to(1)promote complete elimination of some tumours,(2)generate a non-protective immune state to others,or(3)favour the devel-opment of immunologic anergy/tolerance/indifference.Future work is needed to de®ne the molecular basis of the cancer immuno-editing process.M Methods
Mice
RAG2-/-,IFNGR1-/-(ref.20)and wild-type mice,all on a129/SvEv background,were either purchad from Taconic Farms or bred in our speci®c pathogen-free animal facility. STAT1-/-mice,generated in our laboratory,were maintained on a pure129/SvEv background14.For the generation of RAG2-/-´STAT1-/-mice,the status of the RAG2 locus was followed by assaying blood for CD3+cells by FACS as previously described21. Genotyping of the STAT1locus was performed by polymera chain reaction as previously described9.
MCA tumour induction and transplantation
Performed as previously described9.A new preparation of39-methylcholanthrene dissolved in peanut oil was ud in each experiment.Progressively growing mass9mm and larger were scored as tumours and con®rmed by histology.The threshold value of 9mm was chon becau mass of this size invariably continued to increa in size.To eliminate the possibility that the rejection of tumo
urs derived from RAG2-/-mice was due to minor antigenic differences between RAG2-/-mice and129/SvEv mice,RAG2-/-or Taconic129/SvEv tumours were transplanted into immunocompetent mice that were generated by mating male129/SvEv mice(purchad from Taconic)to female RAG2-/-mice.
Determination of retrovirus expression in tumour cells
The feline S+L-focus assay(which tests for live amphotropic retrovirus)and the XC plaque assay(which tests for live B-and N-ecotropic murine retrovirus)were performed on supernatants from12different RAG2-/-tumour cultures by BioReliance.Eighteen different sarcomas from RAG2-/-mice and eleven different sarcomas derived from129/ SvEv wild-type animals were tested for reactivity to the G IX rat antira that reacts with veral murine leukaemia virus(MuLV)proteins16.Primers within conrved regions of the gp70gene were ud to amplify a335-ba-pair band from the rever-transcribed RNA of16different RAG2-/-tumours and7different wild-type tumours.
Received12January;accepted28February2001.
1.Thomas,L.in Cellular and Humoral Aspects of Hypernsitivity States(ed.Lawrence,H.S.)529±532
(Hoeber,New York,1959).
2.Burnet,F.M.The concept of immunologic surveillance.Prog.Exp.Tumor Res.13,1±27(1970).
3.Stutman,O.Spontaneous tumors in nude mice:effect of the viable yellow gene.Exp.Cell.Biol.47,
129±135(1979).
4.Stutman,O.Tumor development after3-methylcholanthrene in immunologically de®cient athymic-
nude mice.Science183,534±536(1970).
5.Stutman,O.Chemical carcinogenesis in nude mice:comparison between nude mice from homo-
zygous and heterozygous matings and effect of age and carcinogen do.J.Natl Cancer Inst.2,353±358(1979).
6.Hunig,T.T-cell function and speci®city in athymic mice.Immunol.Today4,84±87(1983).
7.Maleckar,J.R.&Sherman,L.A.The composition of the T cell receptor repertoire in nude mice.
J.Immunol.138,3873±3876(1987).
8.Dighe,A.S.,Richards,E.,Old,L.J.&Schreiber,R.D.Enhanced in vivo growth and resistance to
rejection of tumor cells expressing dominant negative IFN g receptors.Immunity1,447±456(1994).
9.Kaplan, al.Demonstration of an IFN g dependent tumor surveillance system in immuno-
competent mice.Proc.Natl Acad.Sci.USA95,7556±7561(1998).
10.van den Broek, al.Decread tumor surveillance in perforin-de®cient mice.J.Exp.Med.184,
1781±1790(1996).
11.Smyth, al.Differential tumor surveillance by natural killer(NK)and NKT cells.J.Exp.Med.
191,661±668(2000).
12.Smyth, al.Perforin-mediated cytotoxicity is critical for surveillance of spontaneous
lymphoma.J.Exp.Med.192,755±760(2000).
13. al.RAG-2-de®cient mice lack mature lymphocytes owing to inability to initiate V(D)J
rearrangement.Cell68,855±867(1992).
14.Meraz, al.Targeted disruption of the STAT1gene in mice reveals unexpected physiologic
speci®city in the Jak-STAT signaling pathway.Cell84,431±442(1996).
15.Durbin,J.E.,Hackenmiller,R.,Simon,M.C.&Levy,D.E.Targeted disruption of the mou Stat1
gene results in compromid innate immunity to viral infection.Cell84,443±450(1996).
