Vectors for inserting selectable markers in vector arms and human DNA inserts of yeast artificial chromosomes (YACs)

Gene, 103 (1991) 53-59 0 1991 Elsevier Science

GENE

Publishers

B.V. All rights reserved.

53

0378-l 119/91/$03.50

05026

Vectors for inserting selectable markers in vector arms and human DNA inserts of yeast artificial chromosomes (YACs) (Homologous

recombination;

plasmid;

neomycin

resistance;

LYS2 gene; LI element;

thymidine

kinase;

recombinant

DNA)

Anand K. Srivastava and David Schlessinger Department of Molecular Microbiology and Center for Genetics in Medicine, Washington UniversitySchool of Medicine, St. Louis, MO 63110 (U.S.A.) Received by G.N. Gussin: 17 December Revised: 8 March 1991 Accepted: 13 March 1991

1990

SUMMARY

To facilitate studies of gene expression and homologous recombination, plasmids have been developed which permit the insertion of neomycin resistance-encoding gene (NmR) into either the human DNA insert or the vector arm of a yeast artificial chromosome (YAC). To integrate into the YAC arm, the plasmid pRV1 contains a LYS2 (encoding a-aminoadipate reductase) gene for selection in the yeast host, and a NmR gene for subsequent selection after transfection of mammalian cells. These two sequences are bracketed by fragments of the URA3 gene (encoding orotidine-5’-phosphate decarboxylase) that can disrupt the URA3 gene in the YAC arm by homologous recombination in yeast. To integrate a selectable marker into the insert, the plasmid pRV2 contains a NmR gene and an intact copy of the URA3 gene, bracketed by segments of an LI (LINES) repetitive element. In this case, the vector has been designed for use with YACs that have already been fitted in the vector arm with a different marker (i.e., TK) that has disrupted the URA3 gene in the vector arm. Selection is for the restoration of URA3 gene activity attendant on recombination into an Ll element in the YAC insert. Use of the vectors is illustrated with a YAC clone containing ribosomal DNA.

Transfection studies of DNA constructs have become a standard way to examine the function of gene sequences. The size of DNA fragments that can be accommodated in traditional bacterial and phage vectors has been limited, however. Consequently, for many genes, it was difficult to

achieve constructs with the signals required for a number of regulatory phenomena, including tissue-specific expression and mRNA metabolism (DeChiara et al., 1990; Reid et al., 1990). The introduction of vectors in which large DNA inserts can be maintained has provided potential reagents for the study of even large genes. The YACs (Burke et al., 1987)

Correspondence to: Dr. D. Schlessinger, Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO

LYS2, gene encoding

63110 (U.S.A.)

gene encoding

INTRODUCTION

Tel. (314)362-2744;

Fax (314)362-1232.

cation; encoding

Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); CHEF, contour-clamped hexagonal electrophoresis; EtdBr, ethidium bromide;

Ll (LINES), large interspersed

kb, kilobase(

a-aminoadipate

Nm resistance;

PFGE,

pulsed-field

nt, nucleotide(s); gel electrophoresis;

TK; TK, thymidine

ribosylanthranilate phate decarboxylase;

reductase;

isomerase;

kinase;

repeated

sequence(s);

Nm, neomycin; r, ribosomal;

TRPI, gene encoding

URA3, gene encoding

YAC, yeast artificial

NmR,

on’, origin of DNA repliTK, gene phospho-

orotidine-5’-phos-

chromosome.

