sign in sign up
<<
Home , Gene knockout, Knockout mouse

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 in alcoholism risk have benefited from advances in . Two important tools used by researchers include transgenic mice, in which a foreign 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 . KE Y W O R D S: animal model; transgenic technology; technology; gene expression; DNA; neurotransmitters; GABA receptors; ; receptor ; 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 models tics (also ref e r re d to as ), 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 of 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 (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 , iz ed mouse eggs (i.e., ) 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 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 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 , 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 . 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 (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. 1The source of this gene could be mouse or another mammalian species, including human.

176 Alcohol Research & Transgenic and Knockout Mice

of one of the host animal’s own genes The transgenic mice wer e tested for sensitivity to alcohol. Although initial (i.e., known as insertional mutagenesis), alcohol drinking (Engel et al. 1998) in vitro (i.e., in a test tube) studies inves - pr oducing changes in behavior that and initial sensitivity to alcohol (Eng e l ti g a t i n g the role of the g2L subunit in could mistakenly be attributed to the and Allan 1999). Results indicated that alcohol potentiation of GABAer g i c transgene itself. Also, the number of overe x p r ession of 5–HT3 rec e p t o r s function sh o wed that this subunit was integrated copies of the transgene can- de c r eased alcohol consumption by 46 ne c e s s a r y for alcohol sensitivity; la t e r not be co n t r olled, and having more copies pe r cent. In support of the role of 5–HT3 studies have not found an absolute g2L of a gene does not necessarily in d i c a t e ex p r ession in alcohol consumption, req u i r ement (Grobin et al. 1998; Sap p in c r eased overe x p r ession of the gene. Engel and colleagues (1998) rep o rt e d and Yeh 1998). The inconsistencies in To control for this, the existence of that the level of alcohol consumption these results may be attributable to dif- mo r e than one founder and conse- in the four transgenic lines was rel a t e d fe r ences in the in vitro prep a r a t i o n s quently more than one line of mice for to their levels of receptor overe x p re s - used by the inves t i g a t o r s . each transgene is desirable. The site of sion (i.e., the greater the level of over - To further investigate the function integration and level of expression will ex p r ession, the greater the reduction in of the g2 subunit in a whole animal differ in each founder, and transgenic consumption). In contrast, overe x p re s - model, Wick and colleagues (2000) mice that descend from the same sion of 5–HT3 receptors increased sen- cr eated transgenic mouse lines overe x - founder will share the same chrom o s o - sitivity to the activating effects of low pr essing the g2L and g2S genes. Two mal integration site. If each transgenic doses of alcohol. When some mice are g2L and one g2S lines of transgenic line displays the same changes in behav- ad m i n i s t e r ed low doses of alcohol, their mice wer e tested for responses to alco- io r , it is more likely that it is attributable locomotor activity increases. This phe- hol, including sedation, ataxia (i.e., loss to the transgene. nomenon is ref e r r ed to as alcohol-induced of coordination), withdrawal seizures , hyperlocomotion and is a measure used and acute functional tolerance (AFT) in mice to test for initial sensitivity to (i.e., a measure of tolerance to alcohol Using Transgenic Mice alcohol. The authors have suggested that occurs within one testing session as in Alcohol Research that 5–HT3 receptors play a role in opposed to tolerance devel o p m e n t th a t Transgenic mice have traditionally been in c r eased initial sensitivity that may be occurs over several days of alcohol trea t - used to study devel opmental proc e s s e s related to decreased alcohol consumption. ment and testing). and as models of human diseases. Although The GABA neurotransmitter system None of the transgenic lines of mice pr ovides another av enue for using trans- di s p l a y ed altered responses to alcohol, transgenic lines have been generated for ge n i c s in alcohol res e a r ch. The GABA with the exception of AFT, in which se v eral genes, their use has been some- A receptor family consists of at least 16 dif- tolerance was decreased in both g2L what limited in alcohol res e a r ch. How- fe r ent protein molecules (i.e., subunits) and g2S transgenic lines compared with eve r , this application remains useful for assembled 5 at a time, creating a rec e p - nontransgenic mice. The lack of speci- identifying candidate genes that underlie tor complex that surrounds a channel. ficity of the g2L transgene and lack of specific aspects of alcoholism (Weh n e r When GABA or GABA-like compounds effects on the other measures may have and Bowers 1995). For example, animal bind to the receptor and activate it, this been caused by insufficient overe x p re s s i o n studies have shown that the serot o n e r - channel temporarily opens and allows the of either transgene. Howeve r , the level s gic (5–HT) neurotransmitter system is passage of negativel y charged molecules of expression wer e sufficient to res c u e in vo l v ed in alcohol consumption and (i.e., chloride ions) to pass from the the lethal of gene-targeted other alcohol-related behaviors (Li and ce l l ’s exterior to its interior. This ion flow mutant mice lacking the entire g2 gene McB ride 1995). Theref o r e, Engel and de c r eases the cell’s exci t a b i l it y , which res u l t s In other words, mice lacking the entire colleagues (1998) created transgenic in inhibition. The GABA system is the g2 gene but overe x p r essing the g2L mice overe x p r essing the 5–HT3 rec e p t o r pr i m a r y modulator of inhibition in the transgene survi v ed (Bauer et al. 2000). pr otein to investigate its role in alcohol brain (Bar n a r d et al. 1998). Alcohol The 5–HT3 and g2L and g2S trans- and other drug abuse. The res e a rc h e r s enhances the inhibitory effects of GABA genic lines are two examples of the use a used the CaMKII promoter to direc t at the GABAA receptor; this interaction of this technology in alcohol res e a rc h ex p r ession of the 5–HT3 transgene to of alcohol and the receptor appears to with a focus on initial sensitivity, toler- the forebrain. Exp r ession of the 5–HT3 cause alcohol’s intoxicating and sedat- ance, or consumption. Other trans- receptor was greatly increased in the ing effects. genic mouse lines have been used to transgenic mice; receptor binding of Num e r ous studies have established study diver s e al c o h o l - r elated pheno- a 5–HT3 agonist (i.e., a chemical that that acute and chronic behavioral effects types, such as alcohol’s effects on aggres - mimics serotonin at the 5–HT3 su b - of alcohol are differentially mediated si o n (i.e., transforming growth factor a type) was increased nearly a hundred - th ro u g h GA B A ergic receptor subunits transgenics [TGF a ]); alcohol as a cofac- fold in cortical regions of the brain in (for a rev i e w, see Grobin et al. 1998). tor in HIV disease (i.e., transactivat o r one of four transgenic lines tested. The The g2 subunit, which exists in both a pro t e i n overe x p r ession [Tat]); alcohol’s remaining lines exhibited lower level s long (g2L) and short (g2S) version, has effects on alcohol cardi o m y opathy (i.e., of receptor expres s i o n . been studied for its role in GABAer g i c alcohol dehydr ogenase transgenics

