genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3

8
Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3 Michael L. Robinson, 1 Carl E. Allen, 1 Brian E. Davy, 1 William J. Durfee, 2 Frederick F. Elder, 3 Christopher S. Elliott, 1 Wilbur R. Harrison 4 1 Division of Molecular and Human Genetics, Dept. of Pediatrics, and Children’s Research Institute, The Ohio State University, Children’s Research Institute, 700 Children’s Drive, Rm. W492, Columbus, Ohio 43205, USA 2 Animal Resource Center, Case Western Reserve University, School of Medicine, Cleveland, Ohio, USA 3 Dept. of Pathology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA 4 Dept. of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA Received: 28 May 2002 / Accepted: 21 August 2002 Abstract. The transgenic mouse line OVE459 carries a trans- gene-induced insertional mutation resulting in autosomal re- cessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene in- sertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM ± 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome. Introduction Transgene-induced insertional mutations occur in an estimated 5–10% of transgenic founders created by pronuclear microin- jection of DNA (Palmiter and Brinster 1986). These mutations are most often recessive and are independent of transgene expression. In contrast to spontaneous or chemically induced mutations, transgene-induced insertional mutations are marked by a unique molecular tag, the transgene itself, which can facilitate molecular identification and cloning of the gene responsible for the mutant phenotype. Occasionally, these in- sertional mutations prove to be alleles of previously charac- terized spontaneous or induced mutations for which there is no molecular information (reviewed in Meisler 1989; Woychik and Alagramam 1998), and some of these have been exploited to identify the relevant gene (Woychik et al. 1985). Unfortu- nately, transgene-induced insertional mutations are not always simple insertions of the transgene. Genomic rearrangements including duplications, deletions, and translocations involving the transgene insertion locus have been reported (Woychik and Alagramam 1998) and can complicate the unambiguous identification of the gene responsible for the mutant pheno- type. Despite this caveat, the expanding wealth of mouse ge- nomic information available promises to alleviate some of the difficulties associated with complex rearrangements at the transgene integration site in many insertional mutations. Human hydrocephalus is a significant clinical problem with an estimated incidence of 1 in 1000 live births (Schurr and Polkey 1993). Hydrocephalus in human patients is often multifactorial, but several cases of familial hydrocephalus demonstrating an autosomal recessive inheritance pattern have been reported (Brady et al. 1999; Castro-Gago et al. 1996; Chow et al. 1990; Chudley et al. 1997; Game et al. 1989; Moog et al. 1998; Teebi and Naguib 1988; Zlotogora 1997; Zlotogora et al. 1994). Despite this, the causative genes for autosomal recessive human hydrocephalus have not been identified. Several distinct autosomal recessive mutations leading to hydrocephalus have been reported in mice (Bronson and Lane 1990; Clark 1932; Dickie 1968; Falconer and Sierts-Roth 1951; Gruneberg 1943a, 1943b; Hollander 1976; Punt et al. 1982; Zimmermann 1933). Recently the transcription factors Foxc1 (Mf1) and Lmx1a were identified as responsible for the hy- drocephalus mutations congenital hydrocephalus (ch) (Kume et al. 1998) and dreher (dr) (Millonig et al. 2000), respectively. The genes responsible for the mutations hydrocephalus 13 (hy1, hy2, and hy3), hydrocephalus with hop gait (hyh), ob- structive hydrocephalus (oh), and hop-sterile (hop) remain un- identified. In addition to these spontaneous mouse mutations, hydrocephalus is at least part of the phenotype in several en- gineered mice created by both insertional mutations (McNeish et al. 1990) and targeted mutations (Dahme et al. 1997; das Neves et al. 1999; Fransen et al. 1998; Homanics et al. 1993; Huang et al. 1995; Ibanez-Tallon et al. 2002; Lindeman et al. 1998). Here we describe a new transgene-induced insertional mutation, OVE459, resulting in autosomal recessive hydro- cephalus. This mutation does not complement the spontaneous mutation hy3 and represents a new allele of this gene. Fur- thermore, we report genetic and physical mapping of the transgene insertion locus, providing new clues to the location of the relevant hydrocephalus-inducing mutant gene. Mammalian Genome 13, 625–632 (2002). DOI: 10.1007/s00335-002-2201-8 Correspondence to: M.L. Robinson; E-mail: robinsom@pediatrics. ohio-state.edu

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Page 1: Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3

Genetic mapping of an insertional hydrocephalus-inducing mutation

allelic to hy3

Michael L. Robinson,1 Carl E. Allen,1 Brian E. Davy,1 William J. Durfee,2 Frederick F. Elder,3 Christopher S. Elliott,1

Wilbur R. Harrison4

1Division of Molecular and Human Genetics, Dept. of Pediatrics, and Children’s Research Institute, The Ohio State University, Children’sResearch Institute, 700 Children’s Drive, Rm. W492, Columbus, Ohio 43205, USA2Animal Resource Center, Case Western Reserve University, School of Medicine, Cleveland, Ohio, USA3Dept. of Pathology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA4Dept. of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA

