Animal Models of Human Disease - Europe PMC

American Journal ofPathology, Vol. 135, No. 1, July 1989 Copyright © American Association ofPathologists

Animal Models of Human Disease Pathology and Molecular Biology of Spontaneous Neoplasms Occurring in Transgenic Mice Carrying and Expressing Activated Cellular Oncogenes

Paul K. Pattengale,* Timothy A. Stewart,t Aya Leder,t Eric Sinn,t William Muller,t Isidore Tepler,t Emmett Schmidt,t and Philip Ledertt From the Department ofPathology, Childrens Hospital of LosAngeles, LosAngeles, California,* the Department of

Genetics, Harvard Medical School,t and the Howard Hughes Medical Institute,* Boston, Massachusetts

This present review focuses on spontaneous neoplasms occurring in transgenic mice carrying and expressing activated cellular oncogenes. The historical development of transgenic mice as in vivo disease models is briefly traced, followed by a brief description of the actual technology in such systems. Additional emphasis is placed on the concept of targeting activated cellular oncogenes to specific tissues in transgenic mice. Cumulative experience with activated (Vmyc, ras, and neu (erb-B2)) oncogenes in transgenic mice is considered in detail, with particular attention paid to the observed pathology, as well as to the kinetics of disease occurrence. It is concluded that transgenic mice offer the interested investigator(s) an excellentprospective, in vivo model of oncogenesis. (Am J Pathol 1989,

135:39-61)

Gene transfer into small mammals such as mice can be achieved by microinjecting a foreign gene of interest into the pronucleus of a fertilized egg. The resultant progeny then carry these extra, exogeneously placed genes as stable chromosomal integrants, which are then inherited in Mendelian fashion through subsequent progeny. Furthermore, these transgenes are expressed at both the RNA and protein level in various target tissues of the mouse. These resulting, so-called "transgenic mice," therefore, allow interested investigators to study a variety of activated genes in the context of the living organism. The purposes of this review article are to provide the reader with a background that traces the historical devel-

opment of transgenic mice as in vivo disease models, and to briefly describe the actual technology of such transgenic systems. This will be followed by a brief review of cellular oncogenes, with an emphasis on how such oncogenes can be targeted to specific tissues in transgenic mice. This strategy of targeted oncogenesis will be expanded on in considerable detail using our cumulative experience with activated, tissue-specific cellular oncogenes and their associated pathologic effect(s) on the whole mouse. We will also briefly consider and discuss the cumulative experience of others using similar systems.

Background Historical Development of Transgenic Mice The modification of whole organism genomes, although previously proposed as a potentially powerful experimental model, has only been experimentally accessible and successful in the past 10 to 15 years.1 Although initial genome transfer experiments were somewhat successful using embryo cell mixing2 and nucleus transfer into fertilized eggs,3'- it is now clear that the most successful experimental system involves directly microinjecting the purified gene(s) of interest into the pronucleus of fertilized This work was supported in part by grants from the American Business for Cancer Research Foundation, E. I. DuPont de Nemours and Co., the Leukemia Society of America, the Margaret E. Early Medical Research Trust, and Genetics Training Grant 5-T32-GM07196. In addition, Dr. Pattengale was supported by a Senior Research Fellowship Grant (NIH/CA07968) and the Avenue 50 Foundation, Dr. Muller was supported by a fellowship from the Medical Research Council of Canada, Dr. Tepler was supported by an NIH Physician Scientist Award (K12DK01401), and Dr. Sinn was supported by the National Foundation for Infectious Diseases. Accepted for publication March 31, 1989. Timothy A. Stewart's present address is Genentech, 460 Point San Bruno Boulevard, South San Francisco, CA 94080. Address reprint requests to Dr. Paul Pattengale, Department of Pathology, Childrens Hospital of Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027.

