Testing the Cre-mediated genetic switch for the generation of conditional knock-in mice.

08:00 EDT 13th March 2019 | BioPortfolio

Summary of "Testing the Cre-mediated genetic switch for the generation of conditional knock-in mice."

The Cre-mediated genetic switch combines the ability of Cre recombinase to stably invert or excise a DNA fragment depending upon the orientation of flanking mutant loxP sites. In this work, we have tested this strategy in vivo with the aim to generate two conditional knock-in mice for missense mutations in the Impad1 and Clcn7 genes causing two different skeletal dysplasias. Targeting constructs were generated in which the Impad1 exon 2 and an inverted exon 2* and the Clcn7 exon 7 and an inverted exon 7* containing the point mutations were flanked by mutant loxP sites in a head-to-head orientation. When the Cre recombinase is present, the DNA flanked by the mutant loxP sites is expected to be stably inverted leading to the activation of the mutated exon. The targeting vectors were used to generate heterozygous floxed mice in which inversion of the wild-type with the mutant exon has not occurred yet. To generate knock-in mice, floxed animals were mated to a global Cre-deleter mouse strain for stable inversion and activation of the mutation. Unexpectedly the phenotype of homozygous Impad1 knock-in animals overlaps with the lethal phenotype described previously in Impad1 knock-out mice. Similarly, the phenotype of homozygous Clcn7 floxed mice overlaps with Clcn7 knock-out mice. Expression studies by qPCR and RT-PCR demonstrated that mutant mRNA underwent abnormal splicing leading to the synthesis of non-functional proteins. Thus, the skeletal phenotypes in both murine strains were not caused by the missense mutations, but by aberrant splicing. Our data demonstrate that the Cre mediated genetic switch strategy should be considered cautiously for the generation of conditional knock-in mice.


Journal Details

This article was published in the following journal.

Name: PloS one
ISSN: 1932-6203
Pages: e0213660


DeepDyve research library

PubMed Articles [22673 Associated PubMed Articles listed on BioPortfolio]

Next-generation human genetics for organism-level systems biology.

Systems-biological approaches, such as comprehensive identification and analysis of system components and networks, are necessary to understand design principles of human physiology and pathology. Alt...

Direct-to-consumer genetic testing.

Direct-to-consumer (DTC) genetic testing is a controversial issue although Korean Government is considering to expand DTC genetic testing. Preventing the exaggeration and abusing of DTC genetic testin...

MultiFunctional Alleles: A novel method for the generation of "All-In-One" null and conditional alleles.

The engineering of conditional alleles has evolved from simple floxing of regions of genes to more elaborate methods. Previously, we developed Conditional by Inversion (COIN), an allele design that ut...

Normal mammary gland development after MMTV-Cre mediated conditional PAK4 gene depletion.

p21-activated kinases (PAKs) are serine/threonine kinases functioning as downstream effectors of the small GTPases Rac1 and Cdc42. Members of the PAK family are overexpressed in human breast cancer, b...

Targeted next-generation sequencing detects rare genetic events in pheochromocytoma and paraganglioma.

Knowing the genetic status of patients affected by paragangliomas and pheochromocytomas (PPGL) is important for the guidance of their management and their relatives. Our objective was to improve the d...

Clinical Trials [7431 Associated Clinical Trials listed on BioPortfolio]

NGS for Non-unions

The purpose of this study is to investigate the role of genetic testing to look for possible infection as a cause for failure of non-healing fractures. The study aims to compare the result...

Impact of Predictive Genetic Testing

Analysing Impact of preemptive genetic testing on quality of life, physical activity and eating behavior at baseline and 3 months after testing.

GENetic Education Risk Assessment and TEsting Study

The goal of the GENERATE study is to improve genetic testing and cancer prevention in family members of pancreatic cancer patients with identified genetic mutations (inherited changes). Th...

A Randomized Experiment of Malaria Diagnostic Testing and Conditional Subsidies to Target ACTs in the Retail Sector: the TESTsmART Trial Aim 1

The TESTsmART Trial consists of two main aims. The overall goal of the two aims is to investigate the impact of malaria rapid diagnostic test (mRDT) subsidies and conditional artemisinin c...

Genetic Testing for Men With Metastatic Prostate Cancer

This research study provides genetic testing to men with prostate cancer that has spread to other parts of the body (metastatic prostate cancer) and will look for inherited genetic mutatio...

Medical and Biotech [MESH] Definitions

An inbred strain of mouse that is used as a general purpose research strain, for therapeutic drug testing, and for the genetic analysis of CARCINOGEN-induced COLON CANCER.

Genes that cause the epigenotype (i.e., the interrelated developmental pathways through which the adult organism is realized) to switch to an alternate cell lineage-related pathway. Switch complexes control the expression of normal functional development as well as oncogenic transformation.

One of the first INBRED MOUSE STRAINS to be sequenced. This strain is commonly used as genetic background for transgenic mouse models. Refractory to many tumors, this strain is also preferred model for studying role of genetic variations in development of diseases.

The introduction of functional (usually cloned) GENES into cells. A variety of techniques and naturally occurring processes are used for the gene transfer such as cell hybridization, LIPOSOMES or microcell-mediated gene transfer, ELECTROPORATION, chromosome-mediated gene transfer, TRANSFECTION, and GENETIC TRANSDUCTION. Gene transfer may result in genetically transformed cells and individual organisms.

A CCAAT-enhancer-binding protein found in LIVER; ADIPOSE TISSUE; INTESTINES; LUNG; ADRENAL GLANDS; PLACENTA; OVARY and peripheral blood mononuclear cells (LEUKOCYTES, MONONUCLEAR). Experiments with knock-out mice have demonstrated that CCAAT-enhancer binding protein-alpha is essential for the functioning and differentiation of HEPATOCYTES and ADIPOCYTES.

Quick Search

DeepDyve research library

Relevant Topics

Bioinformatics is the application of computer software and hardware to the management of biological data to create useful information. Computers are used to gather, store, analyze and integrate biological and genetic information which can then be applied...

Polymerase Chain Reaction (PCR)
PCR (Polymerase Chain Reaction) uses the ability of DNA polymerase (enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two ident...

Searches Linking to this Article