Received: 28 May 2002 / Accepted: 21 August 2002

Abstract. The transgenic mouse line OVE459 carries a trans-gene-induced insertional mutation resulting in autosomal re-cessive congenital hydrocephalus. Homozygous transgenicanimals experience ventricular dilation with perinatal onsetand are noticeably smaller than hemizygous or non-transgeniclittermates within a few days after birth. Fluorescence in situhybridization (FISH) revealed that the transgene inserted in asingle locus on mouse Chromosome (chr) 8, region D2-E1.Genetic crosses between hemizygous OVE459 mice and miceheterozygous for the spontaneous mutation hydrocephalus-3(hy3) produced hydrocephalic offspring with a frequency of22%, demonstrating that these two mutations are allelic. Agenomic library was made by using DNA from homozygousOVE459 mice, and genomic DNA flanking the transgene in-sertion site was isolated and sequenced. A PCR polymorphismbetween C57BL/6 DNA and Mus spretus was used to map thelocation of the transgene insert to 1.06 cM ± 0.75 proximal toD8Mit152 by using the Jackson Laboratory Backcross DNAPanel Mapping Resource. Furthermore, sequence analysisfrom a mouse bacterial artificial chromosome (BAC) clone,positive for unique markers on both sides of the transgeneinsertion site, demonstrated that the genomic DNAs flankingeach side of the transgene insertion are physically separated byapproximately 51 kb on the wild-type mouse chromosome.

Introduction

Transgene-induced insertional mutations occur in an estimated5–10% of transgenic founders created by pronuclear microin-jection of DNA (Palmiter and Brinster 1986). These mutationsare most often recessive and are independent of transgeneexpression. In contrast to spontaneous or chemically inducedmutations, transgene-induced insertional mutations aremarked by a unique molecular tag, the transgene itself, whichcan facilitate molecular identification and cloning of the generesponsible for the mutant phenotype. Occasionally, these in-sertional mutations prove to be alleles of previously charac-terized spontaneous or induced mutations for which there is nomolecular information (reviewed in Meisler 1989; Woychikand Alagramam 1998), and some of these have been exploited

to identify the relevant gene (Woychik et al. 1985). Unfortu-nately, transgene-induced insertional mutations are not alwayssimple insertions of the transgene. Genomic rearrangementsincluding duplications, deletions, and translocations involvingthe transgene insertion locus have been reported (Woychik andAlagramam 1998) and can complicate the unambiguousidentification of the gene responsible for the mutant pheno-type. Despite this caveat, the expanding wealth of mouse ge-nomic information available promises to alleviate some of thedifficulties associated with complex rearrangements at thetransgene integration site in many insertional mutations.

Human hydrocephalus is a significant clinical problem withan estimated incidence of 1 in 1000 live births (Schurr andPolkey 1993). Hydrocephalus in human patients is oftenmultifactorial, but several cases of familial hydrocephalusdemonstrating an autosomal recessive inheritance pattern havebeen reported (Brady et al. 1999; Castro-Gago et al. 1996;Chow et al. 1990; Chudley et al. 1997; Game et al. 1989; Mooget al. 1998; Teebi and Naguib 1988; Zlotogora 1997; Zlotogoraet al. 1994). Despite this, the causative genes for autosomalrecessive human hydrocephalus have not been identified.

Several distinct autosomal recessive mutations leading tohydrocephalus have been reported in mice (Bronson and Lane1990; Clark 1932; Dickie 1968; Falconer and Sierts-Roth 1951;Gruneberg 1943a, 1943b; Hollander 1976; Punt et al. 1982;Zimmermann 1933). Recently the transcription factors Foxc1(Mf1) and Lmx1a were identified as responsible for the hy-drocephalus mutations congenital hydrocephalus (ch) (Kume etal. 1998) and dreher (dr) (Millonig et al. 2000), respectively.The genes responsible for the mutations hydrocephalus 1–3(hy1, hy2, and hy3), hydrocephalus with hop gait (hyh), ob-structive hydrocephalus (oh), and hop-sterile (hop) remain un-identified. In addition to these spontaneous mouse mutations,hydrocephalus is at least part of the phenotype in several en-gineered mice created by both insertional mutations (McNeishet al. 1990) and targeted mutations (Dahme et al. 1997; dasNeves et al. 1999; Fransen et al. 1998; Homanics et al. 1993;Huang et al. 1995; Ibanez-Tallon et al. 2002; Lindeman et al.1998).

Here we describe a new transgene-induced insertionalmutation, OVE459, resulting in autosomal recessive hydro-cephalus. This mutation does not complement the spontaneousmutation hy3 and represents a new allele of this gene. Fur-thermore, we report genetic and physical mapping of thetransgene insertion locus, providing new clues to the locationof the relevant hydrocephalus-inducing mutant gene.

Mammalian Genome 13, 625–632 (2002).

