🚀 5 Key Considerations for Designing Flox Alleles | Conditional Knockout Tips

Описание: Designing a floxed allele correctly is essential for a successful conditional knockout experiment. In this video, we’ll cover how to choose the right exon, where to place loxP sites, and how to make screening easier. Understanding exon selection, splicing, and genetic stability will help you create a robust and effective flox allele. Stay tuned until the end for additional resources on mouse colony management and screening strategies!

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Chapters & Key Points

Introduction
Overview of key factors when designing a floxed allele
Importance of exon selection, loxP placement, and screening strategy

1. Exon Selection
(1a) Constitutive Exon: Choose an exon present in all transcript variants to avoid variant-specific depletion
(1b) Premature Stop Codon: Removing certain exons can trigger nonsense-mediated decay (NMD), making the transcript unstable
(1c) Functional Domains: Target exons encoding critical protein functions to ensure loss of function

2. LoxP Site Placement
(2a) Proximity to the 5’ End: Targeting earlier exons ensures smaller, non-functional protein products
(2b) Splicing Considerations: Position loxP sites carefully to avoid disrupting normal splicing
(2c) Conserved Motifs: Avoid disrupting conserved regulatory sequences within introns
(2d) Distance Between LoxP Sites: Optimal spacing ensures efficient Cre recombination

3. Planning for Screening
Designing genotyping strategies to easily validate your flox allele
Considerations for PCR-based screening and Southern blot validation

Outro
Summary & key takeaways
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Check out our series on mouse colony management for more insights

Timestamps:

00:00 Intro
00:18 Select your exon
00:49 Constitutive exon
00:20 Premature stop codon
04:09 Functional domains
04:35 Placing the loxP sites
04:47 Proximal exons
06:26 Splicing elements
08:08 Conserved motifs
08:59 Distance between LoxPs
10:07 Targeting strategy
11:31 Add restriction sites
12:50 Test screening primers
13:12 Outro