54 in particular, can be used to clone most genes in complete form and in normal sequence context. For example, YAC cloning has permitted the recovery of intact genes, including factor VIII (Abidi et al., 1990) factor IX (Brownstein et al., 1989) glucose-6-phosphate dehydrogenase (G6PD gene; D’Urso et al., 1990) and glycineamide ribonucleotide transformylase (GART gene; Gnirke et al., 1990); the last two have been shown to produce active enzyme from YACs upon transfection of mammalian cells (D’Urso et al., 1990; Gnirke et al., 1990). To investigate the expression and fate of YACs in detail, it is necessary to establish them stably in recipient cells. A selective marker can be part of the YAC vector when a library is constructed (Traver et al., 1989); but a large number of currently available YACs contain no such marker including the original and still widely used pYAC4 (Burke et al., 1987). For those, modification of the existing YAC with appropriate markers, a process that has been called ‘retrofitting’, is indispensible. We have reported one vector (TKLU2) which can be used to introduce the selectable TK gene into the vector arm of a YAC (Eliceiri et al., 1991). This facilitates the subsequent selection of TK-deficient mammalian cells that have been stably transformed with the modified YAC (Eliceiri et al., 1991). Use of the TK gene as a selective marker is restricted to cell lines deficient for the gene. Here we describe two additional vectors, pRV1 and pRV2, which extend the use of such a system to mammalian cells in general. Both plasmids contain the NmR gene, a marker selectable in mammalian cells, and can introduce it, respectively, into a dispensible portion of a YAC (the vector arm) or into the insert DNA of a YAC. Adding Nm R to the vector arm with pRV 1 permits direct transfection studies. With a similar aim, NmR has been introduced into a repetitive sequence (Ah) in the human insert of a YAC (Pavan et al., 1990). In contrast, rather than fitting a YAC directly with the NmR gene in the insert, pRV2 is designed to be used in a second step with YACs that have already been retrofitted with the TK gene in the vector arm. As a result of the successive use of the two vectors TKLU2 and pRV2, YACs are recovered that permit one to select for the NmR marker in the insert and against the TK marker in the arm. This imitates in YACs the positive-negative selection system developed by Mansour et al. (1988) (see Thomas and Capecchi, 1990) to promote gene alteration by a homologous copy in a 2 phage.

obviate the need to make YACs with vectors that commit one to the use of only a particular selective marker, and free one from the dependence on available restriction sites for recombinant DNA manipulations. (a) Retrofitting YAC vectors arms with the NmR gene In construct pRV1 (Fig. 1) the NmR and LYS2 genes were inserted at a unique site within the URA3 gene. Construction was begun by subcloning a 3.7-kb EcoRI-BamHI fragment of plasmid pdBPV-MMTneo (Law et al., 1983) containing the NmR gene, between the EcoRI and BamHI sites of pUC18 (Yanisch-Perron et al., 1985). The resulting plasmid, pNm, was linearized with BamHI. The ends were made flush using the Klenow fragment of E. co/i DNA polymerase I and dNTPs, and the terminal phosphates were then removed by calf thymus alkaline phosphatase (Maniatis et al., 1982). A 5.2-kb EcoRI-Hind111 fragment of plasmid YIp333 (Eibel and Philippsen, 1983) containing the LYS2 gene, which had also been rendered blunt-ended

A

B kb 450350 -

I

Nlll

Fig. 1. Targeting ofthe Nmamarker into a YAC vector arm. (A) Diagram of retrofitting vector pRV1. Only unique restriction sites are shown. (B) PFGE

analysis

were grown

lysine

described

of seven clones (Trp + Ura-

in synthetic

liquid medium

and tryptophan,

low-melting-agarose AND DISCUSSION

blocks

gel shows

and

Lys2 + ). Transformants

supplemented

DNA

yeast

DNA transferred

with aa mixture

was immobilized

and fractionated

in a CHEF

by Carle and Olson (1985). A 1% agarose

TBE buffer with switching

The highly recombinogenic nature of yeast facilitates the modification of resident YACs to incorporate markers that can promote their retention in mammalian cells by selection. Standard techniques of gene disruption and insertion

3456769

7

lacking RESULTS

2 1 .

in 0.5”;

apparatus

as

gel was run in 0.5X

time of 15 s to 30 s for 20 h. The EtdBr-stained

chromosomes

and YACs.

to a nylon membrane

(C)Autoradiogram

(Sureblot,

Oncor)

of the

from the gel

in panel B and hybridized to NmR probe DNA. Lanes 1-3 and 5-8, YACs; lane 9, parental YAC; lane 4, marker of concatamerized I genomes. Arrow

indicates

the position

of YACs.

55 as above, was then ligated to the linear blunt-ended plasmid. The EcoRI-Hind111

fragment

of the plasmid

pNm

bearing the

fused NmR and LYS2 genes was made blunt-ended as described above, repurified, and inserted at a unique Stul site within the URA3 gene [contained on the Hind111 restriction fragment of plasmid YEP24; Botstein et al. (1978) and cloned into the Hind111 site of pUCl8 (Yanisch-Perron et al., 1985)]. Digestion of plasmid pRV1 with Hind111 (Fig. 1A) released a 9.5-kb fragment containing the LYS2 and Nm R genes between segments of the yeast URA3 gene.