DOI: 10.1007/s00335-002-2201-8

Correspondence to: M.L. Robinson; E-mail: robinsom@pediatrics.

ohio-state.edu

Page 2: Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3

Materials and methods

Transgenic mouse production and PCR analyses. A transgenicconstruct, aA-BDNF/bFSH, designed to express BDNF in the devel-oping lens, was produced by subcloning a human BDNF cDNA fusedto the 30 UTR of the bovine aFSH gene (obtained from MurrayRobinson, Amgen, Thousand Oaks, Calif.) into the aA-crystallinpromoter vector CPV2, replacing the SV40 intron and polyadenylationsignal of CPV2 (Robinson et al. 1995) (Fig. 1). The 1552-bp micro-injection fragment in paA-BDNF/bFSH was isolated from the vectorby digestion with SStII (Gibco/BRL, Gaithersburg, Md.), purified,and microinjected into FVB/N pronuclear stage embryos as described(Taketo et al. 1991). Transgenic founders were identified by PCRanalysis with primers PR4 (50-GCATTCCAGCTGCTGACGGT-30), asense primer complementary to the murine aA-crystallin promoter,and 11421 (50-ACACCTGGGTAGGCCAAGCCACCTT-30), an an-tisense primer complementary to human BDNF. A diagnostic band of308 bp was amplified from genomic DNA of transgenic mice following30 cycles of standard PCR with an annealing temperature of 58�C.

Fluorescence in situ hybridization (FISH). FISH was done aspreviously reported (Majumder et al. 1998). Briefly, slides were G-banded and metaphase spreads photographed. The slides were thendestained and hybridized with a 1159-bp digoxigenin-labeled probecontaining the BDNF coding region. Previously photographed G-banded metaphase cells were found and used to determine the preciselocation of the transgene insertion. The chromosomes were counter-stained with 0.5 lg/ml propidium iodide in an antifade buffer and thepreparations viewed with an Olympus BX60 epifluorescence micro-scope. Previously photographed G-banded metaphase cells were foundand rephotographed with Kodacolor 100 Gold film.

Southern blot. Ten micrograms of genomic DNA per lane was di-gested with EcoRI, BamHI, or NheI. Digested DNA was electropho-resed through agarose and transferred to a nylon filter as described(Robinson et al. 1998). Blots were probed with the transgene-specific1159-bp XbaI fragment from paA-BDNF/bFSH following randomprime labeling with 32P-dCTP. Washing and exposure to X-ray filmwere carried out as described (Robinson et al. 1998).

Construction and screening of the OVE459 genomic li-brary. Genomic DNA was isolated from the combined liver, spleen,and kidneys of three hydrocephalic OVE459 pups at 14 days of ageand partially digested with BamHI. The BamHI ends of the partiallydigested OVE459 genomic DNA were partially filled in with the Kle-now fragment of DNA polymerase I and ligated to predigested lambdaphage DNA arms according to instructions provided with the LambdaFix II/XhoI Partial Fill-In Vector Kit (Stratagene, La Jolla, Calif.).The ligation reaction was packaged into phage particles using aGigapack III XL packaging extract (Stratagene) according to themanufacturer’s instructions. Approximately 1.5 · 106 recombinantlambda clones were screened by hybridization with a 32P-labeled 1159-bp XbaI fragment containing the BDNF coding region from paA-BDNF/bFSH. Four positive phage clones derived from independenthybridization spots on the primary screen were purified to homoge-neity. DNA from these phage clones was analyzed by restriction di-gestion and Southern blot with a probe made from a 32P-labeled 1552-bp microinjection fragment.

Sequencing. Initial insert end sequence was determined from purifiedDNA from lambda clones BAA and CAA by using T3 and T7 se-quencing primers present in the lambda vector phage arms. The uniquegenomic fragments in lambda clones BAA and CAA were subclonedinto pBluescript KS- (Stratagene) as NotI fragments. The resultantplasmid clones pB19 and pC5 were approximately 14 and 16 kb in size,respectively. Sequence was determined from each end of the plasmidswith M13 and M13R primers. Template was prepared with Qiagenminipreps. Dye terminator chemistries were employed, and the se-quence was determined on an ABI377 automated sequencer. The GPS-1 Genome Priming system (New England Biolabs) was employed tointroduce unique primer-binding sites in each construct per the man-ufacturer’s directions. Internal sequence in each plasmid construct wasdetermined with the PrimerS and PrimerN supplied with the kit. Se-quence was determined as above by using dye terminator chemistries.

Non-transposon sequence was removed and the insert sequence as-sembled with PHRED/PHRAP/CONSED (Ewing and Green 1998;Gordon et al. 1998).

Genetic complementation analysis. Six untested mice (three malesand three females) from the B6CBACa-Aw-J/A-hy3/+ colony at theJackson Laboratory (Bar Harbor, Me), stock number 002703 (theresult of matings between two mice proven, by test breeding, to carrythe hy3 mutation) were purchased and bred to hemizygous transgenicmice from the OVE459 line. Pups from such matings were genotypedfor the presence of transgene by PCR and were anesthetized prior toperfusion with PBS followed by Bouin’s fixative. Fixed brains wereanalyzed by gross inspection for signs of ventricular dilation. For de-termination of hydrocephalic frequency, reported in Table 1, onlymating pairs where hydrocephalic pups were born were included formatings, including presumed hy3 heterozygotes. Also, only those litterswere counted where all pups were accounted for and typed between 10and 21 days after birth. All OVE459 data are from mice maintained onan FVB/N inbred background. The hy3 mutation has been moved toan FVB/N inbred background since purchase from the Jackson Lab-oratory. To facilitate this genetic background change, the molecularmarkers D8Mit248 and D8Mit215 (polymorphic between the originalhy3 genetic background and FVB/N) were used to identify likelycarriers of the hy3 mutation. D8Mit248 and D8Mit215 are approxi-mately 11 and 5 cM, respectively, on either side of where we believe thehy3 mutation to reside. The hy3 data in Table 1 represent a combi-nation of data gathered before and during this genetic backgroundchange.

