Electrotransformer efficiently transfects mRNA into mouse fertilized eggs to form stable mutants

Summary

With the completion of sequencing of mouse genome sequences, many studies have centered on the biological processes involved in functional genes, particularly in the field of developmental and disease research. So stable genetically modified model animals are important for elucidating the role of genes. However, traditional methods, including homologous recombination in embryonic stem cells or construction of mouse chimeras, are time consuming and labor intensive. However, the new genome editing method significantly optimizes this process. The CRISPR/Cas 9 system is the easiest way to construct a modified genome in an efficient genome editing approach.

Although the CRISPR/Cas9 system simplifies the process of forming mutants, the microinjection of DNAs/RNAs into fertilized eggs requires special techniques, and it takes a lot of time to obtain a large number of mutants (injecting hundreds of fertilized eggs). So, to simplify this process, we tried to introduce RNA into the fertilized egg using an electric converter.

Electrotransfers have also been used in mouse fertilized eggs, but the process involves removing the zona pellucida or thinning it with acid, which is very toxic to cells and has only short RNAs (less than 1 kb). Was imported.

Recently, a rat mutant was successfully constructed by transfecting Cas9 mRNA and gRNA into rat fertilized eggs by keeping the zona pellucida intact. However, genome editing is inefficient (less than 9%) and requires a large amount of Cas9 (1000-2000 ng/ul).

Method and result
To optimize the electrical cycling conditions, we used a BEX CUY21 EDIT II electro-converter, as well as a platinum block electrode, 1 mm spacing (Fig. 1abc ). Place under a stereo microscope and connect to an electro-converter. Each time 5 ul of RNA is added , 40-50 embryos can be electroporated .
In order to improve the efficiency of genome editing, the mCherry mRNA and the limbless gene Fgf10 were used to optimize the parameters, and the ratio of the survival of the fertilized eggs to the two-cell stage was 94-95% , which was much higher than the microinjection method 34-35% .
When we used 400 ng/ul of Cas9 mRNA and 200 ng/ul gRNA for transfection, 87% of the embryos showed deletion of the anterior and posterior legs, which is Figure 2c ( type I ); 10% of the mutants are limb defects ( type II ) ; only 3% of the embryos showed normal (type III). The type I mutant was sequenced and it was found that all of the Fgf10 gene alleles were interrupted.
Subsequently, we examined the efficiency of gene editing for different concentrations of Cas9 mRNA and gRNA . 200ng / ul Cas9 mRNA and 50ng / ul of gRNA, 73% of embryos Amelia; 100ng / ul Cas9 mRNA and 50ng / ul of gRNA, 32% showing mutilated (FIG. 2bc); 50ng / ul Cas9 mRNA and 25ng / ul The gRNA , the embryo is mostly normal.


The right box is the electroporator CUY21 EDIT II (BEX Co.Ltd), which generates electric pulsed, and The left is a stereoscopic microscope for embryo manipulation. Two electrodes were placed on the microscope stage and connected to the electroporator.


Figure 2. CRISPR/Cas9 mediated genome editing by electroporation. (b) Examples of embryos from the three classes that exhibit different limb phenotypes: no limbs (type I), limb defects (type II, left hind limb deficiency, right: truncated fore- And hind-limb) , and normal (type III). (c)Graph summarizing the effects of Cas9 and gRNA electro-poration on limb development. The concentrations of RNAs used in each experiment are shown at left. The numbers in each column are The number of embryos exhibiting the phe-notype in each category.


Figure 3. The single-stranded oligodeoxynucleotide (ssODN)-induced generation of HDR-mediated instertions. (b) Representative embryo electroporated with Cas9 mRNA, gRNA, and ssODN.The mCherry florescene completely disappeared in the electroporated embryos, while control (no electroporation Embryos displayed florescent signals.

To determine the efficiency of genome editing, mCherry was transfected into 11 mice and all 11 mice were found to be free of red fluorescence (Fig. 3b ). This result suggests that the method of electroporation can also be used to efficiently generate HDR- mediated knockout mice.

Next, we reveal whether this mutation will be inherited in the next generation. Electroporation 200ng / ul Cas9 mRNA and 100ng / ul of gRNA into the egg cell, gene targeting mCherry. Twenty- five F0 mice were observed , and no red fluorescence was expressed. Moreover, all F1 mice did not have red fluorescence and the mCherry gene was interrupted. This indicates that the gene editing mutant can be stably inherited.

discuss
In summary, we established an electrical rotation condition to ensure efficient and high survival rates for CRISPR/Cas9- based genome editing. Because electroporation does not require microinjection techniques, and 40-50 embryos can be processed simultaneously , this method can obtain a large number of stable genetic mutant mice faster and easier than previous methods. Using this method, it can lay the foundation for the analysis of F0 phenotype and screening for important genes related to development.

Podiatry Water Distiller

Best Water Distiller,Distilled Water Machine,Podiatry Water Distiller,Portable Water Distiller

ZHEJIANG FOMOS MEDICAL TECHNOLOGY CO.,LTD. , https://www.ifomos.com