16.Old,L.J.&Stockert,E.Immunogenetics of cell surface antigens of mou leukemia.Annu.Rev.Genet.
17,127±160(1977).
17.Marincola,F.M.,Jaffee,E.M.,Hicklin,D.J.&Ferrone,S.Escape of human solid tumors from
T-cell recognition:molecular mechanisms and functional signi®cance.Adv.Immunol.74,181±273 (2000).
18. al.TAP expression provides a general method for improving the recognition of
malignant cells in vivo.Nature Biotechnol.18,515±520(2000).
19.Marusina,K.,Iyer,M.&Monaco,J.J.Allelic variation in the mou Tap-1and Tap-2transporter
genes.J.Immunol.158,5251±5256(1997).
20. al.Immune respon in mice that lack the interferon-g receptor.Science259,1742±1745
(1993).
21.Dighe, al.Tissue speci®c targeting of cytokine unresponsiveness in transgenic mice.Immunity
3,657±666(1995).Supplementary information is available on Nature's World-Wide Web site
()or as paper copy from the London editorial of®ce of Nature. Acknowledgements
We thank J.J.Monaco for providing a cDNA encoding TAP1and T.H.Hann for providing TAP1antibo
dies and the cDNA encoding H-2K b.We thank M.La Regina for performing the veterinary pathology and C.Hughley and C.Arthur for expert technical assistance.We also wish to thank P.M.Allen,K.M.Murphy,A.S.Shaw and E.R.Unanue for helpful suggestions during the preparation of this manuscript and J.F.Piccirillo for assistance with the statistical analysis of the data.This work was supported by grants from the NIH and the Cancer Rearch Institute.V.S.was supported and A.T.B.is supported by a pre-doctoral training grant from the Cancer Rearch Institute(CRI)and H.I.is supported by the John Hans and Edna Alice Old Post-Doctoral Fellowship from the CRI. Correspondence and requests for materials should be addresd to R.D.S.
(e-mail:schreiber@immunology.wustl.edu). ................................................................. Somatic activation of the K-ras oncogene caus early ont
lung cancer in mice
Leisa Johnson*²,Kim Mercer*³,Doron Greenbaum*§,
Roderick T.Bronson k,Deni Crowley*³,David A.Tuveson*³¶
&Tyler Jacks*³
*Department of Biology,Massachutts Institute of Technology,and³Howard Hughes Medical Institute,Center for Cancer Rearch,Cambridge,Massachutts 02139,USA
k Department of Pathology,Tufts University Schools of Medicine and Veterinary Medicine,Boston,Massachutts02111,USA
¶Dana-Farber Cancer Institute,Division of Adult Oncology,Boston, Massachutts02115,USA .............................................................................................................................................. About30%of human tumours carry ras gene mutations1,2.Of the three genes in this family(compod of K-ras,N-ras and H-ras), K-ras is the most frequently mutated member in human tumours, including adenocarcinomas of the pancreas(,70±90%inci-dence),colon(,50%)and lung(,25±50%)1±6.To constuct mou tumour models involving K-ras,we ud a new gene targeting procedure to create mou strains carrying oncogenic alleles of K-ras that can be activated only on a spontaneous recombination event in the whole animal.Here we show that mice carrying the mutations were highly predispod to a range of tumour types,predominantly early ont lung cancer.This model was further characterized by examining the effects of germline mutations in the tumour suppressor gene p53,which is known to be mutated along with K-ras in human tumours.This approach has veral advantages over traditional transgenic strat-egies,including that it more cloly
recapitulates spontaneous oncogene activation as en in human cancers.
The effects of ras gene mutations have been studied in transgenic mice;however,most strains expresd H-ras or N-ras(reviewed in ref.7).Traditional transgenic strategies direct expression of the oncogene in all cells of the target tissue and may lead to supra-physiological levels of expression.In an effort to construct a ras-bad mou tumour model that overcomes the limitations,we have ud a variation of`hit-and-run'gene targeting8to create new mou strains harbouring latent,oncogenic alleles of K-ra s capable of spontaneous activation in vivo.
Prent address:²Onyx Pharmaceuticals,Richmond,California94806,USA(L.J.);§Department of Pharmaceutical Chemistry,University of California at San Francisco,San Francisco,California94143, USA(D.G.).