Interspecific backcross. PCR primers C1 (50-CAAAAGAGCTGAGGAAAGATG-30) and C2 (50-TAGGATGCAGGGGGTT-ATT-30) were derived from the sequence of the mouse genomic insert inphage clone CAA approximately 13 kb from the transgene insert. Thisprimer set was tested on FVB/N, C57BL/6JEi, and SPRET/Ei genomicDNA. C57BL/6JEi and SPRET/Ei genomic DNA was purchased fromthe Jackson Laboratory DNA Resource. Genomic DNA from all 188animals in the combined Jackson BSS [(C57BL/6JEi · SPRET/Ei)F1 · SPRET/Ei] and BSB [(C57BL/6J · Mus spretus)F1 · C57BL/6J] interspecific backcrossmapping panels from the JacksonLaboratoryBackcross DNA Panel Mapping Resource were genotyped for the PCRpolymorphism with primers C1 and C2.

Identification of BAC clones. PCR primers B3 (50-GGTCCGA-GAAACCTGCCTGCTATCA-30) and B4 (50-ACCCACGTCGCCTGTGTTCATTATG-30) were derived from the sequence of the mousegenomic insert in phage clone BAA approximately 11 kb from thetransgene insert. Primers B3 and B4 were used to screen a CITBMouseBAC DNA library pool (Research Genetics, Huntsville, Ala.) ac-cording to provided instructions. BAC DNA clones positive for theB3/B4 primer set were screened with primers C1 and C2 by using PCR.Additional BAC clones were identified in the RPCI-23 mouse BAC

Fig. 1. The 1552-bp microinjection construct used to create OVE459consisted of the murine aA-crystallin promoter (aA) fused to the hu-man BDNF coding sequence (hBDNF) linked to the 30 UTR and poly-adenylation signal from the a subunit of the bovine follicle-stimulatinghormone gene (FSH pA). The coding sequence and 30 UTR werecloned into the CPV2 vector as a 1159-bp XbaI fragment, and thistransgene-specific XbaI fragment was used as a probe for Southern andFISH analysis. Restriction enzyme sites shown are SstII (S), NheI(Nh), BamHI (B), XbaI (X), EcoRI (E), and NotI (N).

626 M.L. Robinson et al.: Genetic mapping of an hy3 allele

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library by screening filter arrays obtained from Pieter de Jong (Chil-dren’s Hospital Oakland Research Institute, Oakland, Calif.) by hy-bridization with the unique genomic inserts from phage clones BAAand CAA. The clone RPCI23-21B7 was completely sequenced by theNIH-funded Genome Sequencing Network. The accession number forthe complete genomic sequence of RPCI23-21B7 is AC069308.

Results

Production of OVE459 transgenic mice. The aA-BDNF/bFSHtransgenic construct, designed to express BDNF in the lens,was injected into pronuclei of FVB/N mouse zygotes, resultingin the production of a single male transgenic founder for thetransgenic line designated OVE459. This founder appearedphenotypically normal in all respects including the eye. Threeother transgenic founders were produced with a similartransgenic construct CPV2/BDNF, differing from aA-BDNF/bFSH only by the replacement of the bovine aFSH 30 UTRwith the SV40 intron and polyadenylation signal of CPV2.These other BDNF transgenic founders also failed to exhibitany phenotypic abnormalities (data not shown).

The founder for line OVE459 was bred to female FVB/Nmice and transmitted the transgene to a portion of his progeny.These F1 hemizygous transgenic mice were phenotypically in-distinguishable from their wild-type littermates. When hemi-zygous OVE459 transgenic mice were interbred to producehomozygous transgenic mice, a portion of the pups in the re-sulting litters usually exhibited a failure to thrive and died,typically between the first and third week after birth. Closerexamination revealed that the dying pups could most often beidentified by 4–6 days after birth. The most severely affected ofthese mice died by about 10 days after birth. The less severelyaffected mice began to exhibit enlargement of the head thatprogressed until death, typically by 21 days, but always priorto 42 days after birth (Fig. 2). Gross necropsy revealed that allaffected mice exhibited enlargement of the brain ventricles andcharacteristic thinning and softening of the top of the skullvault consistent with congenital hydrocephalus. Males andfemales appear to be equally affected. No such phenotype wasever observed in pups from matings between hemizygousOVE459 mice and wild-type mice. These observations led to

the hypothesis that the transgenic line OVE459 carried a re-cessive, transgene-induced insertional mutation leading tocongenital hydrocephalus.

