Perinatal lethality (ple): A mutation caused by integration of a transgene into distal mouse chromosome 15

GENOMICS

4,

&i-504

Perinatal

(1989)

Lethality (p/e): A Mutation Caused by Integration Transgene into Distal Mouse Chromosome 15

DAVID R. BEIER, CYNTHIA C. MORTON,* Department

AYA LEDER, RACHEAL WALLACE, AND PHILIP LEDER

of Genetics, Harvard Medical School, Howard Hughes Medical Institute, Boston, Massachusetts *Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115 Received

October

INTRODUCTION

The introduction of new genetic material into the germline of mice by microinjection or retroviral infection is proving to be a powerful means of analyzing gene regulation and function. The stable incorporation of exogenous DNA into mouse chromosomes, however, results in the disruption of the wild-type genome, which has been found to have a variety of consequences. In several cases, these techniques have resulted in the creation of new and interesting mutations. For example, a retroviral insertion found to be a recessive embryonic lethal was shown to have disrupted the aI collagen gene (Schnieke et al., 1983). In our own laboratory, mice homozygous for a transgene insertion generated by microinjection were found to have a defect in limb development. This mutation was shown to be allelic to the previously identified limb deformity (Id) mutation (Woychik et al., 1985). Another limb deformity mutation is associated with a different transgene insertion and is allelic with Sy@ (Overbeek et al., 1986). 498 Inc. reserved.

02115, and

12, 1988

Several other homozygous lethal mutations associated with transgene insertion have been reported (Covarrubias et al., 1985; Mark et al., 1985; Shani, 1986; Soriano et al., 1987; McNeish et al, 1988). Retroviral integration is facilitated by enzymatic activity encoded by the virus and results in minimal disruption of wild-type sequences. In contrast, stable incorporation of microinjected DNA can be associated with deletions, duplications, and complex rearrangements of mouse sequences (Woychik et al., 1985; Covarrubias et al., 1987; Wilkie and Palmiter, 1987). In one case the introduction of a transgene appears to have resulted in a reciprocal chromosome translocation (Mahon et al., 1988). In cases in which a recessive mutation is associated with the integration of exogenous sequences, the transgene provides a means by which the mutant locus can be cloned and analyzed. Moreover, all transgenes provide potential reference points for identifying closely linked loci using chromosome walking or jumping techniques. For this purpose, it is necessary to locate the transgene precisely on the genetic map of the mouse genome. We have been studying a line of mice carrying a transgene that contains an MMTV viral promoter ligated about 1 kb 5’ to a mouse c-myc proto-oncogene (Stewart et al., 1984). In this line, originally called 1479a, the transgene was expressed in many tissues and caused a variety of tumors, including lymphomas, mast cell sarcomas, and Sertoli cell tumors (Leder et al., 1986). Using cytogenetics and recombinational analysis, we have mapped this transgene precisely to the distal portion of mouse chromosome 15. This region contains a number of developmental loci, as well as a proto-oncogene and a homeobox gene. Further, this region appears to be syntenic between mouse and man. Using genetic analysis of noninbred mice, we have demonstrated a significant deficiency in the expected number of homozygous transgenic mice surviving to weaning age. Homozygotes that do survive are smaller than their siblings and breed poorly. When intercrosses

We have used cytogenetic and recombinational analysis to determine the position of a transgene integrated into the mouse genome. The transgene maps to band F on the physical map of mouse chromosome 15 by in situ analysis and is tightly linked genetically to a cluster of loci that include the mutations caracul (Ca) and microcytic anemia (mk). Genetic analysis of the offspring of noninbred animals carrying the transgene and marker loci demonstrates a significant deficiency of homozygous progeny at weaning. When inbred mice heterozygous for the transgene are mated, about one-quarter of their offspring are homozygous; none of these animals survives more than 1 day after birth. It appears likely that a recessive insertional mutation has occurred as a result of transgene integration into a locus required for postnatal viability. We call this mutation fransgenic perinatal lethality U’g.ple) . 0 lSS9 Academic Press, Inc.

