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Applications of Transgenic and Knockout Mice in Alcohol Research

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Applications of Transgenic and Knockout Mice in Alcohol Research Applications of Transgenic and Knockout Mice in Alcohol Res e a rc h Barbara J. Bowers, Ph.D. Multiple genetic and environmental factors contribute to the development of alcoholism. Researchers attempting to elucidate the roles of specific genes in alcoholism risk have benefited from advances in genetic engineering. Two important tools used by researchers include transgenic mice, in which a foreign gene is integrated into an animal’s genetic material, and knockout/knock-in mice, in which targeted genes either are rendered nonfunctional or are altered. Both of these animal models are currently used in alcohol research to determine how genes may influence the development of alcoholism in humans. KE Y W O R D S: animal model; transgenic technology; gene knockout technology; gene expression; DNA; neurotransmitters; GABA receptors; stem cell; receptor proteins; protein kinases lcoholism is a complex disorde r alcohol affects nearly all brain activity, date genes are either inactivated or altered , that encompasses sever a l ph y s i o- including enhancement of inhibition, resulting in a lack of or change in pro- Alogical and behavioral characteris- mediated by the gamma-aminobutyric tein expression. Although mouse models tics (also ref e r re d to as phenotypes), acid (GABA) neurotransmitter system; may seem poor res e a r ch tools for studying including atypical responses to alcohol ac t i v ation of rewa r d pathways that are the genetics of human neurop h y s i o l o g y in initial sensitivity, tolerance, consump- dopaminergic (DA) and serot o n e r g i c and behavior, substantial genetic simi- ti o n , and withdrawal as well as vulnera- (5 – H T ); and effects on enzyme pro- larities exist betwee n mice and humans, bility to the rewa r ding effects of alcohol. teins that are located inside nerve cells and known correspondence exists Human and animal studies have both (i.e., neurons) (Dia m o n d and Gord o n be t w een mouse ch r omosomal reg i o n s sh o wn that these aspects of alcoholism 1997; Grobin et al. 1998). Theref o re , and human chromosomes (Sil v er 1995). ar e often mediated by chemical path- these systems (as well as several others, This article describes the generation ways in the brain, known as neuro- including nonneuronal genes) have of transgenic mice and knockout/ transmitter systems (Diamond and pr oduced many candidate genes to knock-in mice and recent examples of Gor don 1997; Harris 1999). A major in v estigate for their roles in the devel - ho w these techniques have been applied goal for neuroscientists has been to opment of alcoholism. in alcohol res e a rc h . identify genes and proteins in the brain One approach that is parti c u l a r l y that influence the expression of alco- ef f e c t i v e in the hunt for candidate holism. Inc r eased knowledge of chemi- genes is the rev erse genetics approa c h , BAR BA R A J. BOWE R S , PH.D . , is a res e a rc h cals in the brain and receptor prot e i n s in which a gene of interest is altered to associate at the Institute for Beh a v i o ra l (i.e., protein molecules that rec o g n i z e change its expression, or function, in th e Genetics, Uni v ersity of Colorad o , Boulder, and bind neurotransmitters) has pro- en t i r e animal. This approach invol ve s Co l o ra d o . vided scientists with a priori reasons for generating transgenic mice, in which a studying specific genes and prot e i n s , fo r eign gene is integrated into an ani- This work was supported by Nat i o n a l which are sometimes ref e r r ed to as can- ma l ’s genetic material, as well as knock- Institute on Alcohol Abuse and di d a t e genes and proteins. For example, out or knock-in mice, in which candi- Alc o h o l i s m gr ant AA–11275. Vol. 24, No. 3, 2000 175 Transgenics many copies are introduced into ferti l - to term. Mice that carry the transgene, iz ed mouse eggs (i.e., embryos) at the identified using DNA analysis tech- In the context of mouse models, the single-cell stage (see figure 1). To crea t e niques, are called progenitor or founder term transgenic refers to the introd u c - mouse embryos, female mice are hor- mice. These mice are bred with no n - tion of a forei g n 1 gene (known as a monally induced to hyperovulate and transgenic mice, and the offspring are transgene) into the genetic material of then mated to males for ferti l i z a t i o n . tested for the presence of the transgene a mouse in both the rep ro d u c t i v e (i.e., The ferti l i z ed eggs are harvested from to confirm that the transgene has inte- germ) cells and the nonrep ro d u c t i v e the female and injected with the trans- grated into the germ cells. If the integra- (i.e., somatic) cells. This process leads to gene. The injection takes place early after tion has occurred, subsequent bre e d i n g the expression and propagation of th e fe r tilization, when the embryo contains continues between mice carrying the gene across future generations. Oft e n th e two sets of DNA, one from each paren t . transgene, ref e r r ed to as the F1 ge n e r a - purpose of this technique is to create mice Each set of DNA exists in a separate tion, producing lines of transgenic mice that express more than normal amounts st ru c t u r e, called a pronucleus; theref o re , (F 2 generation). For more information of the gene product (i.e., protein). In both male and female pronuclei exist. on creating transgenic mice, see Camper some applications, howeve r , the scien- Copies of the promoter and foreign gene 1987 and Picciotto and Wickman 1998. tific goal is to introduce a differen t co n s t r uct are microinjected directly into Although res e a r chers can control form of the gene in question. This tech- the male pronucleus with a fine glass the expression of the transgene with the nique allows res e a rc h e r s to evaluate the needle. The res e a r cher injects the trans- choice of prom o t e r , one limitation of role of specific genes in the devel o p - gene before the first cell division to ensure the transgenic technique is its inability ment of disease. For example, a line of that the DNA will develop in all cells to target the integration of the transgene transgenic mice generated to study alco- of the adult animal. The pronuclei then to its natural location on the chrom o - holism would carry a gene that is known fuse, and the cell begins to divide normally. some. The site of integration is unique or suspected to have a role in some App r oximately 50 to 90 percent of for each microinjection, and the trans- aspect of the disease. Res e a r chers can the eggs survi v e the pro c e d u r e and are gene can be randomly inserted anywhere then study the animals’ behaviors to implanted into the oviducts of foster on any chromosome. This outcome ev aluate the role of the gene in alcoholism. mo t h e r s , wh e r e the embryos develop can result in the disruption of a sequence Creating Transgenic Mice Basic req u i r ements for creating a trans- genic mouse include (1) identifying and isolating the candidate gene of interes t Foreign gene is injected into fr om its original organism (i.e., from the the cellular structure contain- ing the genetic material from DNA of the organism’s cells) and (2) the father. selecting a suitable promoter that is placed adjacent to the transgene. Prom o t e r s ar e stretches of DNA associated with a specific gene that guide the expres s i o n of the gene to specific areas in the brain Embryo is transferred into a and turn the expression of the gene foster mother for embryonic “on ” either before birth (i.e., pren a t a l l y ) development. or after birth (i.e., postnatally). The choice of promoter by the scientist depends on its location in the brain Mouse pups (i.e., F1 animals) carrying and expressing the and the time in which (i.e., prenatal fo r e i g n gene are identified. or postnatal) the transgene must be ex p r essed. For example, the prom o t e r for the a -calmodulin kinase II (aCa m K I I ) F1 animals carrying the foreign gene are mated with enzyme gene directs postnatal expres - each other. sion to the forebrain. Theref o r e, any gene associated with this promoter would be Each offspring (i.e., F2 found in the forebrain after birth . animal) carries the foreign Once the transgene construct (i.e., gene in all of its cells. the promoter and DNA) is prod u c e d , Figure 1 General procedure for the generation of transgenic mice.
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