Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

Tanimoto, Yoko; Mikami, Natsuki; Ishida, Miyuki; Iki, Natsumi; Kato, Kuniki; Sugiyama, Fumihiro; Takahashi, Satoru; Mizuno, Seiya

doi:10.3791/64161

Cited by 5 publications

(3 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cas9NmC is a fusion protein of Cas9 and part of mouse Cdt1 that we previously reported 26 . The CRISPR–Cas9 ribonucleoprotein complex and donor DNA were microinjected into zygotes of C57BL/6J mice, in accordance with our previous report 27 . Subsequently, microinjected zygotes were transferred into the oviducts of pseudopregnant ICR female and newborns were obtained.…”

Section: Methodsmentioning

confidence: 99%

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

Okamura,

Mizuno,

Matsumoto

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Pronuclear microinjection is the most popular method for producing transgenic (Tg) animals. Because the production efficiency is typically less than 20%, phenotypic characterization of Tg animals is generally performed on the next generation (F1) onwards. However, apart from in rodents, in many animal species with long generation times, it is desirable to perform phenotyping in the founder (F0) generation. In this study, we attempted to optimize a method of Tg mouse production to achieve higher Tg production efficiency using piggyBac transposon systems and established optimal conditions under which almost all individuals in the F0 generation were Tg. We also succeeded in generating bacterial artificial chromosome Tg mice with efficiency of approximately 70%. By combining this method with genome editing technology, we established a new strategy to perform phenotyping of mice with tissue-specific knockout using the F0 generation. Taking the obtained findings together, by using this method, experimental research using Tg animals can be carried out more efficiently.

show abstract

Section: Methodsmentioning

confidence: 99%

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

Okamura,

Mizuno,

Matsumoto

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…An average proportion of about 8% of correctly floxed mice were obtained from floxing 10 different chromosomal regions. Again, the authors noted that the method “is costly and time-consuming when preparing and maintaining an experimental environment in terms of both hardware and software” [ 12 ] despite using the much cheaper dsDNA plasmid template.…”

Section: Introductionmentioning

confidence: 99%

“…environment in terms of both hardware and software" [12] despite using the much cheaper dsDNA plasmid template.…”

Section: Introductionmentioning

confidence: 99%

Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice

McBeath

Fujiwara²,

Hofmann

2023

IJMS

View full text Add to dashboard Cite

Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a small artificial intron to conditionally knockout a gene of interest in mice. However, many other labs are having difficulty getting the technique to work. The key problem appears to be either a failure in achieving correct splicing after the introduction of the artificial intron into the gene or, just as crucial, insufficient functional knock-out (KO) of the gene’s protein after Cre-induced removal of the intron’s branchpoint. Presented here is a guide on how to choose an appropriate exon and where to place the recombinase-regulated artificial intron (rAI) in that exon to prevent disrupting normal gene splicing while maximizing mRNA degradation after recombinase treatment. The reasoning behind each step in the guide is also discussed. Following these recommendations should increase the success rate of this easy, new, and alternative technique for producing tissue-specific KO mice.

show abstract

Strategies for generation of mice via CRISPR/HDR-mediated knock-in

Mathew

2023

Mol Biol Rep

View full text Add to dashboard Cite

CRISPR/Cas9 framework is generally used to generate genetically modified mouse models. The CRISPR (clustered regularly interspaced short palindromic repeat) gene editing technique, can efficiently generate knockouts using the non-homologous end-joining (NHEJ) repair pathway. Small knock-ins also work precisely using a repair template with help of homology-directed-repair (HDR) mechanism. However, when the fragment size is larger than 4-5 kb, the knock-in tends to be error prone and the efficiency decreases. Certain types of modifications, in particular insertions of very large DNA fragments(10 100kb) or entire gene replacements, are still difficult. The HDR process needs further streamlining and improvement. Here in this review, we describe methods to enhance the efficiency of the knock-in through checking each step from the guide design to the microinjection and choice of the oocyte donors. This helps in understanding the parameters that can be modified to get improved knock-in efficiency via CRISPR targeting.

show abstract

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

Cited by 5 publications

References 8 publications

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice

Strategies for generation of mice via CRISPR/HDR-mediated knock-in

Contact Info

Product

Resources

About