What Is A Conditional Allele?

Understanding Animal Model Options and Uses of Alleles in Genetic Research – What Is a Conditional Allele?

Genetically modified animals are the cornerstone of research programs across many fields of study. When choosing a mouse model in particular, one option that should be thoroughly evaluated is a conditional allele. What is a conditional allele and why is so important for research? Read on to learn how a conditional allele can provide more accurate control over genetic modifications.

Definition of an Allele

What is a conditional allele, and what is it used for? In simple terms, an allele is a variant form of a gene. The word allele could refer to the different gene sequences that exist naturally in a population, or in an animal model, it could be a change that was deliberately introduced. Normally a gene’s sequence is the same in essentially every cell of the body.

In the early days of genetically modified mice, this was also true of genetic changes introduced by researchers. Mice in particular continue to be invaluable, as a large number of different mouse models are now already available. This is thanks to genetic modification techniques that have been developed over the past few decades.

However certain research questions require an animal model where a gene has a different allele in different cells. For example it could be necessary for most cells to have a functional copy of a gene while just a few cells contain a non-functioning copy. Conditional alleles therefore are designed to enable control over exactly when and where different alleles will be expressed in an animal model. Conditional alleles in genetically modified mice allow for the deletion of a gene of interest in a target tissue when combined with a tissue-specific Cre recombinase. A conditional allele is achieved by introducing LoxP sites around a critical exon, a gene, or a cluster of genes.

Comparing a Knockout Allele and a Conditional Knockout Allele

In the context of genetically modified animals, the simplest way to answer “what is a conditional allele?” is to directly compare two types of mouse models: knockouts and conditional knockouts. Knockout mice are a type of mouse model where a specific gene has been altered to permanently disrupt its function. This disruption, or knockout, affects the gene in every single cell of the mouse’s body and at all stages of its life. A common way to knock out a gene would be to delete part of its sequence – this is a permanent change that can’t be reversed. These types of animal models have been valuable for many studies but their limitations can get in the way of some experiments.

A conditional knockout allele can achieve the same result, which is the disruption of the gene’s function, but with more control. This is because the conditional knockout allele will initially function just like the natural unmodified gene. Part of the gene can be deleted just like the knockout allele but the deletion has to be triggered by researchers. This principle applies to all conditional alleles: they initially express one sequence, then can be switched to expressing a different sequence. The switch from normal sequence to disrupted knockout sequence is just one example of a conditional allele.

Generating a Conditional Allele in Mice

Precise and specific genetic modifications must be made in order to create conditional alleles and the process is too complex to briefly describe. To focus on the important details, a specific DNA sequence called loxP is the crucial element of what a conditional allele is. For example, a conditional knockout allele requires the placement of two copies of the loxP sequence in a target gene. Other conditional allele designs use the loxP sequence in different ways. There are constant improvements being made to the methods used to create conditional alleles in mice. It’s possible that new developments, such as the CRISPR/Cas9 method, may make it easier to create conditional alleles in the future.

Types of mouse models with conditional alleles:

• Retain wild-type expression and are amenable to conditional, tissue-specific and/or time-dependent deletion. This approach is particularly necessary for manipulating the approximately 30% of genes that affect the viability of homozygous mutants when deleted. For example, embryonic lethality caused by the deletion of the coding regions of Mixl1 (Pulina et al., 2014) or Brca1 (Xu et al., 1999) can be rescued by conditional mutagenesis. This generates models that can be used to investigate specific gene-dependent processes during mammalian embryogenesis, neurodevelopment and breast cancer when combined with an appropriate Cre-expressing line that enables tissue- or developmental-stage-specific gene deletion.

• “Knockout-first” uses a variation of gene targeting to create a highly versatile allele that combines both gene trap and conditional gene targeting to generate a reporter-tagged knockout allele. The “knockout-first” allele is generated by inserting an FRT-flanked gene-trap vector, which contains a splice-acceptor sequence upstream of a lacZ reporter gene and a strong polyadenylation stop sequence, into an upstream intron. This creates an in-frame fusion transcript that will disrupt the expression of the targeted allele. Additionally, an adjacent exon coding sequence is flanked with loxP sites. This allele can then be converted into a null allele by Cre to abrogate gene expression or into a conditional allele by Flp, which can subsequently be converted by Cre into a null allele. The knockout-first strategy is versatile because it uses a single targeting vector to monitor gene expression using a reporter and tissue-specific gene function using Cre, thereby avoiding embryonic lethality. Models using this knockout-first strategy, include models of skin abnormalities (Liakath-Ali et al, 2014) and age-related hearing loss (Kane et al., 2012).

• Conditionals by inversion employ an inverted module that contains a reporter gene (e.g. GFP) flanked by mutant recombinase target sites (lox66 and lox71) positioned in a head-to-head orientation to enable inversion by Cre recombinase inserted into the anti-sense strand of a target gene. Cre “flips” the inverted module into the sense strand, interfering with and inhibiting target-gene transcription while activating the reporter. The inversion approach is particularly applicable to single-exon genes and to genes in which the exon–intron structure is not clearly defined. This approach has been used to model an angiogenesis defect in delta-like 4 (Dll4) knockout mice (Billard et al, 2012) and to generate immunological phenotypes in interleukin 2 receptor, gamma chain (Il2rg) knockout mice (Economides et al, 2013).

References

MV Pulina, KE Sahr, S Nowotschin, MH Baron, AK Hadjantonakis. 2014. A conditional mutant allele for analysis of Mixl1 function in the mouse. Genesis 52(5): 417-23.

K Liakath-Ali, VE Vancollie, E Heath, DP Smedley, J Estabel, D Sunter et al. 2014. Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen. Nat Commun 5: 3540.

AN Economides, D Frendewey, P Yang, MG Dominguez, AT Dore, IB Lobov et al. 2013. Conditionals by inversion provide a universal method for the generation of conditional alleles. Proc Natl Acad Sci U S A 110(34): E3179-88.

MJ Kane, M Angoa-Pérez, DI Briggs, DC Viano, CW Kreipke, DM Kuhn. 2012. A mouse model of human repetitive mild traumatic brain injury. J Neurosci Methods 203(1): 41-9.

X Xu, KU Wagner, D Larson, Z Weaver, C Li, T Ried, L Hennighausen, A Wynshaw-Boris, CX Deng. 1999. Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nat Genet 22(1): 37-43.

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