513-bp

HinfI-SnaBI

fragment

portion

of a human

Ll

repeat

containing

a 3’-conserved

was isolated

from pCD

KpnI-8 (Scott et al., 1987) and cloned into the SmaI site of pUCl8.) Since LI elements are not present in yeast DNA, LI in the vector should selectively recombine into the Ll of the human DNA insert of YACs. LI have several advantages as a target for the introduction of selectable markers in the human DNA inserts of YACs. These include: (i) Their frequency. LI occurs about once every 50 kb in total human DNA (Singer and Skowronski, 1985) and would therefore likely occur several times in typical YACs

Integrative disruption of the URA3 gene in the YAC arm by this fragment produced recombinants that had become

of 100 to 600 kb. Thus, the selective marker can be intro-

Lys’ Ura in yeast and contained the Nm R gene. In pilot experiments, pRV1 was used to introduce

duced at a fixed number of sites, making it flexible enough to yield a probable insertion at a site useful for a variety of

the

NmR marker into several YAC clones. For example, yeast carrying yA16G4, a YAC clone containing human rDNA (Labella and Schlessinger, 1989) was transformed with 0.25, 0.5 and 1.0 pg of gel-purified pRV1 fragment by the spheroplasting procedure (Burke et al., 1987). From several experiments, 96 colonies were selected for growth in the absence of lysine (due to the L YS2 gene in the right arm of the YAC) and tryptophan (due to the TRPI gene in the left arm of the YAC). Of the 96, 69 were also Ura- (the other strains may have undergone recombination into the already inactive chromosomal URA3 gene, and thus retained an intact homologue in the YAC arm). DNA from seven randomly chosen transformants was fractionated by PFGE (Carle and Olson, 1985) (Fig. 1B). The normal distribution of yeast chromosomes in the range of 200 to 500 kb is seen in the EtdBr-stained gel, along with the modified YACs. Five of the seven contained a 160-kb YAC, as expected (lanes 3 and 5-8); lane 9 shows the smaller parental YAC before modification. The DNA was transferred to a nylon membrane and analyzed by hybridization with probes that included a 3.7-kb EcoRI-BamHI fragment of pdBPVMMTneo containing the NmR gene (Law et al., 1985) and labeled using the random primer method of Feinberg and Vogelstein (1983). Fig. 1C indicates that the five YACs of the anticipated size also contained NmR gene sequences. Insertion of the NmR gene into the YAC was also verified by analysis of the products of restriction enzyme digestion (data not shown). (b) Adapting YACs to promote homologous in human cells

recombination

(1) Principles of using Ll element To introduce the mammalian selectable marker (NmR) into the human DNA insert of a YAC, we capitalized on the occurrence of repetitive LINES (LZ) elements (Scott et al., 1987) in human DNA. Vector pRV2 contains a NmR cassette and a URA3 gene between human LI repeats. (A

purposes, but infrequent enough to be relatively easy to map the site of insertion in the YAC. (ii) Their constancy. The part of Ll outside the portion used varies almost as much as Ah sequences (Deininger, 1989) and can be ofhighly variable length (Hutchison et al., 1989); but the segment employed (517 nt at the 3’ end) is highly conserved in the range of Ll in the human genome. This makes it highly likely that the transforming fragment will be inserted at a comparable site in the repetitive sequence in all cases, and with a comparable chance of recombining with every Ll element. (2) Procedures The vector pRV2 is designed to be used in a two-step procedure (Fig. 2A) to put a positive selectable marker into the insert of a YAC and a negative selectable marker into the vector arm. In step one, using vector TKLU2 (Eliceiri et al., 1991), a YAC was first modified by insertion of the TK gene into the URA3 gene in the YAC vector arm. Yeast cells carrying a YAC with the resultant Trp + Ura- Lys + phenotype were selected and the NmR gene was inserted into their human DNA moiety using the vector pRV2. The final recombinants were Trp’ Lys’ and had the Ura+ phenotype restored (Fig. 2A). This strategy was tested on the same YAC (yAl6G4) used in Fig. 1. The presence of LI in this YAC had already been confirmed, making it feasible to insert DNA at those sites. Yeast cells containing the YAC were transformed with the Hind111 restriction fragment of plasmid TKLU2 containing the TK and LYS2 genes. Insertion of the TK gene into the vector arm of several of the Trp + Ura- Lys + YACs was verified by restriction analysis, and by hybridization with a probe that included a 2040-bp PvuII fragment of plasmid TK321 (Majors and Varmus, 1983) containing the TK gene (Fig. 2C, lane 4, and data not shown). A YAC with the TK and LYS2 genes inserted into its vector arm (Trp + Ura - Lys + ) was then used for the second step of recombination. Cells containing this YAC were

56 Insert

LeftArm TRP

YAC

Ri@l

I.1

Arm

Phenolype

URA3

Trp? Ura+, Lys-

-m

Trp: Ura-, Lys+ !

i TK, LYSZ -

B

I

2

3

C

4

kb

kb

350-

350-

250-

250-

150-

150-

50-

50-

I

2

Fig. 2. Adapting

YACs to promote

homologous

restriction

sites are shown.

