Knockout Design

Critical Exon Selection

Since 1998, ingenious targeting laboratory has completed over 2,500 custom gene targeting projects for researchers at universities, pharmaceutical companies, and research institutions worldwide. Our mouse models have supported research published in more than 800 peer reviewed articles including Science, Nature, and Cell.

Selecting which exon to delete for knockout or flanking for conditional control is arguably the most important decision in targeting design. A well chosen exon maximizes loss of function. A poorly chosen exon may leave residual protein activity.

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2,500+

Projects Completed

800+

Publications

26+

Years Experience

100%

Success Rate

Exon Selection Criteria

Criterion	Rationale
Essential for protein function	Deletion must eliminate activity
Present in all splice variants	Ensures all isoforms affected
Deletion causes frameshift	Prevents partial protein production
Early in coding sequence	Minimizes truncated protein

Reading Frame and Deletion Logic

Frameshift Deletions

Exon length not divisible by 3 creates +1 or +2 frameshift in downstream exons.

Outcome: All downstream codons read out of frame. Stop codons terminate translation early. Protein becomes severely truncated.

Preferred—can cause loss of function

In-Frame Deletions

Exon length divisible by 3 maintains reading frame. Protein lacks specific codons but otherwise intact.

Outcome: Risk of partially functional protein if deleted codons are dispensable (linker regions, flexible domains).

Requires functional domain analysis

Functional Domain Analysis

Understanding where functional domains reside within your target gene is critical. Targeting an exon encoding part of an essential domain can effectively cause knockout.

Domain Type	Typical Size	Importance
Kinase domains	250 to 300 amino acids	Essential for all kinase function
DNA binding domains	50 to 100 amino acids	Essential for transcription factors
Transmembrane domains	~20 amino acids per pass	Essential for membrane proteins
Signal peptides	15 to 30 amino acids	Not essential for mature protein

Targeting Early Exons (Exon 1 to 3)

Consider targeting early exons, such as Exon 1, which can cause loss of function in every transcript variant produced from the gene:

All variants affected: Any alternative splicing downstream is eliminated

Maximal compensation needed: Cells cannot compensate through exon skipping

Clear phenotype: No transcript variant escapes the knockout

Experimental Validation of Exon Selection

Transcript Analysis

Reverse transcribe mRNA from expressing tissues, amplify across selected exon, and sequence to confirm exon inclusion and splice boundaries.

Predictive Frameshift Analysis

Use exon length analysis to predict frameshift consequences. Length divisible by 3 = in-frame; not divisible by 3 = frameshift.

Protein Consequence Modeling

Create model of predicted protein after exon deletion. Identify stop codon location and domains lost.

Selected Publications in Exon Selection

Foundational research on knockout design and exon selection:

Wang L, Noyer L, Jishage M, Wang YH, Tao AY, McDermott M, et al. (2025).

CLNS1A regulates genome stability and cell cycle progression to control CD4 T cell function and autoimmunity. ↗

Sci Immunol 10(108): eadq8860

Clausen BE et al. (1999).

Conditional gene targeting in macrophages and granulocytes using LysMcre mice. ↗

Transgenic Research 8(4): 265-277

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What Researchers Say

“I've been working with iTL over the past 5 years in the production of 3 different genetically altered mice. Not only did iTL help in the design of the mice, but the entire process was transparent with the opportunity at any time along the way to discuss my questions or concerns with scientists who had significant insight into the process. The mice were delivered on time, as billed!”

— Raghu Mirmira, MD, PhD

University of Chicago

Start Your Exon Analysis

Critical exon selection is the foundation of successful knockout design. Our team can help you analyze your target gene's exon structure and identify optimal exons for targeting.