The hit-and-run gene targeting procedure has been ud to introduce subtle mutations and typically involves two distinct steps of homologous recombination performed in embryonic stem (ES)cells in culture 8.In our protocol,however,we produced animals carrying the targeted inrtion allele that was created in cultured cells (`hit'step)and allowed the excision (`run')step to occur in vivo (e Fig.1,Methods,and Supplementary Informa-tion).After inrtional targeting of a plasmid carrying the
K-ras exon 1with an activating glycine to aspartic acid mutation at codon 12(G12D),ES cell clones reprenting two different K-ras alleles were identi®ed and ud for blastocyst injection and germline transmission (Fig.1).Hereafter,we refer to the two alleles as K-ras LA1and K-ras LA2,for latent allele types 1and 2.The alleles differ in that K-ras LA2has two mutant copies of exon 1,whereas in K-ras LA1,only the upstream copy of exon 1is mutant (Fig.1).In K-ras LA1,half of the subquent in vivo recombination events would produce an active K-ras G12D allele and half would produce wild-type K-ras ,whereas in K-ras LA2,all such events would generate the oncogenic form of the gene (Fig.1;and Supplementary Informa-tion).Germline chimaeras from two independent clones of both K-ras LA1and K-ras LA2were bred to C57Bl/6females to create C57Bl/6/129/sv F 1mutants that we monitored over time for signs of dia;the mutations were also studied on a 129/sv inbred back-ground (e Supplementary Information).Becau the K-ras LA alleles were expected to be non-functional in the germline con®g-uration,they were maintained in a heterozygous state (as K-ras is an esntial gene in the mou 9).The recombination frequency of duplicated genomic quences,which can occur through intrachro-mosomal recombination or unequal sister-chromatid exchange,has been estimated to range between 10-3and 10-7per cell generation,depending on the locus 8,10.Recombination frequencies in this range would ensure that animals carrying the K-ras LA alleles would have many,presumably widely distributed,cells expressing the K-ras oncogene.
As shown in Fig.2a,both K-ras LA1and K-ras LA2mutations caud signi®cantly reduced survival compared with wild-type controls,with a mean age of death/sacri®ce of around 300days for the K-ras LA1strain and 200days for the K-ras LA2strain.At necropsy,all animals were found to have an extensive tumour burden,and,as anticipated,the K-ras LA2strain developed a more rapid tumour phenotype.As summarized in Fig.2b,the K-ras LA1and K-ras LA2
mutant strains developed a similar spectrum of tumours.The most frequent site of tumour occurrence was in the lung,where 100%of animals developed multifocal tumours at different stages of pro-gression.The tumours were ®rst detectable as small pleural nodules at one week of age (Figs 2c and 3a).Lungs from one-day-old pups did not contain any tumours or pre-neoplastic lesions,consistent with the ®nding that K-ras expression increas
Wild type
K-ras LA1
K-ras LA2
8.9 k b
7.2 k b
6.1 k b
signi®cantly in rodent lungs shortly after birth11.Tumour multi-
plicity and size incread with age(Figs2c and3a,b),ultimately
resulting in respiratory distress and death or sacri®ce of the animal.
With regards to histopathology,K-ras LA lung tumours em to
evolve through a ries of morphological stages from mild hyper-
plasia/dysplasia to overt carcinoma,reminiscent of human non-
small cell lung cancer(NSCLC).The smallest lesions consisted of
hyperplastic alveolar epithelium(Fig.3c),and cloly rembled
human`atypical adenomatous hyperplasia',a propod precursor
dysplastic lesion to invasive lung carcinoma12,13.As the lesions
enlarged,they formed small tumours termed alveolar adenomas,
compod of a monomorphous population of airway epithelium
with minimal cytologic atypia(Fig.3d).Some alveolar adenomas
showed areas of glandular differentiation(Fig.3e)and papillary
architecture(Fig.3f).About5±10%of the tumours showed features
of well differentiated human papillary adenocarcinoma,including
nuclear enlargement,prominent nucleoli and incread mitotic
rate(Fig.3g).In older mice,we obrved infrequent metastas of
连衣裙用英语
the K-ras LA lung tumours to thoracic lymph nodes,the kidney and
other visceral organs(Fig.3i).Immunohistochemical analysis demonstrated that the lung tumours expresd surfactant apopro-
tein-C and-A,but not Clara cell antigen(Fig.3j,k;and data not
shown),indicating an alveolar type II cell lineage.Human lung adenocarcinomas also frequently express markers of alveolar type II
cells14.