088%7543/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

of a

AN

INSERTIONAL

MUTATION

are performed with inbred transgenic animals, about one-fourth of the progeny are homozygous; however, none of these animals survives more than 1 day after birth. These results demonstrate that integration of the transgene has resulted in a deleterious recessive mutation with extreme expressivity in an inbred background. The phenotype we have observed does not correspond to those described for other loci on distal chromosome 15 (Green, 1981) and is probably not allelic with them. Because of the uniform early onset of mortality noted in an inbred background, we have named this locus pie (for perinatal lethality) and we call this transgenic mutant Tg.ple. MATERIALS

AND

METHODS

Mice The construction of the transgenic line and its dominant phenotype have been previously described (Stewart et al., 1984; Leder et al., 1986). Briefly, fertilized ova of a CD-l X C57B1/6J cross were microinjetted with a fusion gene carrying an MMTV promoter 5’ to a mouse c-myc genomic fragment. A founder animal was identified, bred against CD1 animals, and maintained in this outbred background. Strains used for mapping experiments were obtained from The Jackson Laboratory and were as follows: C3HeB/FeJ a/a CaJ SI Hm, Mk/Re mk/mk, C57B1/6JEi Eh; B6C3Fe a/a Dom, and ABP/Le a/a b/b bt/bt p/p se/se wa-l/wa-1. When the mice were about 4 weeks of age, tails were cut for genotyping by Southern hybridization analysis and the animals were analyzed for phenotype by visual inspection (Ca, bt/bt, Dom, and Eh) or blood smears (mk/mk). DNA Analysis Genomic DNA was extracted from l- to 2-cm tail fragments by digestion in 1 ml of 10 mM Tris (pH 8), 100 mitf NaCl, 10 mM EDTA in 0.5% SDS with 0.1 mg/ml proteinase K overnight at 5O’C. A 0.5-ml portion of this was phenol-extracted, ethanol-precipitated, and resuspended in 100 ~1 of dHZO. Then, 30 ~1 was digested with BamHl and Clal, electrophoresed in 0.8% agarose, and transferred to nitrocellulose according to standard techniques (Maniatis et aZ., 1982). Hybridization of a 32P-labeled c-myc cDNA probe identifies a 6.0-kb endogenous locus band and a 5.2-kb transgene band (Fig. 1). Cell Culture The KJ cell line was derived from a mammary tumor which arose spontaneously in the heterozygous transgenie founder female. The cells, which grow as an adherent line, were maintained in DME medium supplemented with 5% fetal calf serum. Southern hybridiza-

IN

MOUSE

CHROMOSOME

15

+ I +

TgITg

Tgl+

499

+ +

c-myc Tg

FIG. 1. Southern blot hybridization analysis of transgenic mice. Genomic DNA prepared from wild-type (+/+I, heterozygous (Tg/ t), and homozygous (Tg/Tg) transgenic mice was digested with BamHl and Clal, electrophoresed, and transferred to nitrocellulose. Two samples of each are shown. Hybridization of a ?-labeled mouse c-myc DNA fragment identifies two bands: the endogenous c-myc gene at 6.0 kb and the transgene at 5.2 kb. Heterozygotes can be distinguished from homozygotes by the apparent abundance of the transgene relative to endogenous c-myc.

tion analysis of genomic DNA prepared from KJ cells revealed no rearrangement or change in copy number of the transgene. In Situ Hybridization Metaphase chromosomes were prepared from the KJ cell line according to standard cytogenetic protocols. Cytogenetic analysis revealed an essentially tetraploid karyotype. Chromosomes appeared to be structurally normal, with the exception of one iso(5) detected in one cell. Chromosomal in situ hybridization was performed as previously described (Morton et al., 1984). The recombinant plasmid, a 2.1-kb Pst-Pst cDNA fragment of the mouse c-myc gene cloned in pBR322, was nick-translated using all four tritiated dNTPs to a specific activity of 1.69 X lo7 cpm/pg. Metaphases were evaluated for hybridization at the microscope by using a combination of incident ultraviolet and transmitted visible lights and were photographed for analysis. RESULTS

In Situ Hybridization Photographs of 15 metaphase spreads were analyzed for localization of silver grains on or directly beside the chromosomes. Of a total of 63 grains counted, 9 (14.3%) were found at 15D2-El, a region that includes the bands to which the c-myc gene has been localized in the mouse (Wiener et al., 1984; Adolph et al., 1987a). An additional peak of hybridization of equal intensity (10 grains, 15.9% of the total number of silver grains

BEIER

FIG. 2. In situ chromosomal analysis. (A) A metaphase Arrowheads identify two sites of hybridization on chromosome The probe was a c-myc cDNA fragment. The endogenous c-myc to band F identifies the transgene.