Vol. 24, No. 3, 2000 177 [ADH]); and alcohol-induced neuro- transgenic techniques, the mutated in s e r t randomly and only one copy of to xicity in neonatal cerebellum (i.e., gene is inserted into its normal location the mutated gene will have integrated. cell rep r essor gene transgenics [bcl-2]) on the chromosome (i.e., the gene is Caution is necessary when interpret i n g (see table for ref e re n c e s ) . “ta r g e t e d ”). This eliminates the prob - results from gene-targeting experiments lems associated with insertional muta- because of two potentially confounding Gene-Targeting Techniques in genesis seen in some transgenic lines. factors. First, other genes may compen- Knockout and Knock-in Mice Gene-targeting traditionally generates sate in response to the disrupted gene, knockout mice in which a candidate which is nonfunctional throu g h o u t Knockout and knock-in mice are cre- gene is ren d e r ed nonfunctional. Rec e n t l y pr enatal and postnatal devel o p m e n t . ated by gene-targeting techniques that this technique has been used to crea t e Howeve r , recent advances in gene-targeting pr oduce animals in which a specific knock-in mice, in which a is techniques that allow res e a rc h e r s to co n - gene has been deleted (i.e., “knocked in t r oduced into a candidate gene and tr ol wh e r e and when the mutated gene is ou t ”) or mutated (i.e., “knocked in” ) . the function of the gene is changed but ne u r ologically expres s e d may overc o m e Ide a l l y , by inference, any differences in not eliminated. this limitation. For example, the dele- phenotype observed in knockout and Req u i r ements of this method include tion of a gene could be programmed to knock-in mice can be due to the non- the following: (1) the identification and occur in the adult mouse after devel o p - functional or altered gene. Howeve r , isolation of the candidate gene from ment is complete, thereb y el i m i n a t i n g adaptations during devel o p m e n t mouse DNA and (2) cultured, mouse pr oblems caused by devel o p m e n t a l co m - attributable to the mutation and the em b r yonic stem (ES) cells. ES cells pensation. For more information about ex p r ession of the background genotype retain the ability to differentiate into all these techniques, see Sauer 1998. may also produce changes in behavior tissues of a developing mouse. Throu g h The second factor is the mouse’s not directly caused by the missing or genetic engineering techniques, the iso- ba c k g r ound genotype. Because many mutated gene. lated gene is mutated either to make it genes regulate complex behaviors and In some instances, when a gene vital nonfunctional or to change its func- dr ug responses (Ban b u r y Conferen c e to is deleted tion. The gene is then introduced into 1997), the expression of any one gene or mutated, knockout or knock-in mice the ES cell (see figure 2, p. 182), rather is influenced by the presence of the lacking the vital gene cannot devel o p than into an embryo, as in the crea t i o n other genes in an organism’s genotype. be y ond a certain stage, demonstrating of transgenic mice. Once in the ES cell, Th e re f o r e, when a gene is knocked out the gene’s req u i r ement for devel o p m e n - the mutated gene changes places with in a particular inbred strain of mouse tal pr ocesses. In this case, the tech- the cell’s normal (wild-type) gene throu g h (i.e., populations of mice that are genet- niques of transgenics and gene-ta r g e t e d homologous rec o m b i n a t i o n (for a rev i e w, ically identical), the background genotype mutagenesis can be combined to pro- see Capecchi 1994; Picciotto and is not necessarily silent and may mask vide added experimental proof that a Wickman 1998). When this occurs, di f f e r ences in behavior because of the deleted or mutated candidate gene is the cell’s wild-type gene is disrup t e d deleted gene. One way to control for actually the gene responsible for the and becomes nonfunctional (or altered this complication is to breed the deleted mutated phenotype. This is accom- if the knock-in strategy is used). ES or mutated gene onto several inbred plished by inserting the wild-type gene cells expressing the mutated gene are strain genetic backgrounds to assess the (i.e., the normal form of the gene) as a identified by growing the cells in a petri mo r e complicated gene-gene interactions. transgene into the host of the dish in a specific medium in which In addition, res e a r chers must con- mutant mouse, as in the g2 knockout only modified cells can survi v e. Pos i t i v e sider the possible confounding effects mice previously described. If the res u l t - ES cells are microinjected into 3.5 day- of the genetic background of the origi- ing phenotype is equivalent to the old blastocysts (i.e., ferti l i z ed embryos nal ES cell used for homologous wild-type phenotype, the mutation is consisting of 8 to 16 cells). Res e a rc h e r s recombination. Some genetic material co n s i d e r ed “rescued,” and pres u m a b l y implant the blastocysts into foster fr om the ES cell will be closely linked the candidate gene is invol v ed in the mothers, where they develop to term. to the region of the mutated version of ne u r ochemical pathway of interes t . Because the ES cells are introduced at the gene. During recombination, when later stages of embryonic cell division, the altered gene replaces the wild-type Creating Knockout the resulting mouse will be chimeric gene, the ES cell’s genetic material will and Knock-in Mice for the mutated gene (i.e., the mouse be carried with the mutated gene because will carry the mutated gene in some, of its close linkage. This association of To create either knockout or knock-in but not all, cells.) Fur ther breeding and ES cell DNA and the targeted gene will mice, res e a r chers use targeted mutagen- DNA analysis are req u i r ed to identify not be disrupted, even after sever a l esis, the site-specific (vs. random) inte- founder mice, which carry the gene in generations of breeding. If a change in gration of a mutated gene using the their germ cells. These mice are used to behavior is observed in a gene-targeted ce l l ’s natural homologous rec o m b i n a t i o n 2 generate lines of knockout mice. This mechanism that occurs during DNA method does not req u i r e multiple lines 2Homologous recombination refers to the exchange of rep l i c a t i o n . In other words, unlike the of mice, because the mutation does not genetic material between complimentary fragments of DNA.

