SAGEspeed Project Types

Gene Knockout

Gene Knockout

Due to the fact that CRISPR-Cas9 and ZFN technologies directly enable targeted mutagenesis at the one-cell stage embryo, embryonic stem (ES) cells are not required — making model generation much faster and without background strain limitation. 

Engineered Nuclease Targeting Overview

Zinc-Finger proteins are a predominant class of well-characterized DNA binding proteins. Through the ZFN technology, a library of Zinc-Finger proteins have been coupled with a Fok1 endonuclease and designed to recognize and cleave specific DNA sequences. Zinc-Finger Nucleases (ZFNs) are utilized to generate sequence-specific double-strand breaks that are repaired by error-prone nonhomologous end joining (NHEJ). 

Figure 1. Knockout strategy using Zinc-Finger Nucleases in embryos

Figure 2. Milestone timeline for generating a knockout rat or mouse 

NHEJ is utilized for generating targeted gene knockout. A ZFN pair targeting the desired gene is microinjected into the pronucleus of fertilized one-cell embryos. The knockouts are guaranteed to be germline transmissible. Knockouts are generated by introducing variable genomic insertions or deletions that result in a frameshift within the open reading frame. The frameshifts result in the introduction of premature stop codons. When the premature stop codon occurs before the last exon, the transcript is degraded via the nonsense-mediated decay pathway and little or no protein is expressed. Efficiency for generating knockout founders is 10-20% in resulting litters. 

Resulting founder animals are screened for mutations by developing PCR primers that flank the ZFN targeting site. The exact deletions or insertions are then determined by sequencing. 

MicroInjection SessionsTotal Pups BornTotal KnockoutsLarge DeletionsInframe Mutations
4 27 13 11 1

Table 1. Example mutation types and founder rate for gene knockout projects

Conditional Knockout

Homologous recombination (HR) pathway is utilized for generating integration of LoxP sites surrounding the appropriate exon or genomic region. Microinjection of two sets of ZFNs and donor vectors carrying the LoxP sequence into the pronucleus of fertilized, one-cell embryos, efficiently generates HR-mediated mutants that are guaranteed germline transmissible. While NHEJ-mediated knockouts occur at a founder rate of 10-20%, generating donor-mediated mutants occurs less frequently and often requires an increased number of microinjected embryos and resulting pups to screen. 

Resulting founders are screened for mutations by developing PCR primers that flank the targeting site. Several sets of primers are designed, flanking the integration site, to screen appropriate integration and rule out random insertion of the donor sequence into the genome. Founders are confirmed by sequencing.

Figure 3.Milestone timeline for generating a conditional knockout rat or mouse 

Figure 4. Strategy for inserting LoxP sites into gene

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Gene Mutation-Replacement

Gene Mutation-Replacement

Due to the fact that CRISPR-Cas9 and ZFN technologies directly enable targeted mutagenesis at the one-cell stage embryo, embryonic stem (ES) cells are not required — making model generation much faster and without background strain limitation.

Engineered Nuclease Targeting Overview

Zinc-Finger proteins are a predominant class of well-characterized DNA binding proteins. Through the ZFN technology, a library of Zinc-Finger proteins have been coupled with a Fok1 endonuclease and designed to recognize and cleave specific DNA sequences. Zinc-Finger Nucleases (ZFNs) are utilized to generate sequence-specific double-strand breaks that are repaired by error-prone nonhomologous end joining (NHEJ) or high-fidelity homologous recombination (HR).

Figure 1. Gene replacement strategy using Zinc-Finger Nucleases in embryos

HR pathway is utilized for generating the targeted integration of the desired DNA sequence. Desired point mutations, humanized sequences or reporter tags are synthesized and cloned into a donor vector backbone. Microinjection of ZFNs and the donor vector into the pronucleus of fertilized, one-cell embryos, efficiently generates HR-mediated mutants that are guaranteed germline transmissible. While NHEJ-mediated knockouts occur at a founder rate of 10-20%, generating donor-mediated mutants occurs less frequently and often requires an increased number of microinjected embryos and resulting pups to screen. 

Resulting founders are screened for mutations by developing PCR primers that flank the ZFN targeting site. Several sets of primers are designed, flanking the integration site, to screen appropriate integration and rule out random insertion of the donor into the genome. Founders are confirmed by sequencing.

Figure 2. Milestone timeline for generating a gene replacement mouse or rat 

Figure 3. Strategy for inserting a donor vector into an endogenous mouse or rat gene

MicroInjection SessionsTotal Pups BornNHEJ KnockoutsReporter InsertionInframe Mutations
3 27 2 1 2

Figure 4. Example founder rate for targeted insertion of a reporter tag into an endogenous rat gene

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Targeted Transgenic

Targeted Transgenic

Due to the fact that CRISPR-Cas9 and ZFN technologies directly enable targeted mutagenesis at the one-cell stage embryo, embryonic stem (ES) cells are not required — making model generation much faster and without background strain limitation.

Engineered Nuclease Targeting Overview

Zinc-Finger proteins are a predominant class of well-characterized DNA binding proteins. Through the ZFN technology, a library of Zinc-Finger proteins have been coupled with a Fok1 endonuclease and designed to recognize and cleave specific DNA sequences. Zinc-Finger Nucleases (ZFNs) are utilized to generate sequence-specific double-strand breaks that are repaired by error-prone nonhomologous end joining (NHEJ) or high-fidelity homologous recombination (HR).

Figure 1. Targeted insertion strategy into Rosa26 locus using Zinc-Finger Nucleases in embryos

Figure 2. Milestone timeline for generating a targeted transgenic mouse or rat

Figure 3. Strategy for inserting a donor vector into the Rosa26 mouse or rat locus

HR pathway is utilized for generating the targeted integration of the desired DNA sequence. Desired promoter and gene or reporter tags are synthesized and cloned into a validated donor vector backbone. Microinjection of Validated Rosa26 ZFNs and the donor vector into the pronucleus of fertilized, one-cell embryos, efficiently generates HR-mediated mutants that are guaranteed germline transmissible. 

Resulting founders are screened for mutations by validated PCR primers that flank the Rosa26 ZFN site. Several sets of primers are designed, flanking the integration site, to screen appropriate integration and rule out random insertion of the donor into the genome. Founders are confirmed by sequencing. 

Custom Model Builder  Transgenic Mice

 
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