39

40

Pattengale et al

AJPJuly 1989, Vol. 135, No. 1

eggs (embryos). The recent renaissance in molecular biology that uses highly refined recombinant DNA techniques to clone, purify, and amplify relevant gene(s) has greatly aided in the success of such transgenic systems. It should be stressed that, although most of the documented transgenic systems have been produced in mice, successful transgenic systems have been achieved in other animals such as rabbits, sheep, cattle, and pigs.6-8 The earlier experiments of Brinster and Palmiter' initially involved the microinjection of a growth hormone containing transgene into the fertilized eggs of C57 X SJL/J F-1 hybrid mice. The transgene consisted of the mouse metallothionein promotor fused to the rat growth hormone structural gene. The resultant transgenic mice demonstrated elevated levels of serum growth hormone, as well as the dramatically accelerated growth of these mice compared with nontransgenic litter mate controls.9 Further exciting experiments demonstrated that genetically defective, growth hormone-deficient dwarf mice could be partially corrected by injection of the same transgenic construct into the germline of these genetically deficient animals.10 Additional and more recent experiments by this same group further demonstrated that bovine and human growth hormone genes functioned in the same way as rat growth hormone genes in transgenic mice.' 11 In addition to alterations in whole animal growth regulation,9'10 certain transgenic mice expressing a variety of different transgenic constructs have manifested a variety of different pathologic states.11 Excluding neoplastic disorders that will be considered later, various nonneoplastic disorders of the pancreas,12 lymphoid tissue, 13 kidney,14 bone, 5 cartilage,16 eye, 17-'9 and nervous system20'21 have been described." It should be stressed that the production of tissue and/ or organ specific pathology in transgenic systems depends on the choice of an appropriate tissue specific promotor/enhancer used in the transgenic DNA construct for microinjection. For example, the choice of a well-characterized rat elastase gene for microinjection, with both its structural gene and 5' flanking regulatory/promotor/enhancer sequences, is important when targeting a transgene for preferential expression in a tissue-specific manner in pancreatic exocrine cells. The digestive enzyme, elastase, and chymotrypsin and trypsin are serine esterases that are preferentially expressed in the exocrine pancreas. The 5' promotor/enhancer sequences of the rat elastase gene regulate the expression of the downstream, 3' flanking, structural gene sequences in a highly tissue specific manner, presumably through tissue specific proteins, which preferentially bind to the 5' promoter/ enhancer. Using the rat elastase gene, transgenic mice were produced that expressed the rat elastase gene at extremely high levels in the mouse pancreas with extremely low levels in all other tissues examined.22 The tis-

sue specificity of the 5' promotor/enhancer was further demonstrated by creating a hybrid transgenic construct with the elastase promoter/enhancer sequences fused 5' to the human growth hormone structural gene. Transgenic mice receiving this hybrid DNA construct, as expected, preferentially express and translate this hybrid gene in the exocrine pancreas only.23 Using immunohistochemical techniques, human growth hormone was demonstrated within acinar exocrine cells only.23 Further important experiments showed that transgenic mice receiving a hybrid DNA construct consisting of the rat elastase promotor/enhancer fused 5' to the diptheria toxin A structural gene, subsequently developed pancreatic agenesis, and ultimately died of exocrine pancreatic deficiency.12 This is an excellent example of targeted pathogenesis involving tissue-specific expression of a toxin gene with resultant tissue-specific ablation. This important strategy of tissue- and/or organ-specific targeting is of considerable importance in transgenic experiments involving targeted oncogenesis using cellular or viral oncogenes, or both.

Transgene Microinjection Technology and Methodology Because highly detailed methods of transgene microinjection are found elsewhere,24 only the essential details of the technology will be described and illustrated here (Figure 1). In brief, fertilized eggs from timed matings are harvested from the uterine tubules (oviducts) of pregnant female mice. Because the fertilized eggs are surrounded by cumulus cells, a brief digestion with hyalouronidase is used yielding single cell embryos at the pronuclear stage of development, which are suitable for injection with hybrid transgenes. Using micromanipulators and an inverted microscope, the male pronucleus is directly injected with roughly 500 linear copies of purified DNA containing the hybrid transgene of interest. The injected eggs are subsequently transferred using microsurgical techniques to the oviducts of pseudopregnant females, which give rise to viable progeny after 19 days of gestation. If a stable chromosomal DNA integration occurs at the single cell embryo level, the transgenic or foreign DNA is present in every cell of the resulting, transgenic founder mouse. Such transgenic founders are identified by the presence of the foreign transgene in tail DNA using standard Southern blotting techniques. When transgenic founders are sexually mature, they can then give rise to further progeny that carry the transgene through subsequent generations.