In addition to lung cancer,the K-ras LA animals were also prone to
both thymic lymphoma and skin papillomas,each occurring in
about30%of the animals collectively(Fig.2b;and Supplementary Information).The papillomas typically aro in areas subject
to abrasion(that is,the ears and snout),were predominantly
a b c d
e f g
h i j
k l m
Figure3Tumour pathology in K-ras LA mutant mice.a,Thirty-day-old mou lungs showing numerous pleural lesions(arrows).b,Advanced lung tumours in the lungs of a 150-day-old mou.c,Alveolar adenomatous hyperplasia2weeks after birth.d,Lung alveolar adenoma.e,Alveolar adenoma showing focal glandular differentiation (arrowheads).f,Papillary adenoma.g,Well-differentiated lung papillary adenocarcinoma displaying mitosis(arrow).h,Poorly differentiated lung tumour from a K-ras LA;p53-/-mou showing tumour giant cells(asterisk).i,Renal metastasis.j,Pro-SP-C-positive lung adenoma(right arrow)and Pro-SP-C-negative bronchiole(left arrow).
k,CCA-negative lung tumours(right arrow)and CCA-positive terminal bronchioles (leftarrow).l,Oral cavity skin papilloma.m,Colonic aberrant crypt foci(ACF)(arrow).
a
W
T
E
S
(
+
+
)
K
-
r
a
s
L
A
2
E
S
(
*
*
)
K
-
r
a
s
L
A
1
E
S
(
*
+
)
K
i
d
n
e
y
C
(
*
*
)
L
u
n
g
T
(
*
*
)
L
u
n
g
T
(
*
+
)
T
穿越完结小说
h
y
m
i
c
l
y
m
p
h
o
m
a
(
*
*
卤鸡爪做法
)
T
h
y
m
i
c
l
y
m
p
h
o
m
a
(
*
+
)
个人网页设计K
i
d
n
e
y
C
(
*
+
)
L
u
n
g
T
(
*
*
)
b
WT: 5' WT (10 kb)
5' mut (8.9 kb)
3' WT (7.2 kb)
3' mut (6.1 kb)
K-Ras
Asp 1221
21
W
T
E
S
(
+
+
)
K
-
R
a
s
L
A
2
E
S
(
*
*
)
K
-
R
a
s
L
A
1
E
S
(
*
+
)
K
i
d
n
e
y
C
(
*
*
1973年多大)
L
u
n
g
T
儿童缺钙的10个表现(
*
+
)
T
h
y
m
i
c
l
y
m
p
h感冒能喝鸡汤吗
o
m
a
(
*
*
)
T
h
y
m
i
c
l
y
m
p
h
o
m
a
(
*
+
)
L
u
n
g
T
(
*
*
)
M r
Figure4Evidence of recombination in K-ras LA tumours.a,b,DNA(a)and protein(b)were prepared from tumours(T)and normal tissues(C).Control samples were obtained from wild-type(WT),K-ras LA2(**)and K-ras LA1(*+)ES cells.a,Southern blot of Hin dIII±Kpn 1-digested DNA that was probed with a59internal probe demonstrates recombination in tumours(e Fig.1).Loss of the wild-type K-ras allele and/or ampli®cation of the mutant allele is shown in some tumours.b,The rearranged K-ras LA alleles are expresd in tumours.Extracts were immunoprecipitated with a pan-Ras antibody, followed by immunoblotting with antibodies speci®c to wild-type K-Ras and mutant
K-Ras G12D.
pedunculated (Fig.3l),and demonstrated limited,if any,progression to squamous cell carcinomas during the lifespan of the animals.The frequency of lymphoma and papilloma differed depending on genetic background (e Supplementary Information).
Despite the frequency of K-ras mutations in carcinomas of the pancreas and colon of humans,we did not detect the tumours in the K-ras LA mice (n =149).Signi®cantly,however,all of the mutant mice
examined (n =21)had multiple aberrant crypt foci (ACF)of the colon (Fig.3m;and Supplementary Information),whereas wild-type littermates had none.ACF are often obrved in patients with colon cancer,and are microscopic lesions consisting of clusters of abnormally large crypts with an irregularly shaped lumen,incread pericryptal zone and elevated thick epithelium 15.
