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E m a n u e l a G u e r r a , R o s s a n o L a t t a n z i o , R o s s a n a L a S o r d a , F r a n c e s c a D i n i , G i a n M a r i o T i b o n i , M a u r o P i a n t e l l i and Saverio Alberti
Supporting Materials and Methods
Gene replacement in embryonic stem (ES) cells
The pFM54 vector was used for gene replacement. This was kindly provided by Dr. L. Ronfani, San Raffaele, Milan, Italy, and is a derivative of pPNT ADDIN EN.CITE Tybulewicz199110599105991059917Tybulewicz, V. L.Crawford, C. E.Jackson, P. K.Bronson, R. T.Mulligan, R. C.Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogeneCellCell1153-63657AnimalsAnimals, NewbornBlotting, SouthernCloning, Molecular*Genes, LethalGenetic Engineering*HematopoiesisHeterozygoteHomozygoteLymphopenia/geneticsMiceMice, Mutant StrainsProto-Oncogene Proteins c-abl/*physiologyProto-OncogenesRecombination, GeneticResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.Spleen/abnormalitiesThymus Gland/abnormalities1991Jun 282065352http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2065352 [1] with positive (neomycin phosphotransferase gene, neo) and negative (thymidilate kinase gene from herpes virus, TK) selection, each expressed from a mouse phosphoglycerokinase (PGK) promoter. The TBV-2 ES cell line, obtained from 129S2/SvPas mice, was used for homologous recombination. ES cells were grown in DulbeccoТs Modified Eagle Medium (DMEM) with 15% (v/v) heat-inactivated ES-grade fetal calf serum (FCS), 2 mM glutamine, 1╫ nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U/ml penicillin and 50 ╡g/ml streptomycin (Invitrogen, Carlsbad, CA) and 1,000 units/ml leukemia inhibitory factor (LIF) (Esgro, Chemicon, Temecula, CA). Cells were cultured over a feeder layer of mitomycin-C-treated mouse embryonic fibroblasts (MEFs). The MEFs were obtained following trypsin digestion (0.05% trypsin in 0.53 mM EDTA at 37 ░C for 30 min) of B6 mouse emb r y o s a t d a y o f g e s t a t i o n ( E ) 1 4 . 5 . T B V - 2 c l o n e s w e r e s e l e c t e d i n 2 0 0 ╝g / m l G 4 1 8 ( I n v i t r o g e n ) , m a n u a l l y p i c k e d , e x p a n d e d , a n d s t o r e d i n l i q u i d n i t r o g e n . R e p l i c a s w e r e g r o w n o n g e l a t i n - c o a t e d 9 6 - w e l l p l a t e s f o r D N A e x t r a c t i o n . T h e D 1 p o s i t i v e c l o n e w a s g r o w n on mitomycin-C-treated MEFs for morphological and flow cytometry analyses and for B6 blastocyst injection. This clone is available to the scientific community.
DNA extraction
DNA was extracted from ES clones as follows: 50 ╡l 100 mM Tris-Cl, pH 8, 10 mM EDTA, pH 8, 10 mM NaCl, 0.5% Sarcosyl, 1 mg/ml fresh proteinase K was added to each well. Plates were incubated at 60 ░C overnight in a humidified chamber. One hundred ╡l cold ethanol, 75 mM NaCl was added, and the plates were incubated at room temperature for at least 60 min. The plates were centrifuged at 1,200╫ g for 5 min, the supernatant was discarded, and the DNA pellets were washed twice with 70% cold ethanol. For Southern blotting, the DNA was resuspended directly in restriction enzyme mix (New England Biolabs, Beverly, MA) and incubated overnight at 37 ░C. Tail biopsies (0.5 cm), were incubated in 500 ╡l 50 mM Tris-Cl, pH 8, 100 mM EDTA, pH 8, 100 mM NaCl, 1% SDS and 1 mg/ml fresh proteinase K at 56 ░C overnight with constant stirring; 200 ╡l 4.2 M NaCl, 0.63 M KCl and 10 mM Tris-Cl, pH 8, was added, with incubation at 4 ░C for 60 min with constant stirring. The tubes were centrifuged for 20 min at maximum speed in a microfuge, the supernatant was tranferred to a clean tube, and the DNA was precipitated with 1 vol. isopropanol, washed with 2 vol. 70% ethanol and resuspended in 100 ╡l 1 mM Tris-Cl, pH 8, 0.1 mM EDTA, pH 8, and left at room temperature overnight. For embryonic tissues, 5 ╡m sections from УfreshФ (embedded in optimal cutting temperature (OCT)аcompound and snap frozen in liquid N2,) or formalin-fixed, paraffin-embedded embryos were collected on glass slides. Tissues of interest were identified, scraped with a sterile needle and transferred to a polymerase chain reaction (PCR) tube. These were then digested in 40 ╡l 100 mM Tris-Cl, pH 8, 2 mM EDTA, pH 8, 1% Tween 20 (v/v) and 1 mg/ml fresh proteinase K at 56 ░C overnight. One ╡l of this crude extract was used for PCR genotyping.