OVE459 mice carry a recessive insertional hydrocephalus-in-ducing mutation. Southern blots of genomic DNA from hy-drocephalic and phenotypically normal OVE459 transgenicmice hybridized with a transgene-specific probe revealedidentical hybridization patterns (data not shown). Therefore,all OVE459 transgenic mice carried the same transgene inser-tion site, and different transgene integration sites could notexplain the phenotypic variation among transgenic mice. Theseanalyses also demonstrated that approximately 12–15 copies ofthe transgene inserted in a single genomic location. In mostcases, the production of transgenic mice by pronuclear injec-tion results in transgenic founders containing multiple copiesof the transgene in a tandem head-to-tail array at a singlegenomic locus (Brinster et al. 1981), but more complex inte-gration patterns are possible.

The presence of multiple copies of the transgene within lineOVE459 facilitated further analysis of this transgenic line byFISH to determine the chromosomal location where theOVE459 transgene inserted. FISH was also used to test thehypothesis that hydrocephalic mice were transgenic homo-zygotes and that transgenic mice without ventricular enlarge-ment were hemizygous for the transgene. Metaphasechromosome spreads were prepared from both a hydroce-phalic and a phenotypically normal OVE459 transgenicmouse, stained with Giemsa and analyzed by FISH with atransgene-specific probe (Fig. 3). As predicted, the hydroce-phalic mouse was homozygous for the transgene, and the non-hydrocephalic mouse was hemizygous for the transgene. FISHalso revealed that the transgene array was within a single sitenear the distal end of mouse Chr 8 (region D2-E1).

OVE459 is an allele of hy3. The spontaneous, recessive, hy-drocephalus-inducing mouse mutation hy3 has also been ge-netically mapped to the distal portion of mouse Chr 8. Thereported phenotype for homozygous hy3 mutant mice closelyparalleled the gross abnormalities in homozygous OVE459transgenic mice. Therefore, we initiated a breeding comple-mentation experiment to determine whether the insertional

Fig. 2. Wild-type (top) and mutant (bottom)littermates from an intercross of hemizygousOVE459 transgenic mice. Mutant mice arecharacteristically smaller than either non-trans-genic or wild-type-appearing transgenic litter-mates. Although most mutant mice die prior toweaning, mice that survive beyond 10 daysexhibit progressive enlargement of the skull.

M.L. Robinson et al.: Genetic mapping of an hy3 allele 627

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mutation in OVE459 represented a new allele of hy3. The generesponsible for the mutation in hy3 is unknown, and there iscurrently no published molecular test to distinguish wild-typefrom hy3 mutation carriers in progeny from known heterozy-gous mutant parents. As all homozygous hy3 mice die prior tosexual maturity, each of the six mice purchased from theJackson Laboratory had a 2/3 probability of carrying themutation. Therefore, according to the binomial distribution,the probability that none of the six mice carried the mutationwas 0.0014. Considering this, we reasoned that if none of thesix matings between potential hy3 carriers and transgenicOVE459 mice produced hydrocephalic offspring, the twomutations were unlikely to be allelic. Four of these six matingpairs produced hydrocephalic offspring. The carrier status ofthese four (two males and two females) potential hy3 hetero-zygotes was subsequently confirmed by interbreeding to obtainhy3/hy3 homozygous pups. Upon gross inspection, the hy-drocephalus in OVE459 homozygotes, hy3/hy3 homozygotes,and double OVE459/hy3 heterozygotes was indistinguishable,both in terms of the kinetics and gross pathology (Fig. 4). Asexpected, each of these hydrocephalic offspring and all sub-sequent hydrocephalic offspring between OVE459 and hy3mice were positive for the transgene (data not shown). Theproportion of hydrocephalic offspring resulting from matingsbetween OVE459 hemizygotes and between OVE459 hemizy-gous and hy3 heterozygous mice was very similar and ap-proached the predicted 25% Mendelian ratio of an autosomalrecessive trait with full phenotypic penetrance (Table 1). Thecomplete failure of the hy3 mutant allele to complement theOVE459 insertional mutation is the best evidence that thesetwo mutations are allelic and very likely result in disruptedfunction in the same gene or set of genes on mouse Chr 8.

Cloning the transgene insertion site. In contrast to the spon-taneous hy3 mutation, where no molecular markers are

known, the insertion of the transgene in OVE459 mice pro-vided a molecular tag into the genomic locus likely to containthe gene relevant to the hydrocephalic phenotype. Tightlinkage of the hydrocephalus-inducing mutation to theOVE459 transgene insertion sight was suggested by the failureof the mutation to segregate away from the transgene in over20 generations. To clone the transgene insertion site, a ge-nomic lambda phage library was constructed with DNA fromhomozygous OVE459 hydrocephalic mice. This library wasscreened with a transgene-specific hybridization probe (seeFig. 1). Both the FISH data and the genomic Southern blotprobed with a transgene-specific probe indicated that thetransgene inserted in a single genomic location, and theSouthern blot furthermore suggested that 12–15 copies of thetransgene inserted in a tandem array. Since the microinjectionconstruct was approximately 1.6 kb, the tandem transgenearray would be expected to be 19.2–24 kb in length. Since thephage clones in the library kit used can incorporate only re-combinant inserts of 9–23 kb, we realized that it was ratherunlikely that we would recover the entire transgene array witha significant length of flanking genomic DNA on both sides.Therefore, we expected to recover three types of transgene-containing phage clones with the transgene-specific probe.The clones expected included those containing only transgeneand those that contained transgene and the flanking genomic

Fig. 3. FISH mapping of the OVE459 transgeneinsertion locus. (A) A portion of a single meta-phase spread from a hydrocephalic OVE459transgenic mouse is shown following G-banding.(B) The same metaphase region after hybrid-ization to the transgene-specific XbaI fragmentprobe. Hybridization was confined to a singlesite near the distal end of mouse Chr 8, arrows.(C) An ideogram of mouse Chr 8 with an arrowmarking the approximate site of the OVE459transgene insertion between G-bands D2–E1.