Panels B and C: Southern in agarose

blocks

(lanes l-3)

or retrofitted

and a TK probe

recombination.

blot and hybridization

was analysed

(A) Strategy.

Recombination

vector pRV2, used in step 2 for introducing

Steps one and two are described by CHEF

analysis

gel electrophoresis

only in the YAC vector

4

TK

Nm Ehceiri et al. (1991). The recombination

3

Trpt Ura*, Lys+

marker

vector

TKLU2,

used for step 1, has been described

into insert DNA of the YAC, is diagrammed.

by

Only unique

in section b2. of DNA from yeast cells containing as described

in Fig. 1B. Results

retrofitted are shown

YACs. DNA from yeast cells immobilized for a YAC retrofitted

arms (step 1 in panel A; lanes 4). The filter was successively

hybridized

using steps

1 and 2

with a NmR probe (panel B)

(panel C).

transformed with the 5.3-kb EcoRI-BumHI fragment of plasmid pRV2. This fragment contains about 207 bp of conserved sequence of the repetitive I.1 at one end and about 306 bp at the other. Recombination of the transformed fragment in yeast should then integrate theNmR and URA3 genes by disruption of a resident Ll in the human DNA insert of the YAC. To test this expectation, Ura+ transformants were analyzed. Of 89 tested, 82 were also Trp + and Lys + , as expected. (The Lys - colonies probably arose by homologous recombination of the fragment into the YAC arm. This would replace the split URA gene (and its interstitial LYS2 gene) with an intact URA gene.) NmR insertion in the human DNA and TX: insertion in the arm of the YAC were then verified. The Trp + Ura + Lys + transformants were grown in medium lacking lysine and trypto-

phan and embedded in agarose blocks. A typical CHEF gel analysis of DNA from several colonies is presented in Fig. 2B. Hybridization with the NmR gene probe detected a YAC of the expected size (lanes 1 and 3). The filter was washed to remove the probe and was then hybridized with the TK probe to confirm the presence of the TK gene in the YAC as well (Fig. 2C). All ofthe lanes show YACs with the TK gene. One YAC gave a signal with the TK probe but not with the NmR gene probe (compare Figs. 2B and C, lane 2). It may have resulted from recombination between the split URA gene of the YAC arm and the intact URA3 gene of the vector insert, but this has not been analyzed further. Finally, to examine the number of insertions in the YAC, DNA from YACs modified to contain both TIC and NmR

57

TRP

YAC

Right Arm

Insert

Lett Arm

URA3

Ll -. )

TRP

Retrofitted YAC

NrnR. URA3

TK. LYS2

NmR, URA3

4

+

c

2

3

4

kb

kb -

I

23. I

”

ba,a-

23.1-

9.4-

6.5

6.5-

4.3-

TK

Nm Fig. 3. Presence

ofNmK and TK gene in a YAC retrofitted

retrofitted

YAC containing

fragments

generated

in both human DNA insert and vector arm. Upper panel: Schematic

a TK, LYSZ and two Nm, UL43 insertions.

by digestion

of Hind111 restriction

sites flanked

an Ll site. Lower panel: Hind111 digests of the YAC with insertion in the arm only (lane 3) and of the parental Fig. 1B. Southern to remove

transfer

the NmR probe

to duplicate

Arrows

and was then hybridized

indicate

both in the human

and subsequent

with the pBR322

putative

by LI; a’ indicates

YAC (lane 4) were fractionated

nylon membranes

PBR

probe present

genes was digested with Hind111 and analysed by conventional agarose gel electrophoresis followed by Southern blot analysis. The DNA was transferred from the gel to duplicate nylon membranes. The filters were separately probed with NmR or TK DNA. Fig. 3 (top) diagrams the expected digestion products of a YAC and a putative doubly modified YAC. Because there is no Hind111 restriction site in the DNA bearing the NmR and TK genes, DNA containing Nm R or TK is released in a fragment bounded by the nearest Hind111 sites. Thus, when treated with HindIII, the YACs shown in Fig. 2 (lanes 1 and 3) released one (Fig. 3A, lane 2) or two (Fig. 3A, lane 1) fragments containing the inserted genes. This suggests that the clones have respectively incorporated one and two copies of the NmR DNA. The presence of the TK gene in the right arm

the faster-moving

fragment

released

the corresponding

from a single insertion

at

DNA and in the vector arm (lanes 1 and 2), of a YAC retrofitted

by conventional

hybridization

of the YAC and of doubly

Hind111 sites. a, b, c, and d indicate

0.6% agarose

CHEF

gel electrophoresis

with Nm (A), and TK (B) gene probes.