Cellular Oncogenes Cellular (proto)oncogenes (termed c-onc) were initially discovered by retrovirologists who demonstrated that

Spontaneous Neoplasms and Activated Cellular Oncogenes

41 AJPJuly 1989, Vol 135, No. 1

I CD-I x C57BL/6J

"12

hrs 12 hrs

~~~Fertilized Oocytes

r

I Hybrid Gene Injection -500 Copies/Mole Pronucleus

'I,,

Eggs Tronsferred to Pseudopregnont Foster Mother

Figure 1. Diagrammatic representation of the procedures used to create transgenic mice. Fertilized oocytes are harvested approximately 12 hours postcoitus and injected with roughly 500 copies of the relevant DNA construct. Injection is generally into the larger, male pronucleus. The injected eggs are returned to the uterus of sham-pregnant foster mothers and at weaningsamples ofDNA takenfrom tail biopsies are assayed for the presence of the inserted transgenes using the Southern blotting procedure.

-21 Doys

vo >0/

I

038

/ I

/3
Tail DNA Analyzed For Exogenous Gene

Normal Gene

acutely transforming retroviruses containe d transforming sequences (or viral oncogenes [termed v-onc]), which shared significant homology with DNA se3quences contained within normal, uninfected, nonmali!gnant, eukaryotic cells. Presumably these promiscuous, and evolutionarily more recent retroviruses, obtained thEese cellular oncogenes through a mechanism of recormbination and capture (or transduction) of these cellular sequences. It was also found that these transforming se'quences were evolutionarily conserved throughout a wide range of both vertebrate and invertebrate species.27.28 S,uch evolutionary conservation of cellular oncogenes amiong a wide diversity of species strongly argues for a baIsic and fundamental role of these genes in normal CE311 growth and

differentiation. Cellular (proto)oncogenes and their asstociated protein products can be classified into four major groups as follows (Table 1): 1) nuclear proteins, 2) mennbrane-associated proteins, 3) growth factors, and 4) gr(owth factor receptors. Representative examples of eaclh major group are 1) myc 2) ras 3) sis and 4) erb B. Sc:me additional examples are given in Table 1. More specifically, c-myce codes for a 4139 amino acid nuclear protein with DNA binding activity thiat presumably has a key function in the regulation of cEell growth and proliferation. Deregulation of an otherwise normal c-myc gene results in continued growth and F)roliferation of affected cells, presumably because the aff ected cells are unable to effectively leave the cell cycle. Prresumably, the family of myc proto-oncogenes, including N-myc and Lmyc, functions through a similar mechaniism of overex-

-

L

-

-0

--

Tr[ns-

C

-C

_

I

-to0% j Positive

pression of a structurally normal gene at both the mRNA and protein levels. Three well-known mechanisms of myc proto-oncogene activation (deregulation) are chromosomal translocation, promoter insertion (insertional activation), and gene amplification.29 The family of ras proto-oncogenes, in contrast to the myc family of proto-oncogenes, codes for an inner cytoplasmic membrane-bound family of proteins, which are somehow involved in the transmembrane signal transduction of growth factors and mitogens.30 Single-point mutations in certain codons of the ras gene convert the ras proto-oncogene into a very potent transforming gene as measured in the in vitro NIH3T3 assay. Ras genes are widely conserved among animal species including yeast, and include three closely related groups: Harvey (H)-ras, Kirsten (K)-ras, and neuroblastoma (N)-ras. One of the most exciting discoveries in recent years was the finding that the human platelet-derived growth factor (PDGF) gene demonstrated shared DNA sequences with the transforming gene of simian sarcoma virus (ie, the v-sis oncogene).31 In fact, the human c-sis protooncogene is virtually indistinguishable from the structural gene coding for the B-chain of PDGF, a 16 kd polypeptide chain. PDGF is stored and released from the alpha granules of blood platelets during hemostasis and is capable of promoting wound healing by stimulating the growth and proliferation of fibroblasts through their PDGF receptors.32 Presumably, cells that express the c-sis or v-sis oncogene or both, and therefore produce and secrete their own PDGF and/or PDGF-like proteins, are able to grow and proliferate in a self-stimulating (autocrine) manner.

42 Pattengale et al AJPJuly 1989, Vol. 135, No. 1

Table 1.

Classification of Cellular Proto-OncogenesAccording to theirAssociated Protein (Gene) Products*

Protein (Gene) Product Nuclear protein(s)

Representative oncogene(s) myc, myb, fos