Table 1. Frequency of hydrocephalus among offspring of OVE459 and hy3matings.

Matings Total offspring Hydrocephalic Percentage

OVE459 · OVE459 829 199 24.0%hy3 · hy3 584 130 22.3%OVE459 · hy3 355 79 22.3%Overall total 1768 408 23.1%

628 M.L. Robinson et al.: Genetic mapping of an hy3 allele

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DNA on either the centromeric or telomeric side of the in-sertion site. Conveniently, in the lambda Fix II vector systemused to make the library, both SalI and NotI digestion releasethe genomic insert from the vector arms. NotI, in contrast toSalI, also cuts the transgene construct once. This allowed usto estimate both the total length of the genomic insert as wellas the approximate proportion of the genomic insert thatconsisted of transgene copies. Of the four lambda clonesanalyzed in detail, one, designated BAA, was a unique cloneconsisting of an insert of 18 kb. Restriction analysis revealedthat clone BAA contained approximately five copies oftransgene and a mouse genomic insert of 11 kb. Anotherlambda clone, BCA, contained a genomic insert consistingonly of tandemly arranged transgene copies. The two re-maining phage clones, CAA and DAA, appeared identical inrestriction digests, consisting of a genomic insert of 16 kb,approximately 3 kb of which was transgene. Further restric-tion mapping and Southern blotting confirmed that clonesDAA and CAA differed substantially from clone BAA, sug-gesting that these represented opposite sides of the transgeneinsertion (data not shown). Phage clones BAA and CAA wereselected for further analysis. Sequence analysis revealed thatthe transgene array was on the long (23 kb) phage arm sideand the mouse genomic DNA was on the short (9 kb) phagearm side in both phage clones (Fig. 5). Unique PCR primersets were designed to amplify mouse genomic DNA near thedistal ends of each phage clone, relative to the transgene in-sertion, by using sequence information obtained with the T3sequencing primer. These primer sets were named C1 and C2,from the genomic region on the phage clone CAA, and B3and B4, from the genomic region of the BAA phage clone. Asexpected, the PCR primers B3 and B4 from the shorter mousegenomic DNA insert in clone BAA failed to amplify a bandin the longer mouse genomic insert in clone CAA, confirmingthat these clones represent opposite sides of the transgeneinsertion.

Genetic and physical mapping of the OVE459 transgene inser-tion locus. The primer sets B3/B4 and C1/C2 were used toscreen C57BL/6JEi and SPRET/Ei genomic DNA for poly-morphisms that could be used to map the genetic location ofthe genomic DNA flanking the transgene insert in OVE459with the Jackson Laboratory Backcross DNA Panel MappingResource (Rowe et al. 1994). According to the sequence in-formation from the OVE459 genomic clones, the B3/B4 andC1/C2 PCR primers should amplify DNA fragments of 327 bpand 415 bp respectively on FVB/N strain genomic DNA. Nopolymorphisms were detected with primers B3/B4. In contrast,while the primer set C1/C2 amplified an apparently identicallysized band in both FVB/N and C57BL/6JEi DNA, a distinctlylarger band was amplified in SPRET/Ei genomic DNA (Fig.6A). This polymorphism was used to map the location of thegenomic insert in clone CAA in both the BSS and the BSBmapping panel from the Jackson Laboratory, consisting of atotal of 188 backcross animals (Rowe et al. 1994). The C1/C2polymorphism mapped cleanly in both mapping panels andwas assigned the nomen D8Mlr1. D8Mlr1 does not recombinewith D8Mit151 and was placed 5.32 ± 1.64 cM distal toD8Mit313 and 1.06 ± 0.75 cM proximal to D8Mit152 (Fig.6B). This position corresponds to approximately 54 cM on themouse genome informatics Chr 8 consensus linkage map(Blake et al. 2002).

The interspecific backcross panels provided a precise ge-netic location for the genomic DNA on the CAA side of thetransgene insertion, but as no mappable PCR polymorphismwas identified on the opposite flank, genetic mapping of theBAA side was not possible. Therefore, we screened a 129/Svbacterial artificial chromosome (BAC) library from ResearchGenetics, using primers B3 and B4 to identify BAC clones thatencompassed the OVE459 transgene insertion site. Two BACclones, 9N1 and 218P4, were positive for the B3/B4 primer set.Of these two clones, 218P4 was also positive for the C1/C2primer set from the CAA genomic insert (data not shown),

Fig. 4. Coronal brain slices demonstrating thehydrocephalic pathology 7 days after birth. (A)A hemizygous OVE459 brain with barely per-ceptible lateral ventricles (arrows) and a thirdventricle that is too small to be seen at thismagnification. (B), (C), and (D) are brains fromOVE459 homozygous, hy3 homozygous, anddouble OVE459/hy3 heterozygous pups,respectively. The gross pathology of thesehydrocephalic brains appears identical withenlarged lateral (asterisk) and third (arrowhead)ventricles.