ET

AL.

chromosome spread of the heterozygous transgenic cell line KJ is pictured. 15. (B) A histogram of the hybridization results for chromosome 15 is shown. gene has been previously reported to hybridize to bands D2-D3. Hybridization

15, recombination analysis with a number of known phenotypic mutants was performed. When doubly heterozygous animals carrying Tgple and mk (microcytic anemia) were backcrossed against homozygous mk/mk animals, only two nonparental recombinants out of 188 offspring were identified (recombination percentage = 1.2 + 0.8, Table 1). These were both nonanemic, nontransgenic animals (++/+mk). In addition, both recombinant offspring were found in one litter (out of 32 analyzed), suggesting the possibility that they reflect mitotic recombination during embryogenesis prior to germ cell development, rather than meiotic recombination. Because homozygous mk/mk animals are less viable due to their anemia (Russell, 1979), however, other recombinant progeny may not have survived. Therefore,

tallied), representing the putative site of transgene integration, was noted at the distal end of chromosome 15 in band F (Fig. 2). This region of mouse chromosome 15 contains the Int-1 gene, which is an MMTV integration site (Nusse and Varmus, 1982; Adolph et al., 1987a). Since the transgene contains the MMTV long terminal repeat, the possibility that it had integrated at Int-1 was analyzed. Hybridization of a 2.5kb Int-1 genomic fragment to Southern blots of DNA prepared from transgenie mice revealed no rearrangement of sequences in an 18-kb region surrounding Int-I (result not shown). Linkage Analysis In order to position Tg.ple more precisely with respect to other genes on the distal part of chromosome TABLE Linkage

Analysis

1

of Tg.ple and mk, Ca, 6t, Dom, and Eh Progeny

Parental

Cross” T&p&+x+

Recombinant

+ mk

Tg.ple + 128

+ mk 36

Tg.ple

+ mk w+x++

++

+ ca 84

T&e

+ Ca TJ&e+x*

Tg.ple + 54

+ bt

+ bt 92

Tgple

+ bt T&p&+x++

Tg.ple + 76

+Dom TJ$&+x++ +Eh

++ 2

166

1.2 + 0.8

Ca

++ 0

139

0.7 f 0.7

bt

++ 6

176

4.6 2 1.6

Dom

++ 8

56

21.4 k 5.5

Eh

++ 0

a7

0

1 2 Tg.ple

++

+ Dom 17 +Eh 45

Tg.ple

++

Tg.ple + 42

4 0

% Recombination

mk 0

Tgpie + 27

Total

a In all crosses doubly heterozygous parents were both male and female, except Tg.ple +/+ Dom, in which only male doubly parents were used. Animals were scored for genotype and phenotype at about 1 month of age. Tg.ple genotyping was accomplished under Materials and Methods. Ca, bt/bt, Dam, and Eh phenotypes were scored by visual inspection; mk/mk anemia phenotypes by examination of blood smears. Percentage recombination is shown plus or minus the standard error.

