Circling behavior exhibited by a transgenic insertional mutant

Molecular Brain Research, 8 (1990) 355-358

355

Elsevier

BRESM 80065

Circling behavior exhibited by a transgenic insertional mutant Anil K. Ratty 1, Lawrence W. Fitzgerald 2, Milt Titeler 2, Stanley D. Glick 2, John J. Mullins 1'* and Kenneth W. Gross I 1Department of Cellular and Molecular Biology, Roswell Park Memorial Institute, Buffalo, N Y 14263 (U.S.A.) and ZDepartment of Pharmacology and Toxicology, Albany Medical College, Albany, N Y 12208 (U.S.A.)

(Accepted 26 June 1990) Key words: Circling behavior; Insertional mutagenesis; Transgenicmouse

We report here of an abnormal circling behavior expressed in the TgX15 transgenic mouse line as a result of insertional mutagenesis. Homozygous transgenic mice expressed the phenotype while heterozygous transgenics were normal. We also found that the dopamine D2 receptor binding sites in the striata of the circling mice were significantlyelevated by about 31% compared to normal heterozygoustransgenic mice. Other transgenic lines constructed with the same transgene appeared normal suggesting that, in the TgX15 line, a genetic locus significant in mammalian motor behavior has been disrupted. In the course of making transgenic mice, integration of the exogenous D N A into the host genome sometimes alters an endogenous function, resulting in a mutation. The phenotype of insertional mutations are usually recessive and therefore only apparent when the transgenic mice are bred to homozygosity. The integrated DNA then provides a molecular 'handle' for the recovery and analysis of the mutated gene, as illustrated by the analysis of the embryonic lethal mutation resulting from the retroviral insertion of the Moloney murine leukemia virus into the 5" end of the al(I)-collagen gene 2°. Very few cases of insertional mutagenesis in transgenic animals have been reported to date and these include two mutations with limb deformities 15'27, one with a defect in spermatogenesis 16 and a number of developmental lethal mutations4,5,11-13,21,23,26. Here we report of an insertional mutation in a transgenic mouse line which resulted in a circling behavior occurring almost continually at night and in response to stress (e.g. hand clapping) during the daytime. This mutant was one of 19 transgenic mouse lines made by microinjection of a 24 kb X h o I genomic D N A fragment containing the mouse Ren-2 d renin gene into BCF 1 (C57BL/10Ros pd x C3H/HeRos) fertilized eggs la. This fragment contained 5 kb of upstream, 10 kb of intronexon and 9 kb of downstream Ren-2 d sequences. Several of these lines were bred to homozygosity but only one of these, designated TgX15, exhibited the circling behavior.

This suggested that the phenotype was specific for a particular integration site rather than the sequence or the expression of the transgene. We investigated transgene expression by primer extension analysis which differentiated expression of Ren-2 a transgene from that of the endogenous Ren-1 c gene. No evidence of transgene expression was found in the brain, liver, kidney and submaxillary glands of mice that were heterozygous or homozygous for the transgene is. The integrant, about 65-70 kb, comprised two and one half copies of the transgene in a head-to-tail tandem array. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA from mice tail biopsies was carried out to identify mice that were either homozygous or heterozygous for the integrant (Fig. 1). Breeding of heterozygous or homozygous mice with each other or with parental strain (BCF1) was carried out to determine if the circling phenotype co-segregated with the insertion site (Table I). The matings indicated that inheritance of the phenotype was consistent with an autosomal recessive mode at a single locus. Mice that expressed the abnormal circling behavior were also found to be homozygous for the integrant while heterozygotes were phenotypically normal. The autosomaUy recessive circling behavior thus segregated with the integration site of the transgene sequences. Animals that were homozygous or heterozygous for the integrant were fertile and there were no significant differences in the litter sizes from the different

*Present address: Pharmakologisches Institute der Universit/it Heidelberg, Im Neuenheimer Feld 366, 6900 Heidelberg, F.R.G. Correspondence: A.K. Ratty, Department of Cellular and Molecular Biology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY

14263, U.S.A. 0169-328X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

356 C NC o bc

CCN NC N N CCNN d e f (j h i j k I m n

NC o p

140" {nser tionol M u t o n t s

Kb 120

23.1 9.4 6.0

~

BO

1500

m

:]200

o

60-

~

dO"

