Proc. Nati. Acad. Sci. USA Vol. 86, pp. 8482-8486, November 1989 Genetics Construction and screening of a genomic library specific for mouse chromosome 16 (somatic cell hybrids/repetitive DNA/chromosome-specific libraries/cDNA screening/chromosome map) UTE HOCHGESCHWENDER*, J. GREGOR SUTCLIFFE, AND MILES B. BRENNAN*t Department of Molecular Biology, MB 10, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037 Communicated by George R. Stark, August 9, 1989 ABSTRACT We have established a protocol for producing source of anonymous RFLP markers, the library could be libraries of specific mouse chromosomes. The mouse DNA- used directly to screen for candidate clones ofspecific genes. containing clones from a genomic library of a hamster-mouse We applied this general strategy in assembling a repre- somatic cell hybrid containing only one mouse chromosome are sentative genomic library of mouse chromosome 16 consis- identified by screening with radiolabeled mouse repetitive ting of 14,200 individual clones, equal to about two "chro- sequences after specifically blocking hamster repetitive se- mosome equivalents." Each clone occupies an individual quences; 95% of the mouse DNA-containing clones are iden- well in a microtiter tray, allowing repeated plating and tified. We have applied this protocol in producing a library of hybridization analysis of the library. mouse chromosome 16, consisting of 14,200 clones or two To exploit fully this or any other chromosome-specific "chromosome equivalents." Each clone occupies an individual library, it is necessary to screen the library with cDNA well in a microtiter tray, allowing the entire library to be probes to identify clones containing expressed sequences. repeatedly and reproducibly plated and analyzed by hybrid- However, cDNA probes contain sequences that are highly ization. Further, we have established a protocol for making repeated in the genome, and the hybridization of these cDNA probes specifically depleted of highly repetitive se- sequences to their cognates in a large fraction of genomic quences for probing libraries of genomic clones. By screening clones obscures the hybridization signal from the high- the chromosome 16 library with cDNA probes from mouse liver complexity fraction of the cDNA. In screenings of the and brain, we demonstrate the feasibility of identifying ex- chromosome 16-specific library with cDNA probes of adult pressed sequences and characterizing their patterns of expres- brain and adult liver, we demonstrate the feasibility of sion. Such chromosome-specific libraries can facilitate the specifically depleting cDNA probes of sequences highly isolation of dermed genetic loci as well as form the basis for the repeated in the genome and of correctly identifying the production of integrated transcriptional, genetic, and physical expression patterns of sequences in genomic clones. maps of entire chromosomes. MATERIALS AND METHODS Two major reasons for obtaining a complete collection of Cells. Baby hamster kidney (BHK) and Chinese hamster genomic clones for a single mammalian chromosome are the ovary (CHO) cells, NIH 3T3 mouse cells, and 9-6Az2 ham- analysis ofthe arrangement ofgenes on the chromosome and ster-mouse hybrid cells (5) were grown in Dulbecco's mod- the isolation of specific genetic loci mapped to the particular ified Eagle's medium with 10% (vol/vol) fetal bovine serum chromosome. With these goals in mind, we devised protocols (BHK, CHO, 9-6Az2) or bovine serum (NIH 3T3). The for producing mouse chromosome-specific libraries and for 9-6Az2 line contains only mouse chromosome 16. The pres- screening these libraries for transcribed sequences. While ence ofmouse chromosome 16 in our cultures was verified by human chromosome-specific libraries can be made from the probing a Southern blot of restriction endonuclease-digested DNA of flow-sorted chromosomes, the similarity in DNA DNA from mouse, hamster, and the hybrid cell line with the content, base composition, and centric heterochromatin of plasmid pMx34 (6)-a probe for the Mx gene known to lie on mouse chromosomes has thus far prevented their purification chromosome 16 (7). The hybrid line exhibited the mouse by this method (1, 2), although it may be possible to make use hybridization pattern superimposed upon the hamster pat- of Robertsonian translocation strains to purify fused chro- tern, thus proving that our hybrid contained mouse chromo- mosomes. The strategy for producing mouse chromosome- some 16 (data not shown). The intensity of hybridization to specific libraries is to screen genomic libraries from somatic the mouse band in the hybrid sample was submolar, consis- cell hybrids, which contain only a single chromosome from tent with the known mitotic instability of mouse chromo- one of the parent species, with repetitive DNA from that somes in hybrid lines. species. A similar approach has been used to identify human Purification of Nucleic Acids. High molecular weight DNA and mouse chromosome-specific restriction