heterozygous as described were scored

AN

INSERTIONAL

MUTATION

Tg.pk was mapped with respect to the dominant coattexture mutation Cu (caracul). This mutation, which results in a curly coat, maps 0.85 CM from mk (McFarland and Russell, 1975). No decrease in viability for heterozygous or homozygous Cu animals has been reported (Green, 1981). When doubly heterozygous Tg.pk +/+ Ca animals were crossed with wild-type animals, only one recombinant offspring, which carried both Tg.pk and Ca, was identified in 139 progeny (recombination percentage = 0.7 & 0.7, Table 1). Linkage of Tg.pk and Cu is maintained in offspring of this recombinant, which provides an easily scored phenotypic marker for the presence of the transgene. Rare recombination between Tg.pk and both Ca and mk suggests that the transgene maps very near these loci. An alternative explanation for the paucity of recombination between these markers would be recombination suppression due to a chromosome rearrangement associated with transgene insertion. Recombination suppression due to chromosome rearrangement has been well documented for the t haplotype of chromosome 17 (Silver and Art&, 1981; Artzt et al., 1982; Herrmann et al., 1986) and probably accounts for the absence of recombination between the Eh (hairy ears) mutation and other distal markers on chromosome 15 (Lane and Liu, 1984; M. Davisson, personal communication). Therefore, it was necessary to map Tg.pk with respect to additional loci on chromosome 15 in order to determine whether recombination occurred normally with markers at a distance from Ca and mk. The coat color mutations bt (belted), which maps 4.25.2 CM from Co, and Don (dominant megacolon), which maps 21 CM away, were analyzed (Green, 1981; Lane and Liu, 1984). Animals doubly heterozygous for Tg.pk and bt were backcrossed against homozygous bt/bt animals. Eight recombinant offspring were identified in 176 progeny; 2 were Tg bt/+ bt and 6 were ++/+bt (recombination percentage = 4.6 + 1.6, Table 1). This may, however, represent an overestimate of the recombination frequency, since the belted phenotype can sometimes be difficult to identify on a light-color background. (The tester strain carrying bt, ABP/Le, carries the unlinked recessive coat color allele pink-eye (p). This gene, which causes a light coat, segregated randomly with respect to Tg.pk (result not shown).) When doubly heterozygous males carrying Tg.pk and Dom were crossed with wild-type females, 12 recombinants out of 56 progeny were identified (recombination percentage = 21.4 f 5.5, Table 1). The Dom mutation is associated with significant mortality (Lane and Liu, 1984); therefore both nonrecombinant and recombinant Dom offspring are underrepresented. Finally, the transgene was mapped with respect to Eh. As noted, this mutation fails to recombine with

IN

MOUSE

CHROMOSOME

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15

several chromosome 15 mutations (including Ca) and probably represents a chromosomal rearrangement. Similarly, no recombinant8 were found in 87 progeny of doubly heterozygous Tg.pk +/+ Eh animals crossed with wild-type mice (Table 1). The results of recombination analysis of Tg.pk with these various mutants demonstrate that the integration site of the transgene is about 1 CM away from the Ca and mk loci (Fig. 3). The data suggest that the map position of the transgene is proximal to these loci (relative to the centromere), but do not conclusively establish this placement. Decreased

Viability

of Homozygous

Transgenic

Mice

The identification of dominant markers tightly linked to Tg.pk provides a genetic means of identifying homozygous transgenic mice. If doubly heterozygous Tg.ple +/+ Ca animals are intercrossed, the genotype of the offspring will be Tg.pk +/Tg.pk +, Tgpk +/+ Co, or + Cal+ Ca. Since Cu is a dominant marker, animals that do not have the caracul phenotype should be homozygous for the transgene. Because this result will not hold true if recombination occurs between the Tg.pk and the marker gene, identification of homo-

bt “an Ht hl *w Sha

Ca VS med

mk Gdc-1 N

Tg.ple

I f-

GmYC

HOX-3

ht-1 Ela-1 G&-l

FIG. 3. The position of the Z’g.pk transgene on the genetic and physical maps of mouse chromosome 15 is depicted. The mutants Dom (dominant megacolon), bt (belted), Ca (caracul), and mk (microcytic anemia) were used in recombinational analysis with Tg.ple and are shown in boldface type. The approximate map distances (in CM) between these four loci are shown. The Eh (hairy ears) mutation (not shown) is a chromosome rearrangement of distal chromosome 15 that extends at least from Dom to Ca (11). The order of loci on the genetic map is according to Green (8) and M. Davisson (personal communication).