178 Alcohol Research & Health Transgenic and Knockout Mice

Transgenic and Models Used in Alcohol Research continued

Candidate Gene/Protein Phenotype Authors Reference

Transgenic models Acetaldehyde (ADH) Exacerbation of alcoholic heart muscle Liang, Q., et al. Journal of Pharmacology and disease in transgenic hearts Experimental Therapeutics 291:766Ð772, 1999

Adenylate cyclase 7 (AC7) Reduced initial sensitivity to alcohol- Yoshimura, M., et al. Alcoholism: Clinical and induced ataxia and sedation in Experimental Research transgenic mice 23:92A, 1999

Bcl-2 (cell death ) Overexpression of bcl-2 protects Heaton, M.B., et al. Brain Research 817:13Ð18, repressor gene cerebellar neurons from alcohol 1999 neurotoxicity

Gama-aminobutyric acidA Decreased acute functional tolerance Wick, M.J., et al. European Journal of (GABAA) g2L and g2S in both transgenic lines, but no differences Neuroscience in press subunits in alcohol-induced sedation or acute withdrawal or alcohol potentiation of GABAergic function

5ÐHT3 receptor Decreased alcohol consumption in transgenics Engel, S.R., et al. Psychopharmacology 140:243Ð248,1998 Enhanced sensitivity to Engel, S.R., et al. Psychopharmacology alcohol-induced hyperlocomotion, 144:411Ð415, 1999 but no difference in sedation

HIV-1 transactivator protein Alcohol exposure enhances the Prakash, O., et al. Alcoholism: Clinical and (HIV-1 Tat) progression of certain HIV-1 disease Experimental Research traits in transgenic mice 22:266SÐ268S, 1998

Human transforming growth Aggression is not reduced after alcohol Hilakivi-Clarke, L., Neuroreport 4:155Ð158, factor a ( TGFa ) in highly aggressive transgenic male mice et al. 1993

Neuropeptide Y (NPY) Decreased alcohol consumption in Thiele, T.E., et al. Nature 396:366Ð369, 1998 transgenics and increased sensitivity to alcohol sedation Knockout models Aldehyde dehydrogenase ADH 1 mutant mice: increased sensitivity Deltour, L., et al. Journal of (ADH) classes 1, 3 and 4 to sedative effects; ADH 1 and 4: reductions 274:16796Ð16801, 1999 in blood alcohol clearance in mutant mice

Cytochrome P450 (CYP)2E1 Alcohol elimination rate unchanged and Kono, H., et al. American Journal of no effect of the null mutation on alcohol- Physiology 277:G1259Ð induced liver injury G1267, 1999

Dopamine (DA) D1 receptor Decreased alcohol consumption El-Ghundi, M., et al. European Journal of in mutant mice Pharmacology 353:149Ð158, 1998

DA2 receptor Decreased alcohol consumption and Phillips, T.J., et al. Nature Neuroscience reduced sensitivity to alcohol-induced 1:610Ð615, 1998 locomotor impairment in null mutant mice