fragment length from tissue culture cells was isolated as described by Brison polymorphism (RFLP) markers from libraries of hamster- et al. (8). DNA from mice was prepared by removing the human and hamster-mouse somatic cell hybrids (3, 4). How- brain, washing it briefly in phosphate-buffered saline before ever, to extend this approach to the production of repre- homogenizing it gently in 10 mM Tris/1 mM EDTA, pH 7.5 sentative mouse chromosome-specific libraries, it is neces- (TE buffer) in a loose pestle glass homogenizer. The homog- sary that the vast majority, rather than only a fraction (4), of enate was lysed by addition of NaDodSO4 to 1% and then the mouse DNA-containing clones be identified. Such a further processed like DNA from tissue culture cells. When quantitative increase in the representation in the library the DNA was to be sheared, it was sonicated extensively at would allow a qualitative difference in its use: rather than a Abbreviation: RFLP, restriction fragment length polymorphism. *Present address: Brookdale Center for Molecular Biology, The The publication costs of this article were defrayed in part by page charge Mount Sinai School of Medicine, One Gustave L. Levy Place, New payment. This article must therefore be hereby marked "advertisement" York, NY 10029. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8482 Downloaded by guest on September 29, 2021 Genetics: Hochgeschwender et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8483 high power until the size of the DNA fragments was about 500 (9) (pH 7.0) at room temperature, heated to 680C in a large nucleotides as shown by electrophoresis in an agarose gel. volume of 99% formamide (16), washed several times with Phage DNA and plasmid DNA preparations were done TE buffer, and stored at 40C until next use. following standard procedures (9). Screening Somatic Cell Hybrid Libraries. Phage lifts were Total cellular RNA was prepared from fresh mouse tissues prepared by using Biotrans membranes (ICN) according to by the procedure of Chirgwin et al. (10). Enrichment for the supplier's recommendations. No more than five filters poly(A)+ RNA was performed by the procedure of Aviv and were hybridized in one plastic bag. For screening the somatic Leder (11). cell hybrid libraries, filters were first prehybridized at 680C Construction of Libraries. Phage libraries of genomic DNA overnight in the general hybridization solution [6x SSC were constructed by following standard procedures (9). DNA containing 0.5% NaDodSO4, 0.2% sodium pyrophosphate, from BHK cells and NIH 3T3 cells was digested to comple- Sx Denhardt's solution (9), and 10 mM EDTA; lx SSC = tion with BamHI and ligated into phage AEMBL3 (12). DNA 0.15 M NaCI/0.015 M sodium citrate, pH 7] supplemented from 9-6Az2 cells was digested partially with the enzyme with salmon sperm carrier DNA to a final concentration of Sau3AI, and the 9- to 23-kilobase (kb) fragments were 100 gg/ml. This first prehybridization was followed by a isolated on DE-81 paper (Whatman) and ligated into phage second overnight prehybridization in which the general hy- ADASH (Stratagene). DNA from NIH 3T3 cells was digested bridization solution was supplemented with sheared hamster to completion with EcoRI and ligated into phage AZAP DNA (to a final concentration of 100 in addition to the (Stratagene). Ligated DNAs were packaged in vitro (Giga- ,ug/ml) pack; Stratagene) and plated on strain LE 392/P2 (AEMBL3 salmon sperm carrier DNA. This second prehybridization and ADASH libraries) or BB4 (ref. 13; AZAP library). Li- step was followed by an overnight hybridization step, for braries were plated and screened as primary libraries, except which the general hybridization solution was supplemented for the AEMBL 3-NIH 3T3 and -BHK libraries, which were with 100 ,ug ofsalmon sperm carrier DNA and ofyeast carrier amplified. Phage clones from the AZAP-NIH 3T3 library were RNA per ml and with 32P-labeled mouse genomic DNA at 1 converted into plasmid clones by the supplier's automatic x 107 cpm/ml. Filters were first washed at room temperature excision protocol. in 2x SSC/0.1% NaDodSO4, followed by two stringent Preparation of Radioactive Probes. High molecular weight washes in 0.5x SSC/0.1% NaDodSO4 at 680C. genomic DNA was labeled with 32P by nick translation (14). Positive plaques were picked into individual wells of mi- DNAs of phage and plasmid clones were labeled by random crotiter plates containing 0.1 ml of SM medium (9). Initially, hexamer oligonucleotide priming (15). Radioactive cDNA the library was stored at 40C. Later, dimethyl sulfoxide was probes were constructed by first synthesizing unlabeled added to 7% (vol/vol), and the plates were stored at -70°C cDNA from 2 ,ug of poly(A)+ RNA by using an oligo(dT) sealed in plastic bags. For plating, trays were thawed, primer (9). The RNA template was removed by treating for aliquots were plated, and trays were refrozen and stored at 20 min at 68°C with 0.1 M sodium hydroxide, the cDNA was -70°C.