Southern blotting
Southern blotting was performed as previously described ADDIN EN.CITE Sambrook19891421421426Sambrook, J.Fritsch, E.F.Maniatis, T.Sambrook,J.Fritsch,E.F.Maniatis,T.Molecular cloning-A laboratory manualMolecular cloning-A laboratory manual2TropGFPPHuPAmethod1989New YorkCold Spring Harbor Laboratory[2]. The probes were PCR fragments of about 300 bp in length, and they were [32P]-labeled by specific priming ADDIN EN.CITE Feinberg198313716137161371617Feinberg, A. P.Vogelstein, B.A technique for radiolabeling DNA restriction endonuclease fragments to high specific activityAnal BiochemAnal Biochem6-1313211983/07/01AnimalsAutoradiographyBase SequenceCytidine Triphosphate/pharmacologyDNA/biosynthesis/*isolation & purificationDNA Restriction EnzymesElectrophoresis, Agar GelHumansPhosphorus RadioisotopesPlasmidsTemplates, GeneticmethodLABELINGsouthern1983Jul 10003-2697 (Print)6312838eng[3]. The probe sequences were chosen using repetitive and low complexity sequence analysis filters (, ). SacI and SacI/KpnI digested genomic DNAs were hybridized at high stringency with the 5Т and the 3Т probes respectively, according to the strategy depicted in Figure S1.
PCR optimization
Primers were designed using Primer3 ( HYPERLINK "http://frodo.wi.mit.edu/primer3/" frodo.wi.mit.edu/primer3/) ADDIN EN.CITE Rozen200017739177391773917Rozen, S.Skaletsky, H.Whitehead Institute for Biomedical Research, Cambridge, MA, USA.Primer3 on the WWW for general users and for biologist programmersMethods Mol BiolMethods Mol Biol365-861321999/11/05Base SequenceDNA Primers*Database Management Systems*InternetMolecular Sequence DataPolymerase Chain ReactionSequence Homology, Nucleic AcidUser-Computer Interfaceref x Primer320001064-3745 (Print)
1064-3745 (Linking)10547847http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10547847eng[4] (Table S1). Different combinations of forward and reverse primers were tested using several different DNA polymerases (AmpliTaq Gold DNA polymerase, Applied Biosystems, Carlsbad, CA; HotMaster Taq DNA Polymerase, Eppendorf, Hamburg, Germany; GC-RICH PCR System, Roche Applied Sciences, Penzberg, Germany; Failsafe PCR System, Epicentre, Madison, WI), together with different reaction buffers (buffers A-I, FailSafe optimization set) and annealing temperatures (from 55 ░C to 68 ░C; gradient thermalcycler Mastercycler ep, Eppendorf).
RNA extraction and reverse transcription (RT)-PCR
RNA was purified by acid guanidinium thiocyanate-phenol-chloroform extraction ADDIN EN.CITE Chomczynski198711835118351183517Chomczynski, P.Sacchi, N.Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extractionAnal BiochemAnal Biochem156-91621AnimalsCell LineChloroformGuanidineGuanidinesHumansHydrogen-Ion ConcentrationMammary Glands, Animal/analysisPhenolPhenolsRNA/*isolation & purificationRatsSolutionsmethodtropchrpcr1987Apr2440339[5] from liquid-nitrogen snap-frozen intestines. One ╡g of total RNA was reverse transcribed with ImProm-II Reverse Transcriptase (Promega, Madison WI) using random nonamers priming according to the manufacturer protocol. Actual cDNA amounts were quantified by ethidium bromide fluorescence in solution ADDIN EN.CITE Bonasera200711861118611186117Bonasera, V.Alberti, S.Sacchetti. A.c.Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using ethidium bromideBioTechniquesBiotechniques173-176432cancertropchrDNACA R I C H <