Comments Gene product(s) binds to nuclear DNA and presumably affects cell

cycle-associated proliferation Membrane-associated proteins Inner

Outer

ras src

Growth factor(s)

sis

Growth factor receptor(s)

erb-B, erb-B2 (neu)

GTP binding protein that presumably affects a signal transduction; viral oncogene very similar to activated cellular oncogene Gene product is an enzyme that phosphorylates tyrosine residues on proteins

Gene codes for the B-polypeptide chain of platelet derived growth factor (PDGF) Gene(s) codes for a truncated epidermal growth factor (EGF) receptor; tyrosine kinase activity

Because only representative examples of cellular proto-oncogenes are given, the above listing is by no means complete. For further details and additional oncogenes, the reader is referred elsewhere.28 For example, the c-mos proto-oncogene codes for a cytoplasmic serine kinase that shares some homology with the proepidermal growth factor (EGF) molecule.28

The human epidermal growth factor (EGF) receptor gene was the first example of a growth factor receptor gene that shared significant sequence homology with the acute transforming gene of the avian erythroblastosis virus (ie, v-erb-B). The avian v-erb-B gene was presumably derived from the chicken EGF receptor gene. It is of further interest that the sequence homology is most conserved for that portion of EGF receptor protein that is inside the cell (ie, the internal carboxyl half of the protein). Therefore, the v-erb gene product is the cytoplasmic, truncated part of the EGF receptor. The exact mechanism of cellular transformation by v-erb-B, c-erb-B, or both is not clear, but may involve mimicing the presence of an occupied and/or activated EGF receptor. There is also a family of erb-B-related oncogenes termed c-neu and/or cerb-B2 that have been observed in various neoplasms both in rodents and man. Amplication of the human cerb-B2 oncogenes in human breast adenocarcinoma is a recent finding of considerable interest that will be discussed here in conjunction with discussion of the observations associated with the production of transgenic mice carrying the MTV/c-neu transgene.33

Targeted Oncogenesis in Transgenic Mice Fusion transgenes, which use cellular and/or viral oncogenes fused to tissue-specific promotor and/or enhancer sequences, essentially redirect oncogenes to targeted tissues (depending on the nature of the tissue-specific promotor/enhancer). For example, a fusion transgene composed of a c-myc cellular proto-oncogene fused to a mouse mammary tumor virus (MTV) promotor/enhancer will result in transgenic mice that overexpress a deregulated c-myc gene in mammary tissue.34 Such mice are prone to develop mammary adenocarcinoma at an early age.34 These, as well as additional in vivo findings in trans-

genic mice containing other fusion transgenes such as MTV/c-neue and MTV/v-Ha-ras,3 will be discussed in more detail in the following section. Other pertinent and illustrative examples include targeting exocrine pancreas cells with the promotor/enhancer sequences of the rat elastase gene fused to the c-Ha-ras cellular oncogene,3 targeting the crystalline lens with promoter sequences of the alpha-A crystalline gene fused to the SV 40 large T antigen,19 and the targeting B lymphocytes with the immunoglobulin heavy chain enhancer fused to the c-myc oncogene.37 38 As expected, a dramatically accelerated and increased incidence of exocrine pancreatic neoplasms, tumors of crystalline lens, and B cell lymphomas is observed in these transgenic mice.

Transgenic Mice Carrying and Expressing Activated Cellular Oncogenes The myc Oncogene Our early experiments34 were originally designed to create transgenic strains of mice in which the level of myc gene expression might be effected by the hormonal environment of the whole organism. To this end, we created four constructions of the mouse c-myc gene in which increasingly larger portions of its putative regulatory region and normal promoters were replaced by the hormonally inducible mouse mammary tumor virus (MTV) promoter (Figure 2). We therefore produced transgenic lines of mice that carried each of the four artificial transgenic constructs (Figure 2) in their germline DNA. We purposefully chose founder animals that were (CD-1 X C57BL/6)F-1 hybrids because both parental as well as F-1 strains of mice have a very low incidence of spontaneous neoplasms. Because the putative expression of the targeted MTV/myc fusion gene in mammary tissue could be induced by pro-


43


GERML INE C-MYC

C1

,E3

A

1I Kb

ATG ...

F

11D2 1

C

z

i

D

3'

Human Homology

HYBRID A

7

ATG...

