Why does allele-specific deletion avoid confounding phenotypes in target validation?

Monoallelic deletion preserves one functional allele, preventing lethality or severe phenotypic disruption that could obscure enhancer-specific effects on gene expression, enabling clearer interpretation of cis-regulatory function.

How does SNP density in hybrid ES cells enable precise genotyping of deletions?

Natural SNPs occurring every ~125 bp between Mus musculus 129 and Mus castaneus alleles allow design of allele-specific primers that discriminate amplification from each haplotype, facilitating accurate zygosity assessment.

What quantitative measurements confirm successful enhancer deletion and allelic specificity?

qPCR using allele-specific inside primers shows CT value differences 5 cycles or no amplification for the deleted allele, while outside primers confirm deletion size; melt curve analysis validates product specificity.

Why is confirmation of no indels on the non-deleted allele required before expression analysis?

Indels on the wild-type allele could alter gene expression independently, confounding results; gRNA-flanking primers ensure observed expression changes are due solely to enhancer deletion.

What statistical thresholds support go/no-go decisions in enhancer target validation?

CT value differences 5 cycles between alleles indicate heterozygous deletion; consistent allelic expression reduction (e.g., Sox2) across multiple monoallelic clones supports dependency and informs advancement decisions.

Generowanie delecji monoallelicznych za pośrednictwem CRISPR/Cas9 w celu zbadania funkcji wzmacniacza w embrionalnych komórkach macierzystych myszy

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Cited by 23

11:31 min

April 2nd, 2016

10.3791/53552-v

April 2nd, 2016

12.5K views

Sakthi D. Moorthy¹ , Jennifer A. Mitchell¹

¹Department of Cell and Systems Biology, University of Toronto

Eksperymentalne potwierdzenie aktywności wzmacniacza najlepiej podejść poprzez analizę utraty funkcji. Przedstawiono tutaj skuteczny protokół, który wykorzystuje delecję za pośrednictwem CRISPR/Cas9 do badania specyficznej dla alleli regulacji transkrypcji genów w komórkach F1 ES, które zawierają genom hybrydowy (Mus musculus¹²⁹ x Mus castaneus).