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showing that these two amplified regions are physically linkedon a single BAC clone. The mouse genomic insert in BAC218P4 was determined by pulsed field gel electrophoresis to beapproximately 120 kb in size (data not shown). An additionalC57BL/6J BAC clone, RPCI23-21B7, positive for markers onboth sides of the transgene insertion site, was identified byhybridization. BAC 21B7 contained a genomic insert of ap-proximately 240 kb and completely encompassed the genomicregion covered by BAC 218P4 (data not shown). From thecomplete sequence of BAC 21B7, we determined that themarkers defined by primer sets B3/B4 and C1/C2 (D8Mlr1) areseparated by approximately 51 kb of wild-type genomic DNA,and that the microsatellite markers D8Mit151 and D8Mit213lie within the intervening sequence.

While the precise nature of the insertional mutation inOVE459 mice remains unknown, we do know that it is not sosimple as a deletion of the DNA between the flanks of thetransgene insertion sites. To date, we have found no evidencefor a deletion of any kind associated with the OVE459 trans-gene insertion. Southern hybridization with transgene flankingprobes suggests that genomic DNA surrounding the insertionlocus has been duplicated, and we have not yet been able to useflanking genomic probes to distinguish hemizygous versushomozygous OVE459 transgenic mice (data not shown).

Discussion

OVE459 is a transgene-induced insertional mutation thatrepresents a new allele of hy3. There are several pieces of ev-idence supporting this claim. First, transgenic hemizygotesdisplay no unusual phenotype, while transgenic homozygotesdemonstrate a pathological course indistinguishable from hy3homozygous mice. Second, the OVE459 transgene insertionsite has been physically and genetically mapped to the distalportion of mouse Chr 8, corresponding to the location of thehy3 mutation. Most compelling, however, is the fact that miceboth heterozygous for the hy3 mutation and hemizygous forthe OVE459 transgene develop hydrocephalus, demonstratingthat these two mutations do not complement each other.

The hy3 mutation was first identified by Hans Grunebergand described as an autosomal recessive inherited phenotypeincluding nasal discharge, runting, and hydrocephalus withvariable postnatal onset and survival time (Gruneberg 1943b).

R. J. Berry further characterized the hy3 mutant phenotypeafter several generations of inbreeding and reported a consis-tent runted phenotype in presumed homozygotes with ac-companying hydrocephalus, but no nasal discharge (Berry1961). Berry also reported that the incidence of hydrocephalusamong offspring of heterozygous hy3 parents as 16.6%, sig-nificantly less than the expected 25% for an autosomal reces-sive trait with complete penetrance (Berry 1961). The inbredmutant stock was obtained and maintained as heterozygotes(stock HyIII/Le) by the Jackson Laboratory until 1995, whenthe original inbred stock stopped breeding, and the mutationwas then recovered and maintained on a mixed C57BL/6 andCBA/Ca hybrid background (Alicia Valenzuela, personalcommunication). No molecular markers have been reported toidentify heterozygous versus homozygous mutant mice, and,therefore, hy3 heterozygotes are traditionally identified by testmating. We observed a frequency of hydrocephalus amongoffspring of heterozygous hy3 parents of 22.3%. This is higherthan the 16.6% observed by Berry and much closer to theexpected 25% for a fully penetrant autosomal recessive phe-notype. We have no evidence that any homozygous hy3 orOVE459 mutants escape hydrocephalus on any of the geneticbackgrounds we have examined (FVB/N, C57BL/6 or mix-tures of FVB/N, C57BL/6, and CBA/Ca). However, in theabsence of a large study to determine this point with clearmolecular tests to distinguish homozygous mutants from hemi-or heterozygotes, we cannot be absolutely certain that 100% ofthe homozygotes succumb to hydrocephalus. We have rarelyobserved mice phenotypically typed as heterozygotes atweaning who developed lethal hydrocephalus by 6 weeks ofage. No nasal discharge among the homozygous hy3, OVE459

Fig. 5. Diagrammatic representation of the lambda phage clones BAAand CAA representing opposite ends of the OVE459 transgene inser-tion site. The location of the T3 primer used for sequencing and thePCR primers B3/B4 and C1/C2 are indicated with arrowheads. Eachphage clone contains tandem copies of transgene (broken gray line)linked to the 23-kb phage arm, and mouse genomic DNA (solid blackline) linked to the 9-kb phage arm. Restriction enzyme sites shown areSalI (S) and NotI (N).