Vol. 24, No. 3, 2000 179 Transgenic and Knockout Mouse Models Used in Alcohol Research continued

Candidate Gene/Protein Phenotype Authors Reference

Knockout models

DA4 receptor Knockout mice display increased sensitivity Rubenstein, M., et al. Cell 90:991Ð1001, 1997 to alcohol-induced locomotor activity

Dopamine b hydroxylase Knockout results in norepinephrine Weinshenker, D., et al. Journal of Neuroscience depletion; mutants display decreased 20:3157Ð3164, 2000 alcohol preference and increased sensitivity to alcohol-induced sedation and hypothermia

Fyn kinase Mutants exhibit increased sensitivity to Miyakawa, T., et al. Science 278:698Ð701, 1997 sedative effects of alcohol and a lack of alcohol-enhanced phosphorylation of N-methyl-D-aspartate (NMDA) receptors

GABAA a 6 receptor subunit No effect of null mutation on alcohol Homanics, G.E., et al. Molecular Pharmacology sedation 51:588Ð596, 1997 No effect of null mutation on acute or Homanics, G.E., et al. Alcoholism: Clinical and chronic alcohol tolerance or Experimental Research withdrawal hyperexcitability 22:259Ð265, 1998

GABAA b3 receptor subunit Knockout mice do not differ from controls Quinlan, J.J., et al. Anesthesiology 88:775Ð780, in alcohol-induced sedation 1998

GABAA g2L subunit Mutant mice display normal responses to Homanics, G.E., et al. Neuropharmacology alcohol potentiation of GABAergic receptor 38:253Ð265, 1999 function, sedation, anxiolysis, AFT, with- drawal seizures, and hyperlocomotor activity

GABAA d receptor subunit Null mutants consume less alcohol Bowers, B.J., et al. Alcoholism: Clinical and than controls Experimental Research 24:97A, 2000

Neuropeptide Y (NPY) Knockout mice consume more alcohol Thiele, T.E., et al. Nature 396:366Ð369, 1998 than controls and are less sensitive to alcohol sedation

NPY Y5 receptor No effect of null mutation on alcohol Thiele, T.E., et al. Alcoholism: Clinical and consumption Experimental Research 23:61A, 1999

Protein kinase A (PKA) Increased alcohol consumption and Thiele, T.E., et al. Journal of Neuroscience regulatory II b (bII) subunit decreased initial sensitivity to sedation 20:RC75, 2000 in knockout mice

Protein kinase C g subunit Decreased development of rapid and Bowers, B.J., et al. Alcoholism: Clinical and (PKCg) chronic tolerance to sedation and Experimental Research hypothermia in mutant mice but 23:387Ð397, 1999 dependent on genetic background Bowers, B.J., et al. Addiction 5:47Ð58, Decreased initial sensitivity to sedation, 2000 hypothermia, and alcohol potentiation Harris, R.A., et al. Proceedings of the National

of GABAA receptor function Academy of Sciences 92:3658Ð3662, 1995

180 Alcohol Research & Health Transgenic and Knockout Mice

Transgenic and Knockout Mouse Models Used in Alcohol Research continued

Candidate Gene/Protein Phenotype Authors Reference

Knockout models

Protein kinase C e subunit Null mutant mice exhibit increased Hodge, C.W., et al. Nature Neuroscience (PKCe) sensitivity to alcohol sedation and 2:997Ð1002, 1999 hyperlocomotion and alcohol potentiation

of GABAA receptor function Decreased alcohol consumption

Serotonin 1B (5-HT1B) receptor Elevated alcohol consumption in null Crabbe, J.C., et al. Nature Genetics 14:98Ð101, mutants, but see text (Crabbe et al., Science 1996 284:1670Ð1671, 1999) Lack of alcohol-induced conditioned place Risinger, F.O., et al. Alcoholism: Clinical and preference in knockouts; normal alcohol- Experimental Research induced taste aversion 20:1401Ð1405, 1996 Modest increase in rate of responding Risinger, F.O., et al. Behavioral Brain Research for alcohol in operant paradigm by 102: 211Ð215, 1999 knockout mice

