Animal Models for Disease: Knockout, Knock-In, and Conditional Mutant Mice

LePage, David F.; Conlon, Ronald A.

doi:10.1385/1-59745-213-0:41

Cited by 15 publications

(13 citation statements)

References 26 publications

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“…Short single stranded DNAs can be procured as oligonucleotides (primers from any commercial source such as IDT, Operon etc.). Larger double stranded targeting DNA preparation, on the other hand involve careful designing strategies and complex construction steps that are described elsewhere (Hall et al, 2009; LePage and Conlon, 2006). The primers used as repair oligonucleotides (commercially synthesized) are preferably reconstituted on the first injection session day and frozen as multiple aliquots at −80°C for later injection sessions (if necessary).…”

Section: Basic Protocolmentioning

confidence: 99%

Mouse Genome Editing Using the CRISPR/Cas System

et al. 2014

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The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much faster than the previously used techniques and more importantly multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one cell mouse embryos to create knockout or knock-in mouse models.

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Section: Basic Protocolmentioning

confidence: 99%

Mouse Genome Editing Using the CRISPR/Cas System

et al. 2014

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“…BAC DNA was linearized with SgrAI within the BAC backbone and was transfected into undifferentiated ES cells by electroporation (30). Drug selection (250 g/ml G418) was initiated at 24 h, and resistant colonies were picked after 8 days.…”

Section: Bac Modification and Transfectionmentioning

confidence: 99%

Escape from X chromosome inactivation is an intrinsic property of the Jarid1c locus

Carrel²

2008

Proc. Natl. Acad. Sci. U.S.A.

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Although most genes on one X chromosome in mammalian females are silenced by X inactivation, some ''escape'' X inactivation and are expressed from both active and inactive Xs. How these escape genes are transcribed from a largely inactivated chromosome is not fully understood, but underlying genomic sequences are likely involved. We developed a transgene approach to ask whether an escape locus is autonomous or is instead influenced by X chromosome location. Two BACs carrying the mouse Jarid1c gene and adjacent X-inactivated transcripts were randomly integrated into mouse XX embryonic stem cells. Four lines with single-copy, X-linked transgenes were identified, and each was inserted into regions that are normally X-inactivated. As expected for genes that are normally subject to X inactivation, transgene transcripts Tspyl2 and Iqsec2 were X-inactivated. However, allelic expression and RNA/DNA FISH indicate that transgenic Jarid1c escapes X inactivation. Therefore, transgenes at 4 different X locations recapitulate endogenous inactive X expression patterns. We conclude that escape from X inactivation is an intrinsic feature of the Jarid1c locus and functionally delimit this escape domain to the 112-kb maximum overlap of the BACs tested. Additionally, although extensive chromatin differences normally distinguish active and inactive loci, unmodified BACs direct proper inactive X expression patterns, establishing that primary DNA sequence alone, in a chromosome position-independent manner, is sufficient to determine X chromosome inactivation status. This transgene approach will enable further dissection of key elements of escape domains and allow rigorous testing of specific genomic sequences on inactive X expression.epigenetics ͉ dosage compensation ͉ transgene

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“…protein), or the consequences of a loss-of-function mutation in a gene in one or both chromosomal copies (LePage & Conlon, 2006). Thus, researchers can identify the effects of a homozygous and/or heterozygous mutant genotype(s) on one or more histopathological or behavioral outcomes of interest.…”

Section: Considerations For Researchersmentioning

confidence: 99%

Animal models in genomic research: Techniques, applications, and roles for nurses

Osier

Pham

Savarese

et al. 2016

Applied Nursing Research

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Animal research has been conducted by scientists for over two millennia resulting in a better understanding of human anatomy, physiology, and pathology, as well as testing of novel therapies. In the molecular genomic era, pre-clinical models represent a key tool for understanding the genomic underpinnings of health and disease and are relevant to precision medicine initiatives. Nurses contribute to improved health by collecting and translating evidence from clinically relevant pre-clinical models. Using animal models, nurses can ask questions that would not be feasible or ethical to address in humans, and establish the safety and efficacy of interventions before translating them to clinical trials. Two advantages of using pre-clinical models are reduced variability between test subjects and the opportunity for precisely controlled experimental exposures. Standardized care controls the effects of diet and environment, while the availability of inbred strains significantly reduces the confounding effects of genetic differences. Outside the laboratory, nurses can contribute to the approval and oversight of animal studies, as well as translation to clinical trials and, ultimately, patient care. This review is intended as a primer on the use of animal models to advance nursing science; specifically, the paper discusses the utility of preclinical models for studying the pathophysiologic and genomic contributors to health and disease, testing interventions, and evaluating effects of environmental exposures. Considerations specifically geared to nurse researchers are also introduced, including discussion of how to choose an appropriate model and controls, potential confounders, as well as legal and ethical concerns. Finally, roles for nurse clinicians in pre-clinical research are also highlighted.

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Animal Models for Disease: Knockout, Knock-In, and Conditional Mutant Mice

Cited by 15 publications

References 26 publications

Mouse Genome Editing Using the CRISPR/Cas System

Mouse Genome Editing Using the CRISPR/Cas System

Escape from X chromosome inactivation is an intrinsic property of the Jarid1c locus

Animal models in genomic research: Techniques, applications, and roles for nurses

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