Evidence for recombination of the latent allele in tumours was obtained through the analysis of DNA,mesnger RNA,and protein (Fig.4;and Supplementary Information).Southern blot analysis showed loss of one duplicate copy of exon 1from the K-ras LA allele in tumour DNA (Fig.4a).Some of the lung tumour samples contained residual signal from the latent allele,probably owing to macrophage in®ltration (Fig.4a;and data not shown).Notably,50%of all thymic lymphomas examined (n =33),as well as some of the lung tumours,showed a greater than 1/1ratio of mutant/wild-type alleles (Fig.4a;8.9-kiloba (kb)and 10-kb bands,respec-tively).Furthermore,in four of the ®ve lung tumours tested,the mutant K-ras mRNA was expresd at higher levels than the wild-type allele (data not shown).This may re¯ect ampli®cation of the mutant allele and/or loss of the wild-type allele in a fraction of tumour cells,as has been demonstrated for ras oncogenes in human and murine tumours 3,16±19.Lar capture microdisction 20and polymera chain reaction (PCR)was ud to show that recombi-nation had occurred within the tumours (data not shown).The expression of the m
utant K-ras allele was demonstrated by PCR with rever transcription (RT±PCR)analysis of mRNA (e Sup-plementary Information)and immunoblotting using polyclonal antibodies speci®c to the K-Ras G12D protein (Fig.4b).
In human adenocarcinomas of the lung,colon and pancreas,K-ras mutations are frequently associated with alterations in the p53pathway 5,6,21.In addition,ectopic expression of oncogenic ras can cau p53-dependent growth arrest and apoptosis in cell-culture systems 22,23.We therefore crosd the K-ras LA1strain to a strain carrying a germline deletion in p53(ref.24)and examined mice with the genotypes K-ras LA1/+;p53+/-and K-ras LA1/+;p53-/-.As shown in Fig.5,compound p53and K-ras LA1mutant mice had reduced lifespans compared with tho of either single mutant parental strain.Notably,lung tumours in the K-ras LA /p53double mutants showed more malignant features than the K-ras LA tumours,including marked cellular pleomorphism and anaplasitc changes
with giant-cell formation (Fig.3h).Furthermore,mice with the genotype K-ras LA1/+;p53-/-had a broader tumour spectrum com-pared with the parental strains,with about 30%of the double-mutant animals developing haemangiosarcomas or ®brosarcomas (Table 1).
Here we describe a new,general strategy for constructing cancer-prone mou strains and,in particul
ar,the development of strains with profound predisposition to lung cancer rembling NSCLC in humans.Through an adaptation of the hit-and-run gene targeting protocol,we have created a situation in which multiple cells of the lung and other tissues of the mou independently acquire onco-genic K-ras mutations and,thereby,initiate neoplastic progression.We have also demonstrated that mutation of p53cooperates in the progression of lung tumours in K-ras LA mice,furthering the relevance to human lung cancer development.Future studies will focus on determining what other germline mutations can cooperate with the K-ras LA mutation to accelerate (or inhibit)tumour devel-opment in the lung as well as to characterize what mutations occur spontaneously in this model.Of note,preliminary analysis of tumour DNA and protein indicates that p16Ink4A ,which is deleted or epigenetically silenced in up to 60%of human NSCLC (ref.21),is not a frequent mutational target in this model (J.Sage,unpublished obrvations).
There are two general explanations to account for the tumour spectrum in the K-ras LA mou strains.First,the activating re-arrangement event may occur at varying frequencies in different tissues of the mou,a possibility that we are unable to address experimentally with available reagents.Alternatively,cells of the lung (as well as thymus and skin)may be especially nsitive to the proliferative effects of oncogenic K-ras ,such that a tumour will more readily ari when the recombin
ation event occurs in such cell types.Indeed,K-ras mutations occur frequently in both sponta-neous and chemically induced lung tumours in mice (reviewed in ref.25).In other cell types,expression of oncogenic K-ras may have no effect on its own or may produce an alternative cellular phenotype,such as abnormal differentiation,cell cycle arrest,nescence or apoptosis.
The possibility of cell-type-speci®c respons to oncogenic K-ras expression is strongly supported by the development of non-progressing ACF in K-ras LA mice.In humans,K-ras mutations have also been linked to the development of benign ACF;however,in the context of a pre-existing APC gene mutation,K-ras mutations are associated with tumour progression 26.Thus,the relative order of ras gene mutations may also be important in determining their potential tumorigenic effects.In this regard,we have crosd the K-ras LA strains with the Apc min strain 27to asss a possible genetic interaction between the two genes in the mou.However,we failed to obrve a change in the intestinal polyp phenotype associated with Apc min (data not shown),possibly owing to the relatively low frequency of the K-ras rearrangement event given the number of animals examined (n =54).