Vesicular monoamine Lethal in homozygous mutants; increased Wang, Y-M., et al. Neuron 19:1285Ð1295, 1997 transporter 2 (VMAT2) alcohol-induced hyperlocomotion in heterozygotes mouse, the ES cell’s DNA, and not the tion, tolerance, or withdrawal res p o n s e s knockout mice previously described; al t e r ed gene, could be the cause. There- (i.e., a 6 and g2L) (Homanics et al. 1997, ho weve r , the d knockout mice wer e fo r e, when interpreting behavioral data, 1998, 1999). In addition, alcohol tested for alcohol drinking. When d res e a r chers should consider the pheno- enhancement of GABAA receptor inhi- mutant and wild-type mice wer e offered type of the inbred strain that is the source bition, alcohol-induced reduction in a choice between water and alcohol of the ES cell. an x i e t y , and alcohol-induced hyper- solutions ranging from 3 to 11 perce n t , locomotion wer e not found to be dif- mice lacking the d subunit drank sig- fe r ent in g2L mutant mice compared nificantly less alcohol (Bowers et al. Using Knockout and Knock-in Mice d in Alcohol Research with control mice (Homanics et al. 20 0 0 b), suggesting that the subunit is 1999). Although b3 mutant mice differed one factor invol v ed in alcohol pref e r - Genes that encode receptor proteins are in their response to general anesthetics ence. The results of drinking studies fr equently selected as candidate genes co m pa r ed with nonmutant mice, they using several other receptor knockout in the gene-targeting approach. Seve r a l did not differ from the control mice in mice, some of which are described later rel e v ant receptor subunit knockout alcohol-induced sedation (Quinlan et in this article, indicate that this behav- mouse lines have been used in alcohol al. 1998). Explanations for these unex- ior is multigenic. res e a r ch to evaluate contributions of a pected results may invol v e some of the Both the dopaminergic and serot o n - specific receptor subunit in alcohol’s confounding factors previously dis- ergic neurotransmitter systems have behavioral actions, such as sedation, cussed. For example, the lack of effects been implicated in alcohol-induced initial sensitivity, ataxia, withdrawal, in the a 6 knockout mice may be caused hyperlocomotion and the rei n f o rc i n g tolerance, or consumption. For example, by in t e rf e re n c e fr om the mice’s genetic effects of alcohol. Receptor proteins for four lines of mice lacking the GABAA ba c k g r ound or from overc o m p e n s a t i o n by each of these transmitters are derived receptor subunits a 6, b3, g2L, and d other GABAA subunits. The authors fr om families of genes, several of which ha v e been specifically created to test for also suggest that the g2S subunit may ha v e been selected as gene-targeting responses to alcohol and anesthetics substitute for the missing g2L su b u n i t , candidates. The D2 dopamine rec e p t o r (B o wers et al. 2000b; Mihalek et al 1999; masking a potential role of g2L in alco- knockout mice wer e created based on Homanics et al. 1997, 1998, 1999; hol sensitivity. human studies that implicated var i a n t s Quinlan et al. 1998). Sur p r i s i n g l y , The amount of alcohol consumed in of the D2 receptor in alcoholism, mutant mice lacking a 6, b3, and g2L a free-choice drinking paradigm is one although this association has not been subunits failed to show different res p o n s e s me a s u r e of the rei n f o r cing effects of found in ever y study (Goldman 1995). to alcohol compared with control mice, alcohol. Alcohol consumption was When D2 mutant and wild-type con- as measured by alcohol-induced seda- not evaluated in the GABAA su b u n i t tr ol mice wer e offered a choice of

Vol. 24, No. 3, 2000 181 alcohol or water, the D2 mutant mice may appear counterintuitive; howeve r , Alcohol res e a r ch with knockout mice co n sumed significantly less alcohol (Phi l l i p s the 5–HT receptors are heterog e n e o u s has also targeted certain enzyme pro- et al. 1998). A similar study of D1 and have different functions in the teins known as kinases. Protein kinases receptor mutants also rep o r ted a decrea s e brain. For example, 5–HT1B rec e p t o r s ar e enzymes that activate or deactivat e in consumption that may have been regulate release of serotonin from the the function of proteins, including associated with higher levels of dopamine ne u r on, whereas activation of 5–HT3 receptor proteins, by attaching phos- in some brain regions (El- G hundi et al. receptors produces a rapid excitation of ph a t e gr oups to the proteins. For exam- 19 9 8 ) . the neuron. Num e r ous other studies ple, protein kinase C (PKC) may mod- Dec r eased initial sensitivity has been ha v e rep o r ted the effect of single-gene if y , and thus affect the function of, the associated with alcoholism in human on alcohol drinking, indicating GA B A A rec e p t o r . The activation and populations (Schuckit 1988); theref o re , that many genes regulate this phenotype. de a c t i v ation of specific proteins are two this phenotype is frequently tested in (S ee the table for ref e r ences to th e s e components of a signaling mechanism mice using alcohol-induced increases in and other receptor protein knockout th r ough which chemical signals are locomotor activity as the measure of st u d i e s . ) rel a y ed from the cell’s surface to its interior. se n s i t i v i t y . Dopamine is thought to reg - ul a t e basal as well as alcohol-induced hyperlocomotion. Contrary to human studies, howeve r , the D2 knockout mice, which consumed less alcohol, also Normal (+) gene is isolated. demonstrated reduced sensitivity to the ac t i v ating effects of alcohol. On the other hand, mice lacking the D4 rec e p t o r A mutation to create a defective wer e supersensitive to alcohol-induced (-) gene is introduced. locomotion; to date, these mice have not been tested for alcohol consump- The (-) gene is introduced into tion (Rubinstein et al. 1997). embryonal stem (ES) cells in In contrast with the decrease in tissue culture; not all ES cells alcohol drinking observed in dopamine will incorporate the (-) gene into their DNA. receptor knockout mice, initial studies of mice lacking the 5–HT1B rec e p t o r Cells are grown in a medium that allows only cells with the (-) sh o wed that they consumed twice as gene to multiply. much alcohol as wild-type control mice (C rabbe et al. 1996). Howeve r , the in c r eased consumption demonstrated Cells with the (-) gene are by the mutant mice was not rep l i c a t e d injected into mouse embryos. in later studies. This is most likely attributable to latent genetic back- gr ound interactions between the ES cell genotype and the genotype of the Embryos develop into chimeric in b r ed strain on which the mutation mice expressing the (-) gene in was bred. This interaction did not appear some cells and the (+) gene in until after several generations of bree d - other cells; one of the chimeric mice is mated with a normal ing had been conducted (Crabbe et al. mouse. 1999). Additional tests of alcohol- induced behaviors indicated that initial Mouse pups (i.e., F1 animals) sensitivity to the ataxic effects of alco- carrying a (+) and a (-) gene copy are identified and mated hol was also increased in 5–HT1B mutants, whereas tolerance to chron i c with each other. tr eatment developed more slowly and me a s u r es of withdrawal sensitivity wer e The offspring (i.e., F2 animals) are not affected by the deletion of the gene. analyzed; about 25 percent will have inherited the (-) gene from These results are similar to those rep o rt e d both parents and will completely for the overe x p r essing 5–HT3 tr a n s g e n i c s lack the (+) gene. described earlier. The res e a r ch conclu- sions indicating that both underex p re s s i n g Figure 2 General procedure for the generation of knockout mice. and overe x p r essing 5–HT rec e p t o r mutants would have similar phenotypes