~~~~kXXX~~~~~~~~~~~~~~

I1

~~11::

1

1

2

-

r

HYBRID

lactogenic stimuli, glucocorticoids or both, and because causally associated with the development of histologically diverse neoplasms such as carcinomas, sarcomas, and lymphomas,39 42 we reasoned that pregnant and/or lactating female mice would be likely to develop an increased incidence of mammary tumors compared with nontransgenic control litter mates. Consistent with this prediction, we observed that multiparous, pregnant females from two of the founder strains, one carrying hybrid construct C and one carrying hybrid construct D (Figure 2), developed mammary tumors at approximately 7 months of age. Both the tumors and breast tissue of these founder animals expressed RNA transcripts corresponding to the fusion transgene. Histopathologic analysis demonstrated that the tumors were moderately well-differentiated breast adenocarcinomas, which were capable of local invasion, distant metastasis, or both (Figure 3). Further detailed observation of transgenic mice carrying hybrid construct C demonstrated that all the available F-1 female progeny that inherited the MTV/myc gene also developed mammary adenocarcinoan activated myc gene has been

1 |

a-x

H

L

I

I

_

121

131I

I

I

H_

_-

YBRID C

ATG...

___________~

MONINI,1h

mW

ATG...

.lvwNMN Figure 2. Diagrammatic representation of the mouse c-myc genfe and a series of constructs containing the mouse mammary tumor ivirus (MTV) promoter/enhancer. The top diagram represents the c-myc gene. The open, numbered boxes represent the three c-myc exons. The two arrows represent the two major transcription initiation sites. The diagram under the figure represetnts the initial transcript with the heaiy segments representing the exons. The letters A, B, C, and D represent the poitits at which the c-myc sequence was replaced by the MTV promoter/enhancer (showtn as a striped rectangle) in each of the hybrids. The large arrow represents the transcriptionial initiation site of the MTV promoter. The lightljy striped rectangle is a segment of genomic DNA present in the original plasmid from which these constructs were derived.47

..1-

3

I \ 5

.

Wa I I

.

.

.

.

1 2

1

1 3

1

HYBRID D KATG... 2 \MTV | 2 |

--AI

_

II

| 37|I

during their second or third pregnancies (see pediin Figure 4). Further observation of this transgenic line (now termed TG.M) has clearly demonstrated the Mendelian inheritance pattern of this constitutionally deregulated myc gene and its close association with the accelerated development of mammary adenocarcinomas in multiparous female mice.3435 It is of further interest that TG.M transgenic mice carrying hybrid construct C and other transgenic mice carrying constructs A, B, or D (Figure 2), express high levels of transgene-specific mRNA in the salivary gland, but do not develop salivary gland neoplasms. Thus, one can use the pattern of tissue expression and the development of neoplasms in these tissues to define better the tissue-transforming spectrum of the myc oncogene. With regard to this latter point, transgenic mice carrying hybrid construct A (Figure 2) expressed the transgene in the salivary gland only and remained tumor-free. In keeping with the latter point, it was fortunate that the MTV/myc transgenic founder strain, carrying hybrid fusion construct B (Figure 2) in its germline, expressed mas gree

i;*|zt


X

. -

.e.{

;.:

;X

..