Becau of its frequent involvement in human cancer,the Ras pathway is considered an attractive target for chemotherapeutic
P e r c e n t s u r v i v a l
Age (days)
Figure 5Cooperation between K-ras LA and p53mutations.The combination of K-ras LA with a p53loss-of-function mutation accelerated the ont of cancer,resulting in
statistically signi®cant decread survival as compared with either mutation in isolation.
Table 1Effect of p53status on the tumour spectrum in K-ras LA mice
Tumour type
K-ras LA1+/-;p53+/-K-ras LA1+/-;p53-/-..............................................................................................................................................................................
Lung adenocarcinoma 100100Thymic lymphoma 3739Papilloma 244Fibrosarcoma
328Haemangiosarcoma
233Duodenal adenocarcinoma 02Undifferentiated sarcoma 10Histiocytic sarcoma 14Medullablastoma 02Osteosarcoma 02Teratoma
2
..............................................................................................................................................................................Percentage of animals with a given tumour type.Numbers were combined for animals from both K-ras LA1and K-ras LA2.K-ras LA1+/-;p53+/-(n =54);K-ras LA1+/-;p53-/-(n =87).
intervention28.The K-ras LA mou strains may be uful in asssing the ef®cacy of Ras pathway-directed therapies,especially given potential differences in the tumorigenic conquences of K-ras mutations versus other ras gene mutations,as well as the relative resistance of K-Ras to farnesyl-transfera inhibitors(reviewed in refs29,30).The spontaneous nature of K-ras activation in this model may more accurately mimic the interaction of tumour cells and their normal environment,which may be relevant to the activity and ef®cacy of chemotherapeutic agents.Becau the tumour phenotype of the K-ras LA mice exhibits such short latency and high penetrance,the strains could also be uful in screening potential chemopreventative agents.M Methods
Construction of targeting vector
We ud the K-ras genomic clone to construct the targeting vector as described9.A1.9-kb Sal I±Xho I fragment containing exon1quences was isolated and subcloned into pGEM-7Z+(Promega).Site-directed mutagenesis was performed using the Amersham Sculptor kit to create p K-ras±Ex1D12.The quence of the oligonucleotide ud to create the Hin dIII and Asp12mutations was59-ATGACTGAGTATAA G CTTGTGGTGGTTGGAGCTG A T GGCGTAGGC-39(the two nucleotide alterations are indicated in bold).All clones were quenced to insure that only the desired mutations were incorporated into exon1.Next, p-39-K-ras D12was created by ligating the following fragments:a1.9-kb Sal I±Xho I fragment from p K-ras±Ex1D12;a1.7-kb Xho I±Xba I fragment from p K-ras-3';and a3.0-kb Sal I±XhoI fragment from pKSII+(Stratagene).A vector carrying the desired lectable marker was®rst generated by subcloning a1.8-kb Xho I±Sac I fragment from pPGKRN containing a wild-type neo gene into a5.5-kb Xho I±Xba I fragment from pPNT to create pPRNT.Next,pKSII+-R neo was created by subcloning a1.9-kb Eco RI±Acc65I neo fragment from pPRNT into Eco RI±Acc65I-digested pKSII+.The®nal targeting construct, p K-ras D12-LA,was created by ligating a2.8-kb Not I±Sal I fragment from p K-ras-59,a3.6-kb Sal I±Xba I fragment from p-39-K-ras D12and a4.8-kb Xba I±Not I fragment from pKSII+±R neo.Exon1quences in the®nal targeting vector were con®rmed by quencing.
Targeting and characterization of ES cell clones
D3ES cells were cultured,electroporated,and lected as described9.To identify targeted clones,DNAs were digested with Bam HI+Kp nI,resolved on0.8%agaro gels,and Southern blot analysis using a59external probe was performed as described9.Targeted clones were analyd further using Southern blotting to determine the integration pattern and copy number.We performed quence analysis to con®rm the status of the Asp12 mutation in each of the correctly targeted events.We ud PCR to amplify K-ras exon1 quences.The59primer(59-GGGTAGGTGTTGGGATAGCTGTCGACAAGC-39)was located in intron0and the39primer(59-CCTTTACAAGCGCACGCAGACTGTAGAGC-39)was located in intron1.The520-bp fragment was phenol-chloroform-extracted, puri®ed in a Microcon100microconcentrator(Amicon),and then quenced(US Biochemical)using a primer(59-TCTTGTGTGAGACATG-39)located immediately59to exon1.All ES clones were maintained in the prence of G418to prevent the growth of cells that had undergone recombination before in vivo analysis.