182 Alcohol Research & Health Transgenic and Knockout Mice

Res e a r ch suggests that PKC func- models used in alcohol res e a r ch (see SIB L E Y , D.R.; WES T P H A L , H.; AN D O’ D OW D , B.F. tion is associated with alcohol’s effects table). The techniques of rev erse genet- Disruption of D1 receptor gene expression attenu- ates alcohol-seeking behavior. European Journal of on the brain (Stubbs and Slater 1999). ics continually evol v e, and as new mod- Ph a r m a c o l o g y 353:149–158, 1998. PK C is comprised of a family of enzyme els are developed, scientific and clinical subtypes (Par ker 1994). Rec e n t l y , understanding of the neurobiology of ENG E L , S.R., AN D ALL A N , A.M. 5- H T 3 re c e p t o r knockout models of two of these sub- over-expression enhances ethanol sensitivity in alcoholism will most certainly continue mice. Ps y c h o p h a r m a c o l o g y 144: 411–415, 1999. types have been tested for alcohol behav- to advance. ■ iors. PKCgis excl u s i v ely expres s e d in the ENG E L , S.R.; LYO N S , C.R.; AN D ALL A N , A.M. 5- brain, including regions associated with HT 3 receptor over-expression decreases ethanol sel f - administration in transgenic mice. alcohol sedation and ataxia. Tests of References Ps y c h o p h a r m a c o l o g y 140:243–248, 1998. initial sensitivity to the sedating effects of alcohol demonstrated that mice lack- Banbury Conference on Genetic Background in GOL D M A N , D. Candidate genes in alcoholism. Mice. Mutant mice and neuroscience: Recom- Clinical Neuroscience 3:174– 181, 1995. in g PK Cgwer e less sensitive than wild- mendations concerning genetic background. type control mice. This may be rel a t e d Ne u r o n 19:755– 759, 1997. GRO B I N , A.C.; MAT T H E W S , D.B.; DEV A U D , L.L.; to PKCg’s action at the GABA rec e p t o r , AN D MOR R O W , A.L. The role of GABAA re c e p t o r s A BAR N A R D , E.A; SKO L N I C K , P.; OLS E N , R.W.; in the acute and chronic effects of ethanol. Ps y c h o - as alcohol-enhanced inhibition of the MOH L E R , H.; SIE G H A R T , W.; BIG G I O , G.; pharmacology 139:2–19, 1998. receptor function was absent in brain BRA E S T R U P , C.; BAT E S O N , A.N.; AN D LAN G E R , S.Z. tissue from the null mutant mice com- International union of pharmacology. XV. Subtypes HAR R I S , R.A. Ethanol actions on multiple ion pa r ed with the wild-type controls (Har r i s of g-aminobutyric acidA receptors: Classification on channels: Which are important? Al c o h o l i s m : the basis of subunit structure and receptor function. Clinical and Experimental Research 23 : 1 5 6 3 – 1 5 7 0 , et al. 1995). In addition, rapid and Pharmacological Reviews 50:291–313, 1998. 19 9 9 . ch r onic tolerance to alcohol was decrea s e d in these mice, albeit dependent on BAU E R , K.; ESS R I C H , C.; BAL S I G E R , S.; WIC K , M.J.; HAR R I S , R.A.; MCQ U I L K E N , S.J.; PAY L O R , R.; HAR R I S , R.A.; FRI T S C H Y , J.M.; AN D LÜS C H E R , B. ABE L I O V I C H , A.; TON E G A W A , S.; AND WEH N E R , J. ba c k g r ound genotype (Bowers et al. Rescue of g2 subunit-deficient mice by transgenic M. Mutant mice lacking the gisoform of protein e 1999, 2000a). PKC is also highly overexpression of the GABAA receptor g2S or g2L kinase C show decreased behavioral actions of ex p r essed in the brain and is localized subunit isoforms. European Journal of Neuroscience ethanol and altered function of g-a m i n o b u t y r a t e in some but not all of the same brain 12(7):2639–2643, 2000. type A receptors. Proceedings of the National Academy of Sciences US A 92:3658– 3662, 1995. regions as PKCg. Tests of alcohol co n - BOW E R S , B.J.; OWE N , E.H.; COL L I N S , A.C.; sumption have indicated that mice lack- ABE L I O V I C H , A.; TON E G A W A , S; AN D WEH N E R , HOD G E , C.W.; MEH M E R T , K.K.; KEL L E Y , S.P.; ing PKCe in c re a s e d their drinking 200 J.M. Decreased ethanol sensitivity and tolerance MCMAH O N , T.; HAY W O O D , A.; OLI V E , M.F.; pe r cent over that of control mice and, development in gamma-PKC null mutant mice is WAN G , D.; SAN C H E Z -P ER E Z , A.M.; AN D MES S I N G , dependent on genetic background. Alcoholism: R. O . Supersensitivity to allosteric GABAA re c e p t o r in contrast with PKCgmutants, demon- Clinical and Experimental Research 23:387–397, 1999. modulators and alcohol in mice lacking PKCe. st r a t e d an in c re a s e d sensitivity to alcohol- Nature Neuroscience 2:997–1002, 1999. induced sedation (Hodge et al. 1999). BOW E R S , B.J.; COL L I N S , A.C.; AN D WEH N E R , J.M. Background genotype modulates the effects of g- HOM A N I C S , G.E.; FER G U S O N , C.; QUI N L A N , J.J.; Tests of alcohol sensitivity in kinase PKC on the development of rapid tolerance to DAG G E T T , J.; SNY D E R , K.; LAG E N A U R , C.; MI, Z.P.; knockout mice are not limited to the ethanol-induced hypothermia. Addiction Biology WAN G , X.H.; GRA Y S O N , D.R.; AN D FIR E S T O N E , PK C family of enzymes. Rec e n t l y , 5:47–58, 2000a. L. L . Gene knockout of the a 6 subunit of the g- res e a r ch has been conducted using the aminobutyric acid type A receptor: Lack of effect BOW E R S , B.J.; MIH A L E K , R.M.; HOM A N I C S , G.E.; on responses to ethanol, pentobarbital, and general fo l l o wing types of mice: mice lacking WEH N E R , J.M. Decreased ethanol consumption in anesthetics. Molecular Pharmacology 51:588–596, 1997. the protein kinase A reg u l a t o r y subunit GA B A A delta subunit knockout mice. Alcoholism: bII (i.e., PKAbII), mice lacking the Clinical and Experimental Research 24:97A, 2000b. HOM A N I C S , G.E.; LE, N.Q.; KIS T , F.; MIH A L E K , R.; HAR T , A.R.; AN D QUI N L A N , J.J. Ethanol tolerance ty r osine kinase Fyn (i.e., a kinase of the CAM P E R , S.A. Research applications of transgenic and withdrawal responses in GABAA receptor alpha amino acid tyrosine), dopamine beta mice. Bi o T e c h n i q u e s 5:638– 650, 1987. 6 subunit null mice and in inbred C57BL/6J and strain 129/SvJ mice. Alcoholism: Clinical and hyd r oxylase knockout mice (i.e., mice CAP E C C H I , M. Targeted gene replacement. Sc i e n t i f i c lacking the dopamine beta form of the Am e r i c a n 270:52–59, 1994. Experimental Research 22:259–265, 1998. hyd r oxylase enzyme), and mice lacking HOM A N I C S , G.E.; HAR R I S O N , N.L.; QUI N L A N , J.J.; CRA B B E , J.C.; PHI L L I P S , T.J.; FEL L E R , D.J.; HEN , forms of the gene for aldehyde dehy- KRA S O W S K I , M.D.; RIC K , C.E.M.; DE BLA S , A.L.; R.; WEN G E R , C.D.; LES S O V , C.N.; AN D SCH A F E R , dr ogenase (i.e., an enzyme invol v ed in G.L. Elevated alcohol consumption in null mutant MEH T A , A.K.; KIS T , F.; MIH A L E K , R.M.; AUL , J.J.; AN D FIR E S T O N E , LL. Normal electrophysiological alcohol metabolism) (see table). mice lacking 5-HT1B serotonin receptors. Na t u r e Ge n e t i c s 14:98–101, 1996. and behavioral responses to ethanol in mice lacking From these investigations of knock- the long splice variant of the g2 subunit of the g- out and transgenic mice, res e a rc h e r s CRA B B E , J.C.; WAH L S T E N , D.; AN D DUD E K , B.C. aminobutyrate type A receptor. Ne u r o p h a r m a c o l o g y can easily appreciate the complexity of Genetics of mouse behavior: Interactions with labo- 38:253–265, 1999. al c o h o l ’s actions and the difficulty in ratory environment. Sc i e n c e 284:1670–1672, 1999. LI, T.K., AN D MCBRI D E , W.J. Ph a r m a c o g e n e t i c de f i n i t i v ely interpreting the contribu- DIA M O N D , I., AN D GOR D O N , A.S. Cellular and models of alcoholism. Clinical Neuroscience 3: 1 8 2 – tions of individual proteins to alco- molecular neuroscience of alcoholism. Ph y s i o l o g i c a l 188, 1995. holism. The examples discussed in this Re v i e w s 77:1–20, 1997. MIH A L E K , R.M.; BAN E R J E E , P.K.; KOR P I , E.R.; ar ticle are by no means an exhaustive EL-G HU N D I , M.; GEO R G E , S.R.; DRA G O , J.; QUI N L A N , J.J.; FIR E S T O N E , L.L.; MI, Z.P.; rep r esentation of all mutant mouse FLE T C H E R , P.J.; FAN , T.; NGU Y E N , T.; LIU , C.; LAG E N A U E R , C.; TRE T T E R , V.; SIE G H A R T , W.;