502

BEIER

zygotes by this method is only provisional. Noncaracul progeny of this type of intercross were found to be homozygous by breeding or Southern analysis (result not shown). The use of genetic markers also permits us to quantitate the number of homozygous and heterozygous progeny. In an intercross such as described above, of 104 offspring, 32 were caracul and not transgenic (+ Cu/+ Cu), 60 were caracul and transgenic (Tg.ple +/+ Cu), and 12 were not caracul and were transgenic (Tg.ple +/Tg.ple +) (Table 2). The deficiency of presumptive homozygous animals relative to the predicted number (based on a Mendelian distribution) is significant (X 2 = 10.15, P < 0.01). Since the animals were analyzed at weaning, we cannot report at what stage of development loss of homozygotes occurs. Similar results were found for an intercross using animals doubly heterozygous for Tg.ple and Eh (Table 2). Because the homozygous Eh/Eh mutation is lethal, in this case there are only two classes of offspring: Tg.ple +/+ Eh, which have the hairy ears phenotype, and Tg.ple +/Tg.ple +, which appear as wild-type. Again, the deficiency of presumptive homozygous progeny in this cross is significant (X2 = 10.0, P < 0.01, Table 2). To test the effect of the transgene in a homogeneous genetic background, we are making congenic C57Bl/ 6J Tg.ple mice by serial backcrosses. Seventh-generation (N7) heterozygous animals were mated and genotypes of their progeny assessed. In these mice, which do not carry genetic markers, homozygous transgenic animals can be presumptively identified by the abundance of the transgene as determined by analysis of bands on a Southern blot autoradiograph (Fig. 1). Of 64 mice observed to be born, 23 died during the first day of life. Of these, 20 were genotyped and 13 found to be homozygous (Table 3). Of the mice that survived the first day of life, 29 were eventually genotype4 among these none homozygous for the transgene were identified. Thus, at least 23% of the total number of mice born were homozygous, but none that survived more than 1 day were found. As noted previously, in a noninbred background a TABLE Frequency

of Homozygous

Intercross Genotype: Progeny:

A: Tgple

+ Ca/+ Ca 32

+/-I Tg.ple

2 Transgenic Ca X Tg.ple i-/+ 60

Ca

Offspring +/+

Ca

Tg.ple

+lTg.pie 12

+

+/Tg.ple 4

+

x* = 10.15, P < 0.01 Intercross Genotype: Progeny:

+ Eh/+ Lethal

B: Tg.ple Eh

+/+ Tgple

Eh X Tg.ple +/+ 37

Eh

x2 = 10.0, P < 0.01

+/i-

Eh

Tg.ple

ET

AL.

TABLE Intercross Survival Less than 1 day More than 1 day Total (IV = 64)

3

of N7 Heterozygotes

+I+

Tg.ple/+

4 13 17

3 16 19

Tg.ple/Tg.pk 13 0 13

ND 3 12 15

Note. Seven N7 heterozygous females were mated with four N7 heterozygous males and nine litters of progeny were analyzed. Genotypes were determined using Southern blot hybridization analysis (see Fig. 1). The apparent deficiency of heterozygotes is not statistically significant (X2 = 3.2, P > 0.05). ND, not determined.

few homozygous mice do survive. Of the 16 presumptive homozygotes identified by genetic analysis with Ca or Eh, only 5 have reproduced. These putative homozygous animals are smaller than their littermates and show increased morbidity and mortality. Only 4 have survived longer than 6 months. In the outbred CD-l background, putative homozygous animals identified by Southern analysis were also observed in general to be smaller than their littermates. When mated, these putative homozygous animals also showed poor reproductive performance, although fertile animals of both sexes have been identified. Matings of outbred homozygous animals to each other in an effort to generate a homozygous Tg.ple line were remarkably nonproductive; the rare successful intercross resulted in an average litter size of three or four offspring. DISCUSSION

We have used cytogenetic and recombinational analysis to map precisely the location of a transgene to the distal portion of mouse chromosome 15. The Tg.ple transgene lies very close to a cluster of loci that include the mutations Ca (caracul) and mk (microcytic anemia). The position of the transgene proximal to these loci is suggested by its recombination frequency with respect to the bt (belted) mutation. Cytogenetic analysis reveals that the Tg.ple transgene lies in band F on the physical map of chromosome 15. Absence of recombination between markers can be due to either close genetic linkage or recombination suppression. The latter has been shown to occur when intrachromosomal rearrangements disrupt the homology of paired chromosomes, interfering with recombination. On the basis of genetic and molecular analyses, this has been shown to account for the suppression of recombination found for markers on chromosome 17 in mice carrying the t haplotype allele (Silver and Artzt, 1981; Artzt et al., 1982; Herrmann et al., 1986). Because transgene insertion is associated with interand intrachromosomal rearrangement, it was necessary to investigate this possibility as responsible for the rare