Generation of K-ras LA mice
C57BL/6blastocyst-stage embryos were injected with10±15K-ras LA ES cells and subquently tran
sferred to pudopregnant Swiss Webster females for further development.Chimaeric mice were mated to C57BL/6and129/Sv animals and F1agouti offspring were genotyped.Germline transmission of the mutant allele was detected by either Southern blot or PCR analysis of tail DNA obtained at weaning.PCR protocols for genotyping are available on request.
RT±PCR/oligonucleotide Southern analysis
Procesd mRNA(Poly(A)+RNA)was isolated from ES cells or tissue using Qiagen's Oligotex Direct mRNA kit as per the manufacturer's recommendations.Rever tran-scription was performed at708C for15min using Perkin Elmer's Rever Transcription RNA PCR kit.Brie¯y,250ng of Poly(A)+RNA was reverd transcribed using recombinant thermostable rever transcripta(r Tth)DNA polymera in the prence of10mM Tris-HCl,pH8.3,90mM KCl,1mM MnCl2,200m M each deoxynucleotide triphosphate,and the 39primer,K-Ras Ex.3-39A:59-ACGGAATCCCGTAACTC-39.Complementary DNA was puri®ed over Qiagen Qiaquick columns,eluted with10mM Tris-HCl,pH8.8,and then ampli®ed by PCR using Clontech's Klentaq/GC melt kit according to the manufacturer's recommendations.The PCR primers ud were K-Ras Ex.3-39A and K-Ras Ex.0-59B (59-CATTTCGGACCCGGAGCGAGC-39),with an annealing temperature of608C and40 rounds of ampli®cation.PCR products were isolated on2.5%agaro gels and transferred onto Hybond(Amers
ham Life Science)for oligonucleotide-speci®c Southern analysis. Filters were hybridized overnight at378C in5´Denhardts,5´SSPE,0.5%SDS and100mM sodium pyrophosphate,pH7.5containing200ng of the oligonucleotide probe labelled with 32P at the59end.The oligonucleotide probes ud were as follows:wild type
(59-GGAGCTGGTGGCGTAGGCAA-39)and Asp12(59-GGAGCTGATGGCGTAGGCAA-39).Filters were washed in6´SSC at room temperature,followed by successive washes in 3M tetramethylammonium chloride,50mM Tris-HCl,pH8.0,2mM EDTA,and0.1%SDS at room temperature and then at618C.Histological analysis and immunohistochemistry
All animals showing obvious tumours or other signs of distress were killed and subjected to full necropsy.For histological analysis,all tissues and tumours were®xed in either Bouin's®xative or10%neutral buffered formalin,paraf®n procesd,ctioned at4m m, and stained with haematoxylin and eosin.Immunohistochemistry was performed using the Vectastain ABC kit(Vector)on4-m m cut paraf®n ctions.Brie¯y,endogenous peroxida was quenched using3%H2O2in distilled water.Sections were blocked for2h at room temerature in PBS containing0.2%Triton X-100and normal goat rum.They were then incubated overnight at48C in PBS supplemented with0.2%Triton X-100and the appropriate primary antibody.The following primary
antibodies and their respective dilutions were ud:anti-pro-SP-C at1:750;anti-SP-A at1:1,000;and anti-CCA at1:1,000. The next day,ctions were allowed to equilibrate to room temperature for1h and were then extensively washed with0.2%Triton X-100in PBS.All subquent steps were followed as per the manufacturer's recommendations.
Lar capture microdisction
According to the NIH guidelines,ctions were cut at8m m,dried at608C for15min and stained with haematoxylin and eosin.After lar capture using the Arcturus PixCell Lar capture microdisction instrument,DNA was extracted by lysis in30m l of LCM buffer (10mM Tris-HCl,pH8.0,1mM EDTA,1%Tween-20,and0.04%Proteina K)at378C overnight.We then heat-inactivated Proteina K at858C for10min.One to®ve microlitres of this was then ud as PCR template.
K-Ras Asp12peptide and antibody generation
Wild-type and Asp12K-Ras peptides(residues5±17,SynPep)were coupled to keyhole limpet haemocyanin and ud to produce high-titre,antigen-speci®c,rabbit polyclonal antibodies(Pocono Rabbit Farms and Lab).Af®nity puri®ed or IgG fractions detected wild-type and K-Ras G12D by immunoblotting,but were incapable of detecting K-Ras immunohistochemically.