-

w

t+.

h^

04.

Figure 3. Histologic appearance ofspontaneous mammary tumors occurring in TG.M transgenic mice. Virtually identical histologies were observed in TG.K mice. A: Low-power view of the mammary tumor showing a moderately well-differentiated, locally invasive adenocarcinoma arising in and involving subcutaneous mammary tissue. Note the residual, uninvolved, non-neoplastic breast tissue. B: Lower power view of the lung showing a large subpleural nodule of metastatic adenocarcinoma. C: Medium-power view ofa malignant mammary tumor showing a moderately well-differentiated adenocarcinoma of the breast. D: High-power view of the primary mammary tumor, showing malignant epithelialglands. Note thepiled and heaped up appearance ofthe dysplastic epithelial cells, which also exhibit nuclear atjpia, prominent large nucleoli, and a conspicuous mitotic rate. Mouse TG.Mfounder and subcutaneous mammary tissue (A, H&E, X 70); lung (B, X 70); subcutaneous mammary tissue (C, X 135); and subcutaneous mammary tissue (D, X640).

transgene specific mRNA in a wide variety of tissues.43 The basis for these tissue expression differences among MTV/myc transgenic strains may reside in the structure of the integrated genes or in their randomly integrated chromosomal location. The transgenic founder strain carrying hybrid fusion construct B (now termed the TG.K strain), interestingly, expressed mRNA corresponding to the hybrid gene in the breast, salivary gland, testis, pancreas, lung, spleen, liver, heart, muscle, and thymus. In the most carefully studied pedigree, 14 of 35 transgenic (TG.K) mice (40% incidence) spontaneously developed a wide variety of malignant neoplasms at a mean age of 14 months compared with a control background incidence of 1 % at a mean age of 30 months.43 Interestingly, the inheritance pattern of the myc transgene in TG.K as well as TG.M transgenic mice was that of autosomal dominance with variable penetrance. This Mendelian inheri-

tance pattern has held up over time (data not shown). The histologic tumor types observed in TG.K animals, which are depicted in Figures 3, 5, and 6B were as follows: mod-

erately well-differentiated adenocarcinomas of the breast (Figure 3); low grade in situ neoplasms of testicular Sertoli cells (Figure 5); malignant lymphomas of the immunoblastic, follicular center cell (Figure 5) and lymphoblastic (Figure 6B) types; and malignant mast cell neoplasms (Figure 5). Although the TG.K testicular neoplasms morphologically resembled interstitial cell testicular neoplasms as described by a number of investigators,45 in vitro cell cultures derived from these tumors were responsive to Sertoli cell growth factors (Dr. Anthony BelIve, personal communication, 1985). Three TG.K animals each had two separate and distinct histologic types of malignant neoplasms, an extremely rare occurrence in mice of any age. It was also very interesting that other tissues in TG.K mice,


45


141- 3

(FOUNDER)

OT Litter I

EL- Litter 2 1 LI}_. V k UUy VV 1 )TO 1)T K T 36 37 38 39 40 Tl llAtnD

Figure 4. Occurrence of mammary adetnocarcinoma among the progeny of transgeniic founider TG.M (141-3). Squares refer to males and circles to females. Half-filled symbols refer to animals carrying the transgeniic construction on a single chromosome. The letter T beside a symbol indicates that the animal (founder, 36, 38, 40) developed a breast tumor; K indicates that the animal ( 39) was killedfor analyses before any pregnancy. Litters 1 and 2 of the founder animal did not survive the initial postpartum period because of maternal neglect. Litter 3 was raised by fostering to a CD1, outbredfemale. In most cases, including the analyzed litter3, the male parent was an outbred CD1 mouse. The wildtype progeny of this litter (37, 41, 42, 43) were not keptfor analysis.

