an embryo lysis solution, which comprises a component A and a component B. The component A comprises sylvite, ethylenediamine tetraacetic acid, magnesium salt, gelatin, ethyl phenyl polyethylene glycol and Tween 20 according to a concentration ratio of (4-6) mmol: (0.2-2) mmol: (0.05-0.5) mmol: (9-11) mg: (400-500) [mu] l: (400-500) [mu] l, and the component B comprises protease K and streptavidin according to a mass ratio of (15-25): 1. The invention also discloses application of the embryo lysis solution in lysis of fertilized eggs or early embryos of decayed animals. The invention also discloses a method for detecting the activity of the gene editing reagent for the non-diagnostic and non-therapeutic purposes. The method comprises the following steps: microinjecting the gene editing reagent into a fertilized egg or an early embryo of a decayed animal for gene editing and culture; splitting the cultured fertilized eggs or early embryos by using an embryo splitting solution; and carrying out gene detection on the cracked product. The invention also discloses a detection kit for the activity of the gene editing reagent.

Description

technical field [0001] The invention relates to the technical field of cell lysis, in particular to an embryo lysate and its application. Background technique [0002] It is well known that CRISPR-Cas9 gene editing technology is a hot topic in the field of life science recently, and the rise of this technology has made the field of gene editing develop by leaps and bounds. The researchers designed a single guide RNA (sgRNA) based on the structural characteristics of the TracrRNA and CrRNA complex. The sgRNA has the function of the TracrRNA:CrRNA complex, which can be recognized by the Cas9 protein and guide the Cas9 protein to bind to the target site, thereby Play the function of fixed-point editing. The key to this technology is the design of guide RNA to achieve the modification of specific target DNA sequences such as knockout, insertion and site-directed mutation. The main factors affecting the efficiency of CRISPR-Cas9 technology include the following points: firstly,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/89C12N9/22
CPCC12Q1/6806C12N15/89C12N9/22C12Q2527/125C12Q2521/537
Inventor 李琴邵壮潘讴东茅伟杨兴林杨佳丽
Owner OBIO TECH SHANGHAI CORP LTD
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