Immunoprecipitation and western analysis
Cell lysates were prepared,immunoprecipitated with Y13-259(Santa Cruz Biotechnol-ogy),and analyd by western blotting as described9.The anti-K-Ras D12and anti-K-Ras G12 polyclonal rabbit ra were each ud at a1:1,000dilution,whereas the F234anti-K-Ras mou monoclonal antibody(Santa Cruz Biotechnology)was ud at1:200.Secondary goat anti-rabbit or goat anti-mou antibodies were both ud at1:7,000dilution.
Received14November2000;accepted23January2001.
1.Bos,J.L.ras oncogenes in human cancer:a review.Cancer Res.49,4682±4689(1989);erratum Cancer
Res50,1352(1990).
2.Khosravi-Far,R.&Der,C.J.The Ras signal transduction pathway.Cancer Met.Rev.13,67±89(1994).
3.Bos, al.Prevalence of ras gene mutations in human colorectal cancers.Nature327,293±297
(1987).
4.Mills,N.E.,Fishman,C.L.,Rom,W.N.,Dubin,N.&Jacobson,D.R.Incread prevalence of K-ras
oncogene mutations in lung adenocarcinoma.Cancer Res.55,1444±1447(1995).
5.Pellegata, al.K-ras and p53gene mutations in pancreatic cancer:ductal and nonductal tumors
progress through different genetic lesions.Cancer Res.54,1556±1560(1994).
6. al.Genetic alterations during colorectal-tumor development.N.Engl.J.Med.319,
525±532(1988).
7.Adams,J.M.&Cory,S.Transgenic models of tumor development.Science254,1161±1167(1991).
8.Hasty,P.,Ramirez-Solis,R.,Krumlauf,R.&Bradley,A.Introduction of a subtle mutation into the
Hox-2.6locus in embryonic stem cells.Nature350,243±246(1991);erratum Nature53,94(1991).
9. al.K-ras is an esntial gene in the mou with partial functional overlap with N-ras.
Genes Dev.11,2468±2481(1997);erratum Genes Dev.11,3277(1997).
10.Seperack,P.K.,Strobel,M.C.,Corrow,D.J.,Jenkins,N.A.&Copeland,N.G.Somatic and germ-line
rever mutation rates of the retrovirus-induced dilute coat-color mutation of DBA mice.Proc.Natl Acad.Sci.USA85,189±192(1988).
11.Thrane, al.Differential distribution and incread levels of ras proteins during lung
development.Exp.Lung Res.23,35±49(1997).
武汉什么时候封城12.Nakanishi,K.Alveolar epithelial hyperplasia and adenocarcinoma of the lung.Arch.Pathol.Lab.Med.
114,363±368(1990).
13.Kitamura,H.,Kameda,Y.,Ito,T.&Hayashi,H.Atypical adenomatous hyperplasia of the lung.
Implications for the pathogenesis of peripheral lung adenocarcinoma.Am.J.Clin.Pathol.111,610±622(1999).
14.Linnoila,R.I.,Mulshine,J.L.,Steinberg,S.M.&Gazdar,A.F.Expression of surfactant-associated
protein in non-small-cell lung cancer:a discriminant between biologic subts.J.Natl Cancer Inst.
Monogr.13,61±66(1992).
15.Pretlow, al.Aberrant crypts:putative preneoplastic foci in human colonic mucosa.Cancer Res.
51,1564±1567(1991).
16.Bos,J.L.The ras gene family and human carcinogenesis.Mutat.Res.195,255±271(1988).
17.Finney,R.E.&Bishop,J.M.Predisposition to neoplastic transformation caud by gene replacement
of H-ras1.Science260,1524±1527(1993).
18.Bremner,R.&Balmain,A.Genetic changes in skin tumor progression:correlation between prence
of a mutant ras gene and loss of heterozygosity on mou chromosome7.Cell61,407±417(1990).
19.Yokota,J.,Tsunetsugu-Y okota,Y.,Battifora,H.,Le Fevre,C.&Cline,M.J.Alterations of myc,myb,
and rasHa proto-oncogenes in cancers are frequent and show clinical correlation.Science231,261±265(1986).
20.Bonner, al.Lar capture microdisction:molecular analysis of tissue.Science278,1481±1483
(1997).
21.Salgia,R.&Skarin,A.T.Molecular abnormalities in lung cancer.J.Clin.Oncol.16,1207±1217(1998).
22.Serrano,M.,Lin,A.W.,McCurrach,M.E.,Beach,D.&Lowe,S.W.Oncogenic ras provokes
premature cell nescence associated with accumulation of p53and p16INK4a.Cell88,593±602(1997).