:

Amu On

ELI- Litter I 0 |TUMOR @,day

L

LJI

} LITTer 4ttor I

I

I

: }

I-

-TUMOR @ day 174 Pregnant

which constitutionally expressed high levels of transgenespecific mRNA, such as tissues from the salivary gland, pancreas, lung, liver, muscle, heart, and thymus, did not undergo malignant transformation. Therefore, not all tissues that express the myc transgene exhibit neoplasms. These findings, therefore, begin to define the in vivo transforming spectrum of the myc oncogene. Furthermore, with the exception of the immediate consequences of spontaneous neoplasms, no developmental abnormalities and/or anomalies were seen at autopsy in either healthy or tumor-bearing TG.K mice. Although the malignant cells from spontaneous tumors arising in TG.M or TG.K transgenic mice expressed high levels of transgene-specific mRNA, and furthermore had not undergone additional structural changes in the exogenous and/or endogenous c-myc gene(s), it should be stressed that normal, nonneoplastic cells from these transgenic strains also expressed transgene-specific mRNA. This observation, coupled with the observation that spontaneous tumors arose as solitary masses amidst normal cells, led us to conclude that elements in addition to an activated c-myc gene are required to induce malignancy in the whole organism. Additional evidence for the multi-hit or multiple-step hypothesis for oncogenesis is provided by the stochastic (random) nature of tumor incidence in these tumor-prone transgenic strains, as well as by the unequivocal demonstration of genotypic monoclonality among the B cell-derived neoplasms using B cell gene rearrangement methodology.43-"

42

41

3

43

131

Litter 2

Litter 2

ELE[}

Litter

I

I-

I -i i tgar TTer iI

I

Litter 2 1 *-TUMOR@ day 168

Pregnant I

Although it was of interest that TG.K transgenic mice developed a relatively high incidence of malignant lymphoid cell neoplasms (ten B cell-derived tumors and one T cell-derived tumor), which were presumably due to the activity of the MTV (promoter/enhancer)/myc transgene in lymphoid cells, more precise and consistent targeting of the myc gene to the B cell lineage can be achieved using a mouse immunoglobulin enhancer in the hybrid fusion construct. Earlier studies using this approach in transgenic mice demonstrated that a deregulated c-myc gene can give rise to pre-B cell and B cell malignancies.3746 In keeping with these earlier findings, we established a somewhat different mouse lymphoma model by taking an otherwise normal human c-myc gene with its intact promoter and introducing an isolated mouse immunoglobulin heavy chain (IgH) enhancer into the c-myc gene's first intervening sequence (intron) (Figure 6A). This construct was then used to create a line of transgenic mice that express this human gene in immature pre-B cells.38 Mice that inherit this activated oncogene stochastically develop a particularly narrow range of lymphoblastic lymphomas and related leukemias that are pre-B cell derived (Figure 6B). These findings38 and earlier findings3746 may provide a valuable model for subtypes of childhood acute lymphoblastic lymphoma and related leukemia. Conversely, targeting of a myc transgene to T lineage cells can be achieved using promoter/enhancer elements in the fusion transgene, which are constitutionally active in T cells. Using this approach, we have been able


Figure 5. Histologic appearance ofspontaneous neoplasms occurring in transgenic mice of the K strain (TG.K). Mammary tumors were adenocarcinomas and had virtually identical histologies to those observed in TG.M mice (see Figure 3). A: Lowpower view ofSertoli cell neoplasm of testes (TG.K mouse, H&E staining, testis X50). B: High-power view of Sertoli cell neoplasm of testis. Note the characteristic organoid, trabecular, and cord-like appearance of this testicular stromal cell neoplasm. Also note the mild nuclear atypia (TG.K mouse, H&E staining, testis, X300). C: High-power view of B cell-derived immunoblastic lymphoma showing malignant B immunoblasts with conspicuously amphophilic cytoplasm and plasmacytoid features (TG.K mouse, H&E staining, lymph node, X460).

lvm}£AFJI!_Ls/:8|uMng§)1X^&4tBh%.,q;-ibka'o9KjD5Nwer*zW