The construction of transgenic and gene knockout/knockin mouse models of human disease

Abstract

The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research, including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual’s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care.

Insights into the Neurobiology of the Nicotinic Cholinergic System and Nicotine Addiction from Mice Expressing Nicotinic Receptors Harboring Gain-of-Function Mutations

“…The knock-in approach uses homologous recombination in embryonic stem (ES) cells to replace one WT allele of the nAChR gene of interest with an allele containing the hypersensitive point mutation (Doyle et al, 2012). This approach has the advantage of being highly precise, only modifying the endogenous nAChR gene and usually leaving behind only one 34-base pair loxP sequence (Doyle et al, 2012). The knock-in approach is more time-consuming, however, usually requiring 1 to 3 years to produce a usable strain.…”

“…Creation of a transgenic mouse is the alternative approach to a knock-in. In this method, exogenous DNA that directs expression of the hypersensitive nAChR subunit of interest is randomly inserted into the genome (Gong et al, 2003;Doyle et al, 2012). Because the ectopic DNA is capable of incorporating into undesired loci in the genome, possibly disrupting expression of other important genes, several founder strains must be studied at the outset, and misexpression of the nAChR gene of interest in ectopic brain areas must be mitigated.…”

“…The use of bacterial artificial chromosomes (BACs) containing the nAChR gene of interest along with important regulatory sequences can reduce expression artifacts (Gong et al, 2003). Although the transgenic approach suffers from imprecision, the time to product is much shorter (ϳ3 months), and creation of a targeting vector is simpler compared with the knock-in approach (Gong et al, 2003;Doyle et al, 2012). Regardless of the molecular biological route taken to produce the desired mouse strain, the resulting hypersensitive mutation should produce a substantial leftward shift in the concentration-response curve for the particular receptor of interest (Fig.…”

Methods to Study Long Noncoding RNA Biology in Cancer

“…These mice are called transgenic mice. The basic principle of constructing transgenic mouse is that the reconstructed target genes (or the genome fragments) are injected into the donor mouse's fertilized eggs (or embryo cells before implantation) by microscopic injections, and then this fertilized egg (or embryo cells before implantation) is implanted into recipient animals' oviduct or uterus in order to develop into transgenic animals with foreign genes [ 94 ]. This technique not only reveals the function of exogenous gene but also serves for the mass production of engineered animals through analysis of the relationship between the transgene and the phenotype of experimental mice.…”

Generation of Transgenic Mouse Model Using PTTG as an Oncogene

“…Figure is redrawn from Doyle et al 2012 [ 7 ] Skilled and experienced people are required for successful transfers of microinjected zygotes to pseudopregnant recipient mice. Locating the infundibulum (opening of an oviduct) into which the microinjected zygotes need to be transferred is critical and is difficult for beginners.…”