BOO

5.2 4.0 Fig. 1. Diagnostic test for transgene integrant. Southern blot analysis of RFLPs from Bg/II-digested tail DNAs with a DNA fragment isolated from the 3" host flanking sequence. Lanes a-n are DNA from progeny of a heterozygous transgenic × homozygous transgenic mating; lane o, non-transgenic BCF DNA; and, lane p, circling insertional mutant DNA. C, mice with circling phenotype; and N, no circling phenotype. Homozygous transgenic animals are identified by the presence of a 10 kb band and the absence of a 6 kb band. Non-transgenic animals have the 6 kb band but not the 10 kb band while heterozygous transgenics have both the 6 and 10 kb bands. The 23 kb band was due to a BgllI restriction site further downstream of the integrant.

sets of parents. However, we did not find the expected 1:1 segregation of circling mice and normal mice from the homozygous transgenic male and heterozygous transgenic female parents and we cannot explain this anomalous result. The circling phenotype was observed as early as 1-2 weeks of age. A TgX15 pedigree was established and the integrant was found to be transmitted for at least 6 generations without any apparent genetic rearrangements in the integrant and the host flanking sequences. TABLE I

0

20"

', 20

0

1 40

60

BOUND

8 0 'tOO 1 2 0 1 4 0 ( f rna l / r a g )

J

0 .I5

1

1 [3H-NMSP]

2

2.5

3

tqM

Fig. 2. Saturation isotherms of the specific binding of [3H]Nmethylspiperone to striata from circling mutants and normal littermates. Results are shown from 1 of 3 independent experiments. Inset: Scatchard transformation of the data. Method: the binding analysis was performed as described previously9 but with some minor modifications. The striata were homogenized with a Dounce homogenizer at 4 °C in 50 mM Tris-HC1, 05 mM N%EDTA, 140 mM NaC1, 10 mM MgSO4 and 0.1% (w/v) ascorbic acid. The homogenate was centrifuged at 14,000 rpm at 4 °C for 15 rain. The pellet was resuspended in homogenate buffer and recentrifuged twice before being stored at -80 °C. Radioligand binding assays were performed in triplicate in a volume of 2 ml homogenate buffer containing [3H]N-methylspiperone (NEN; 83.4 Ci/mmol) and 10-5 M pargyline. 10 ~tM (-)-sulpride was used to define non-specific binding. Incubations were initiated by addition of 1 mg wet weight striatal tissue and carried out at 37 °C for 30 min followed by rapid filtration over glass-fiber filters (Schleicher and SchneU) which had been pre-soaked in 0.1% polyethyleneimine. The filters were washed with ice-cold 50 mM Tris-HCl, pH 7.4 and the radioactivity retained was counted by liquid scintillation spectroscopy. Data were analysed by the non-linear regression analysis EBDA 1° and RS/1 (Bolt, Beranck and Newman; Boston, MA) programs.

T h e circling b e h a v i o r was q u a n t i t a t e d in a cylindrical

Circling phenotype segregates as a recessive trait

rotometer

which

detects

quarter

turns

by photocell

activation and full turns w h e n f o u r q u a r t e r turns are

Parents

m a d e in the s a m e direction. This a p p a r a t u s discriminates

I~lx@ No. of parents No. of litters Total no. of pups Litter size No. of pups with circling phenotype % pups with circling phenotype x2(l) Segregation tested

6

6 12

BxO 2 2 4

Bx(~ 6 6 26

77 6.4 (0.6)

23 6.0 (1.5)

150 5.8(0.5)

18

23

45

BxO 2 4 4 32 7.5(0.7)

0

b e t w e e n r o t a t i o n s and r a n d o m m o v e m e n t and b e t w e e n r o t a t i o n s in d i f f e r e n t directions 8. T h e circling b e h a v i o r was q u a n t i t a t e d

as net

rotations

and

%

preference

d e f i n e d by the f o l l o w i n g f o r m u l a e :

TABLE II Quantitation of spontaneous nocturnal (16 h) circling

23 0.108 1:3

100

37 26.7

0

No. of animals

Net rotations of individual mice

%preference (means + S.E.M.)

4

903,4464,658,60*

90.3 + 6.1"*

4

2,55,51,103

67.9 -+ 8.4

1: 1

Number of parents refers to the number of different individual wild-type, heterozygous or homozygous transgenic animals which were used to generate the numbers within each column of data. Circles represent females, squares males. Litter size expressed as mean (S.E.M.).

