Genotyping is generally performed by PCR + sequencing methods. See the following figure for details:
图10. A.PCR products sequencing chromatogram of wild-type mouse.B. PCR products sequencing chromatogram of F0 generation positive mice.After the action of CRISPR/Cas9, repair via non-homologous end joining (NHEJ) may also occur along with the repair via homologous recombination (HR).Therefore, noise peaks may occur near the point of digestion.C. PCR products sequencing chromatogram of F1 generation positive mice.In the genome of F1 generation positive mice, one copy is a wild type and the other copy is a mutant. The sequencing of the region in the vicinity of the Cas9 action site shows two peaks near the site of mutation (two peak shapes are produced for the wild type and mutant genotypes, one for each genotype). C' and C" are the results of monoclonal sequencing of PCR products in Figure C. C' is a wild type and C‘' is a mutant. The comparison of peak shapes reveals that the mutation in C" is GTT, compared to the CCT mutation at the target mutation site in C', while the peak shapes of C' and C" are consistent with that of C.
For knockout mice obtained by random repair via NHEJ, their genotypes can generally be determined by PCR+ sequencing.See the following figure for details:
Figure 9. A. PCR products sequencing chromatogram of wild-type mouse. B. PCR products sequencing chromatogram of F0 generation positive mice. After the action of CRISPR/Cas9, a number of different genotypes can be generated due to repair by NHEJ, thus producing noise peaks near the digestion point of Cas9. C. PCR products sequencing chromatogram of F1 generation positive mice. In the genome of F1 generation positive mice, one copy is a wild type and the other copy is a mutant. The sequencing of the region in the vicinity of the Cas9 action site shows two peaks (two peak shapes are produced for the wild type and mutant genotypes, one for each genotype). C' and C" are the results of monoclonal sequencing of PCR products in Figure C. C' is a wild type and C' is a mutant. The comparison of peak shapes reveals that C" loses two bases compared to C'. The peak shapes of C' and C" are consistent with that of C.
Refer to the following figure for primer design.
Figure 8. A. To identify whether the Neo gene in Flox mice has been removed; B. To identify whether the target gene in specific tissues has been knocked out.
Refer to the following figure for primer design.
Figure 7. A. Three-primers genotyping protocol based on ESC targeting; B. Two-primers genotyping protocol based on ESC targeting; C. genotyping protocol based on CRISPR/Cas9.
During the PCR of genotyping, genomic DNA is usually extracted from mouse tail tissues by crude extraction. The genomic DNA obtained by this extraction method contains a high amount of impurities, which may affect the efficiency of subsequent PCR. Consider improving the extraction method to ensure the quality of extracted genomic DNA. In the PCR system, the concentration of genomic DNA can also affect the efficiency of PCR. Too much or too little genomic DNA can lead to the failure of PCR amplification. Adjusting the PCR system might be a solution. If PCR shows non-specific bands, consider increasing the annealing temperature to enhance the specific binding of the primers.
The basic principle for the genotyping of genetically-engineered mice by PCR is to utilize the sequence difference between the genome of genetically-engineered mice and that of wild-type mice. Using the mouse genomic DNA as a template for PCR, different genotypes of genetically-engineered mice can be determined with gel electrophoresis based on the band size of specific products generated by the genotypes.
Studies have shown that Cre expression in mouse models may also exert toxic side effects, such as infertility and decreased viability. This is especially true for the Cre homozygous mice of certain strains. An excessively high Cre activity may induce the recombination at unknown loxP sites in the genome, thus triggering knockout or translocation and subsequently affecting mouse survival. In addition, in the Cre transgenic strains obtained by microinjection, the insertion of Cre gene may interfere with the functions of certain important endogenous genes.
In most of the strains studied so far, Cre expression is not affected by the source of Cre inheritance. However, for some strains, such as the EIIa-Cre strain, the recombination efficiency of Cre is higher when the Cre gene is derived from maternal inheritance.
Not in all strains. Some Cre-expressing strains may show large variation in terms of Cre expression even in littermates. In addition, caution should be taken for the strains with a mosaic pattern of Cre expression. You may see large experimental variations when using these mice, and hence it is difficult to repeat experimental results. In order to obtain valuable experimental conclusions, it may be necessary to analyze the phenotypes of more mice.
Since Cre is driven by a brain-specific promoter, does that mean Cre is only expressed in the brain? This is not true unfortunately. Studies have shown Cre activity in other tissues of most strains. For example, in addition to the brain, Cre is also expressed in the kidneys and thymus of Emx1-Cre mice.