AN INSERTIONAL

MUTATION

recombination observed between the Tg.ple transgene and the Ca and mk markers. There appears to be no evidence for gross chromosomal rearrangement by cytogenetic analysis. The preservation of normal chromosome 15 structure is supported by in situ mapping, which agreed with localization of the endogenous cmyc gene to its previously reported position in bands D2-D3 (Wiener et al., 1984; Adolph et al., 1987a). Furthermore, genetic analysis with the more proximal markers Dom and bt reveals that the Tg.ple transgene shows the amount of recombination that would be predicted for a gene mapping near the Cu and mk loci, suggesting that no large rearrangement has occurred. The identification of dominant genes tightly linked to the transgene insertion site has allowed us to use genetic analysis to identify and characterize homozygous transgenic animals. When noninbred doubly heterozygous animals carrying the transgene and marker loci in repulsion were intercrossed, there was a significant deficiency in the expected number of homozygous progeny. Homozygous Tg.ple/Tg.ple animals that do survive to weaning age have decreased size, viability, and fertility compared to their siblings. When Tg.ple animals backcrossed into the C57B1/6J background for seven generations were intercrossed, one-fourth of their progeny were homozygous; all of these mice died within 1 day of birth. These results suggest that the transgene has disrupted a locus required for normal viability, resulting in a deleterious recessive mutation. This insertional mutation does not appear to be allelic with any of the known mutants that map to the distal portion of chromosome 15, since its phenotype does not correspond to those previously reported (Green, 1981). At present, the nature of the presumptive defect in the homozygous animals is unknown. However, the uniform and rapid onset of mortality in an inbred background is suggestive of a severe metabolic or neurological derangement. The evidence that noninbred homozygous mice do survive suggests that there are unlinked genes that can compensate for the deleterious effect of the Tg.ple insertion. An important caveat to note is the fact that the transgene in this case carries an intact c-myc proto-oncogene that has been shown in the CD-l background to be transcribed in a variety of tissues. However, because of the early onset of mortality, we do not think that the phenotype we have noted is due to an increased dosage of the c-myc transgene in homozygotes. The region of chromosome 15 identified by Tg.ple is interesting for several reasons. The transgene lies near a cluster of tightly linked loci identified by mutations that have a diversity of phenotypes, including effects on epidermal (Cu, Ve, N, Shu), neural (SW), and embryonic (Ve) development (Green, 1981). The mapping of Tg.ple to band F in the physical map of chromosome 15 is also of note. The homeobox gene Hex-3 has been

IN MOUSE

CHROMOSOME

15

503

found to be tightly linked to the Cu locus (Hart et al., 1987) and has also been mapped cytogenetically to chromosome 15, band F (Adolph et uZ., 1987b). Several other cloned genes, including Int-1, G&-l, and EZu-1, have been localized to this region using in situ hybridization (Adolph et al., 1987a,b). Moreover, the human homologs of these genes all map to human chromosome 12; Hox-3 and Int-1 have been mapped more precisely to q12-q13 and q12-q14, respectively (Cannizzarro et al., 1987; Arheden et al., 1987; Turc-Care1 et al., 1987). Thus, this portion of chromosome 15 appears to be syntenic in both mouse and man. That is, the linkage relationship of genes in this region has been conserved during evolution. Many chromosomal regions have been found to be similarly syntenic (Searle et al., 1987). In most cases, it is not known whether these linkage relationships are functional or fortuitous. It would be interesting to determine whether the locus identified by Tg.pZe also has a conserved homolog on human chromosome 12. ACKNOWLEDGMENTS We gratefully acknowledge Dr. Muriel Davisson for helpful discussions and for assistance in reviewing murine karyotypes. D.R.B. was supported by a Clinical Scientist Fellowship from the Damon Runyon-Walter Wmchell Cancer Research Fund (DRG-042). C.C.M. was supported by an NIH Postdoctoral Fellowship (CA-07511). This work was supported in part by a grant from E. I. DuPont deNemours & Co., Inc.

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