Fig. 6. (A) PCR primers C1 and C2 from clone CAA were used toscreen genomic DNA from FVB/N (FVB), C57BL/6JEi (B6), andSPRET/Ei (SP) for polymorphisms to use for mapping. Note that theDNA amplified from SPRET/Ei with C1 and C2 primers is larger thanthe band amplified from FVB/N and C57BL/6JEi with the sameprimers. MW and NT represent DNA size markers and no template,respectively. (B) Haplotype figure combining data from The JacksonBSB and BSS backcrosses showing part of Chr 8 with loci linked toD8Mlr1. Loci are listed in order with the most proximal at the top. Theblack boxes represent the C57BL6/JEi allele, and the white boxes theSPRET/Ei allele. The number of animals with each haplotype is givenat the bottom of each column of boxes. The percentage recombination(R) between adjacent loci is given to the right of the figure, with thestandard error (SE) for each R. Raw data from The Jackson Labo-ratory were obtained from the World Wide Web address http://www.jax.org/resources/documents/cmdata.

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or double heterozygous mice was observed. It is possible thatthe lack of nasal discharge and increased penetrance of thehydrocephalic phenotype relate to different sets of modifierloci present in the original and current hy3 stocks.

It is our belief that this work provides the most preciselocation to date for the hydrocephalus-inducing gene in hy3mutant mice. The hy3 mutation was originally mapped bythree-point test cross byMargaret C. Green and communicatedto Roy Robinson of the Jackson Laboratory by a personalletter in 1970 (Jeff Ceci, personal communication). In this letter,Green placed hy3 approximately 17 cM distal to Os (oligosyn-dactylism) and 11 cM proximal to e (recessive yellow) on mouseChr 8. This information was used to place the location of hy3 at57 cM on the consensus Chr 8 linkage map (Blake et al. 2002).Using molecular markers, we have determined that the trans-gene insertion site does not recombine with D8Mit151 in 188backcross animals in the combined BSB and BSS interspecificbackcross mapping panel from the Jackson Laboratory. This isconsistent with the sequence information obtained from BACRPCI23-21B7 indicating that D8Mlr1 is only 47.8 kb fromD8Mit151. While transgene-induced insertional mutations caninduce genomic rearrangements making cloning of the relevantgenes difficult, our observations suggest the gene responsiblefor inducing hydrocephalus in hy3 homozygous mice will befound in close proximity to D8Mit151. Further experimentswill be necessary to determine whether the relevant mutationlies between the flanks of the OVE459 transgene insertion siteor adjacent to the insertion locus. To our knowledge, there areno genes or family members of genes that have been previouslyassociated with mammalian hydrocephalus near this region ofmouse Chr 8.

The region encompassing D8Mit151 on mouse Chr 8 cor-responds to human Chr 16q21-23. Haploinsufficiency of thisregion of 16q has been associated with general growth failure,perinatal death, facial bone dysgenesis, shortened limbs, andhydrocephalus (Fryns et al. 1981; Naritomi et al. 1988; Riveraet al. 1985). Interestingly, hydrocephalus has also been re-ported in an infant with a balanced translocationt(4;16)(q35;q22.1) including this region (Callen et al. 1990;Taysi et al. 1978). Furthermore, Sakuragawa and Yokoyamamapped the Chr 16 breakpoint of this or a similar hydro-cephalus-associated t(4;16)(q35;q22.1) translocation betweenhaptoglobin and calretinin (Sakuragawa and Yokoyama 1994).This group also discovered a rearrangement of genomic DNAwithin 1.2 Mb of calretinin with genomic DNA from fibro-blasts carrying this translocation. Interestingly, according tothe Mouse Genome Sequencing Consortium V3 Assembly,D8Mlr1 is approximately 376 kb distal to calretinin on mouseChr 8. The relationship between these human conditions as-sociated with loss or rearrangement of 16q21-23 and hydro-cephalus in hy3 homozygotes is unclear, but represents aninteresting avenue for further investigations.

Acknowledgments. The authors acknowledge Dr. Paul A. Overbeek,Molecular and Cellular Biology, Baylor College of Medicine for hissupport of this project; Katrina Waymire of Emory University forpronuclear injection; and Jason Wirth, William Olson, and ChristianM. Rizo for technical assistance. DNA sequencing was accomplishedwith the excellent technical assistance of Huachun Zhong with theadvice and assistance of Dr. Robert Munson. The DNA SequencingCore Facility at Children’s Research Institute is supported in part bythe NIH grant HD34615. We also acknowledge the NIH-funded Ge-nome Sequencing Network for sequencing the BAC clone RPCI23-21B7 and the Mouse Genome Sequencing Consortium for provingmouse genome assembly information. The authors received valuablenonpublished information from both Alicia Valenzuela of the JacksonLaboratory, Bar Harbor, Me., and Dr. Jeff Ceci at the University ofTexas Medical Branch, Galveston, Tex. The authors also thank Dr.Gail E. Herman, Dr. Heithem El Hodiri, Dr. Michael Weinstein, and

Jamie S. Depelteau for critical review of the manuscript, and bothLucy B. Rowe and Mary E. Barter of The Jackson LaboratoryBackcross DNA Panel Mapping Resource for their invaluable assis-tance with the genetic mapping of the OVE459 mutation. This workwas supported by a grant from the March of Dimes Birth DefectsFoundation, 6-FY00-280.

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