Insertionalmutants Heterozygous littermates

*Animal got disconnected from the recording device. **P < 0.05.

357 TABLE III Binding characteristics of [3H] N-methylspiperone in mouse striatum

Results presented as mean + S.E.M.

Bma x (fmol/mg protein)

Kd (nM)

Insertional mutants

Heterozygous littermates

120.5 + 8.8* 0.081 + 0.013

91.7 + 9.3 0.073+ 0.008

*P < 0.05.

Net rotations = (full turns in preferred direction) - - (full turns in non-preferred direction) % Preference = (full turns in preferred direction)/(total full turns) × 100

heterozygous mice. Similar measurements were also made in the nucleus accumbens and cortex, structures which are known to modulate striatal mechanisms involved in circling. The levels of these neurochemicals in the circling mutants did not differ from those in their normal heterozygous littermates (data not shown). Dopamine D 2 receptor binding sites in the striatum were assayed using radioligand binding techniques. The density of D 2 binding sites in the striatum assayed by maximal binding of [3H]N-methylspiperone (nmax) was significantly increased by about 31% (P < 0.05) in the insertional mutants compared to heterozygous littermates (Fig. 2 and Table III); there was no significant difference between the two groups in the dissociation constants (Kd) of the D 2 ligand with the receptor. These data suggest that an up-regulation of dopamine D 2 receptors may be correlated with the circling phenotype. There are a number of spontaneous mouse mutants that express the 'shaker-waltzer' syndrome, which is characterized mainly by a tendency to run in circles, hyperactivity, jerking head movements and abnormal responses to changes in position 24. All of these mutants have gross deformities and degeneration in the bony and membranous labyrinths of the inner ear. While our insertional mutant displayed circling, it did not exhibit the other behaviors characteristic of the 'shaker-waltzer' syndrome. Furthermore, there was no evidence of any hearing loss or inner ear deformities or degeneration in our mutant 19, thus supporting the hypothesis that a central, rather than a vestibular, derangement caused the circling phenotype. In conclusion, the transgene integration in the TgX15 mouse line has apparently disrupted an endogenous genetic locus affecting motor function. This is the first instance in which insertional mutagenesis has resulted in a well-characterized behavioral abnormality. Further neurochemical and neuroreceptor analysis of the mutant should help in the understanding of the molecular mechanisms of motor function.

The % preference parameter enables comparisons between mice independent of the total activity. Table II shows the results obtained for the nocturnal circling behavior of representive mutant mice and normal heterozygous littermates. Spontaneous nocturnal rotation by rodents has been reported to be a normal phenomenon 6. However, the net rotations and % preference of the circling phenotype was significantly increased over the normal littermates. The 'rotating rodent' had been used for more than 15 years as a model in studies addressing the function of the dopaminergic nigrostriatal component of the basal ganglia. Ungerstedt 25 demonstrated that high rates of circling behavior can be elicited by amphetamine, a dopamine releaser, or apomorphine, a dopamine agonist, in rats that had been unilaterally depleted of dopamine by 6-hydroxydopamine lesions. Circling phenomena have been extensively investigated in rats 7"17 and in humans 2, particularly in patients with hemi-parkinsonism 3 and schizophrenia I and are understood to be mediated by components of the dopaminergic system. Since circling has been associated with an imbalance of nigrostriatal function, as measured by striatal amine content, metabolism and receptor mechanisms, we next determined, by high performance liquid chromatography 22, the levels of the neurotransmitters, dopamine, serotonin and norepinephrine, and their metabolites, dihydroxyphenylacetic acid, homovanillic acid and 5hydroxyindoleacetic acid, in the striata of the mutant and

We thank Dr. R.W. Keller for his help in conducting the HPLC assays, Ms. Laura Fitzgerald for help in doing the radi01igand assays and Drs. Craig Jones, Curt D. Sigmund, William Held, Dennis Stephenson, Ken Abel and Mr. John Fabian for critical reading of this manuscript. A.K.R. is a recipient of the Human Frontier Science Program Organization fellowship. Part of this work was aided by Institutional Research Grant IN-54-30 of the American Cancer Society. The transgenic mouse lines were made under the auspices of NIH Grants GM 30248-07 and HL 35792-04.

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