Introduction Genetically Engineered Mouse Models Strengths and Caveats

Reliable methods to generate specific DNA constructs targeted to specific regions of the mouse genome, in combination with advances in mouse embryonic stem cell technology, make it now possible to create lesions of any mouse gene locus of interest (for reviews, see Bonasera and Tecott 2000; Gaveriaux-Ruff and Kieffer 2007; Miyoshi and Fishell 2006). Mouse models generated using these kinds of genetic "dissecting tools" provide powerful and unique insights into the functional roles of specific genes at many different scales of organization. In fact, recently developed approaches allow the investigator to limit transgene expression to a specific developmental line of cells or switch transgene expression in response to drugs or introduction of viral vectors. These techniques offer a nearly limitless palette of tools for mechanistic studies associating gene expression with gene function. As long as one keeps in mind the limitations of these approaches, transgenic mouse models will remain important tools to elucidate the role that specific molecules contribute in organism development, cellular and tissue physiology, whole organ function, and organization of animal behavior.

Our current understanding of how serotonin-2C receptors (5-HT2CRs) influence central nervous system (CNS) development, neuronal physiology, and whole animal behavior owes much to the above-mentioned transgenic mouse technology. Currently, intense study has focused on the first 5-HT2CR transgenic mouse model initially developed in the laboratory of Laurence H. Tecott (Tecott et al. 1995). In this model, a targeting vector complementary to the htr2c 5th-exon coding region containing an in-frame stop mutation was introduced into mouse embryonic stem cells. Mice derived from these embryonic stem cells were screened for animals that had the mutation present in germ line cells. These chimeras were the founders of the

Department of Medicine/Division of Geriatrics, University of Nebraska Medical Center, Omaha, NE, 68190-5039, USA e-mail: [email protected]

G. Di. Giovanni et al. (eds.), 5-HT2C Receptors in the Pathophysiology of CNS Disease, The Receptors 22, DOI 10.1007/978-1-60761-941-3_4, © Springer Science+Business Media, LLC 2011

htr2c- mutant mouse line. Chimeras in turn were initially bred onto a hybrid mouse background B6D2 (an F1 hybrid between C57BL/6 and DBA/2) to take advantage of the hybrid's better breeding success. Later mice arising from this colony were bred to a pure C57BL/6 background, with the first mice congenic to C57BL/6 being produced around 2001. Since the mutation introduced into these transgenic mice produced a truncated 5-HT2CR protein with no biological function, mutant mice descending from this line are characterized by having a complete lack of 5-HT2CR function throughout their entire life span.

To fully appreciate the strengths and limitations of data coming from transgenic 5-HT2CR mouse models, a few aspects of important mouse biology should be mentioned. Since the htr2c locus is X linked, the only genotypes possible when breeding a heterozygous female to a wild type male will be either wild-type or 5-HT2CR mutant (in male mice) and wild-type or 5-HT2CR heterozygotes (in female mice). To obtain female 5-HT2CR mutant mice, one must cross 5-HT2CR heterozygous female mice with 5-HT2CR mutant males (this cross will also yield 5-HT2CR mutant males and 5-HT2CR heterozygous females). Thus, standard mouse breeding practices will readily produce populations of wild-type and littermate 5-HT2CR mutant male mice, wild-type and littermate 5-HT2CR heterozygous female mice, and heterozygous and 5-HT2CR mutant female mice. However, no single cross can produce both wild-type and 5-HT2CR mutant female mice. Thus, studies of 5-HT2CR receptor function in female mice must address potential issues of either differential maternal care or cross-fostering of mouse pups. Additionally, inactivation of X-linked genes in females occurs in a mostly stochastic manner (Ohlsson et al. 2001). This leads to mouse-to-mouse differences in the precise 5-HT2CR dose in female heterozygotes, and it is thus an additional source of experimental variation to be addressed in studies of 5-HT2CR function in females.

Finally, the mouse genetic "background" chosen to carry the mutant allele may be a particularly important factor in interpreting experimental data. Currently available 5-HT2CR mutant mice have undergone a rigorous backcrossing to a C57BL/6 genetic background, and most of the studies published regarding this mouse employ animals more than 20 backcrosses from the original F1 hybrids. While this degree of backcrossing is ample to remove any residual genetic background contributed by ES cells, DNA sequences flanking the transgene and included in the initial recombination event may remain even after 20 backcrosses. Controlling for this issue requires obtaining a second independent transgenic mouse line (that would certainly not have integrated the transgene in exactly the same way as the first line), and repeating behavioral assessments in this line. This highly rigorous control is often not available for a variety of reasons.

Alternatively, concerns about flanking DNA effects behavioral phenotypes can be allayed by comparing observed mutant phenotypes with those obtained by pharmacological inhibition or activation of the relevant gene target. Thus, most of the literature describing the phenotypes of 5-HT2CR mutant mice evaluate animals that are otherwise mostly congenic to C57BL/6. C57BL/6 is a mouse strain particularly appropriate for studies of mouse behavior. Compared with most strains, C57BL/6 demonstrates better learning and memory task acquisition on available cognitive batteries, lower indices of trait anxiety-related behaviors, greater propensity for ethanol and cocaine ingestion, and less sensitivity to factors that change sensorimotor gating (Crawley et al. 1997; Holmes et al. 2002). C57BL/6 mice also have a relatively high seizure threshold. However, with age these mice demonstrate sen-sorineural hearing loss, diet-related obesity, type II diabetes, and atherosclerosis.

Defeat Drugs and Live Free

Defeat Drugs and Live Free

Being addicted to drugs is a complicated matter condition that's been specified as a disorder that evidences in the obsessional thinking about and utilization of drugs. It's a matter that might continue to get worse and become disastrous and deadly if left untreated.

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