Vol. 24, No. 3, 2000 183 ANA G N O S T A R A S , S.G.; SAG E , J.R.; FAN E S L O W , QUI N L A N , J.J.; HOM A N I C S , G.E.; AN D FIR E S T O N E , SCH U C K I T , M.A. Reactions to alcohol in sons of alco- M.S.; GUI D O T T I , A.; SPI G E L M A N , I.; LI, A.; L.L. Anesthesia sensitivity in mice that lack the b3 holics and controls. Alcoholism: Clinical and DELOR E Y , T.M.; OLS E N , R.W.; AN D HOM A N I C S , subunit of the g- aminobutyric acid type A recep- Experimental Research 12:465–470, 1988. G. E . Attenuated sensitivity to neuroactive steroids tor. An e s t h e s i o l o g y 88:775–780, 1998. in g-aminobutyrate type A receptor delta subunit SIL V E R , L.M. Mouse Genetics. New York: Oxford knockout mice. Proceedings of the National Academy RUB E N S T E I N , M.; PHI L L I P S , T.J.; BUN Z O W , J.R.; University Press, 1995. of Sciences USA 96:12905–12910, 1999. FAL Z O N E , T.L.; DZI E W C Z A P O L S K I , G.; ZHA N G , G.; FAN G , Y.; LAR S O N , J.L.; MCDOU G A L L , J.A.; CHE S T E R , STU B B S , C.D., AN D SLA T E R , S.J. Ethanol and pro- PAR K E R , P.J. Protein kinase C: History and per- J.A.; SAE Z , C.; PUG S L E Y , T.A.; GER S H A N K , O.; LOW , tein kinase C. Alcoholism: Clinical and Experimental spectives. In: Kuo, J.F., ed. Protein Kinase C. Ne w M.J.; AN D GRA N D Y , D.K. Mice lacking dopamine D4 Research 23:1552–1560, 1999. York: Oxford University Press, 1994. pp. 3–15. receptors are supersensitive to ethanol, cocaine, and WEH N E R , J.M., AN D BOW E R S , B.J. Use of transgen- PHI L L I P S , T.J.; BRO W N , K.J.; BUR K H A R T -K AS C H , S.; methamphetamine. Ce l l 90:991–1001, 1997. ics, null mutants, and antisense approaches to study WEN G E R , C.; KEL L Y , M.A.; RUB I N S T E I N , M.; SAP P , D.W., AN D YEH , H.H. Et h a n o l - G A B A A ethanol’s actions. Alcoholism: Clinical and GRA N D Y , D.; AN D LOW , M.J. Alcohol preference Ex p e r i m e n t a l Re s e a r c h 19:811–820, 1995. and sensitivity are markedly reduced in mice lack- receptor interactions: A comparison between cell lines and cerebellar purkinje cells. The Journal of ing dopamine D2 receptors. Nature Neuroscience WIC K , M.J.; RAD C L I F F , R.A.; BOW E R S , B.J.; MAS C I A , Pharmacology and Experimental Therapeutics 1:610–615, 1998. M.P.; LÜS C H E R , B.; HAR R I S , R.A.; AN D WEH N E R , 284:768–776, 1998. PIC C I O T T O , M.R., AN D WIC K M A N , K. Us i n g J.M. Behavioral changes produced by transgenic knockout and transgenic mice to study neurophysi- SAU E R , B. Inducible in mice using overexpression of g2L and g2S subunits of the ology and behavior. Physiological Reviews 78 : 1 1 3 1 – the Cre/lox system. Methods: A Companion to GA B A A receptor. European Journal of Neuroscience 1163, 1998. Methods in Enzymology 14:381–392, 1998. 12(7):2634–2638, 2000.

Brainwaves, gene scans, and street maps: What can they tell us about alcoholism?

he answer to this and other questions can be found in Alcohol Ale rt , the quarte r l y bulletin published by the National Institute on Alcohol Abuse and Alcoholism. TAlcohol Alert provides timely information on alcohol res e a r ch and treatment. Each issue addresses a specific topic in alcohol res e a r ch and summarizes critical find- ings in a brief, four-page, easy-to-read format. Our most recent issue—From Genes to Geography—The Cutting Edge of Alcohol Research (No. 48)—demonstrates how com- puterized data analysis can integrate vast quantities of disparate data to support alcoholism prevention and treatment efforts.

For a free subscription to Alcohol Alert, write to: National Institute on Alcohol Abuse and Alcoholism, Attn.: Alcohol Alert, Publications Distribution Center, P.O. Box 10686, Rockville, MD 20849–0686. Fax: (202) 842–0418. Alcohol Alert is available on the World Wide Web at http://www.niaaa.nih.gov

184 Alcohol Research & Health