as described

in

Filter A was then washed

in the right arm of the YAC (C).

of the YAC was reconfirmed by the finding of the expected fragment of similar size, corresponding to the right arm of the YAC, detected by both the TK and pBR DNA probes (Figs. 3B and C, lanes 1 and 2 compared with lane 3). The control parental YAC released a somewhat faster moving fragment ‘d’, as detected by a pBR probe (1691-bp BamHIPvuII fragment of pBR322) specific for the right vector arm (Fig. 3C, lane 4, and schematic at the top of Fig. 3). These vectors are in current use in a number of laboratories. For example, in one adaptation, YACs from human X chromosome 1~36 were modified with pRV1 and introduced into human melanoma and neuroblastoma cells. Stable NmR transformants were then selected to test for putative tumor suppressor genes in the region (T.S. Upper and S. Sarkar, personal communication).

58 (3) Applications The ease and simplicity of the manipulations in these examples and the generality of the G418/Nm selection make these vectors potentially useful for a variety of transfection studies. Other markers, targeted sites, and ways to transfer genes into mammalian cells are also coming into use. Some groups have used electroporation

of cells to introduce

DNA

(Doetschman et al., 1988) or have selected for activity of hypoxanthine-gu~ine phosphoribosyl transferase (HPRT; Szybalska and xanthine-guanine

Szybalski, 1962; Huxley et al., 1991), phosphoribosyl transferase (gpt; Jasin

and Berg, 1988), or NmR (Dorin et al., 1989) genes to promote the homologous recombination of a transfected gene at a homologous site. These techniques may also be adaptable to the retrofitting ofYACs. Also, in recent experiments, the NmR marker has been used to select for mouse embryonal carcinoma (Pavan et al., 1990) or L cells (Pachnis et al., 1990) that had incorporated a YAC by spheroplast fusion. Pavan et al. { 1990) used a vector analogous to pRV2, but which inserts the selectable marker into DNA at an Ah rather than an LI element. Ah sequences are much more frequent, and therefore provide many more potential insertion sites, although unlike the LI loci used here, the sites may not be equally homologous with the vector fragment. The selection system reported here is analogous to that of Capecchi (1989), in that both select for a clone insert and against the vector arm, enriching the transformants for homologous recombinants. However, in the earlier work, lambda recombinants that contained only portions of genes were used, and the experimental design aimed at the inactivation of a chromosomal gene. Instead, a YAC modified by pRV1 and pRV2 would likely contain even a large gene intact and in normal context, with the NmR marker in an Ll element in a non-translated region. Thus, selection for

REFERENCES Abidi, F.E., Wada,

M., Little, R.D. and Schlessinger,

chromosomes tion and

containing

human Xq24-Xq28

representation

of probe

D.: Yeast artificial

DNA: library construc-

sequences.

Cenomics

7 (1990)

363-376. Botstein,

D., Falco,

Stinchcomb, (SHY):

SC.,

Stewart,

D.T., Struhl,

a eukaryotic

Burke,

D.T., Carle,

tors

for recombi-

Isolation

of single-copy

chromosome

clones.

M.: Cloning

of large segments

of artificial

chromosome

of vec-

236 (1987) 806-812. the genome

by homologous

recombination.

244 (1989) 1288-1292.

G.F. and Olson,

Proc. Natl. Acad. T.M.,

deficiency growth

M.V.:

of yeast artificial

G.F. and Olson,

M.R.: Altering

DeChiara,

Olson,

DNA into yeast by means

Science

Science Carle,

containment

244 (1989) 1348-1351.

exogenous Capecchi,

S.,

Little, R.D., Burke, D.T., Korsmeyer,

D, and

genes from a library

Science

M., Scherer,

R.W.: Sterile host yeasts

Gene 8 (1979) 17-24.

B.H., Silverman,G.A.,

S.J., Schlessinger, human

Brennan,

system of biological

nant DNA experiments. Brownstein,

SE.,

K. and Davis,

M.V.: An electrophoretic

Efstratiadis,

phenotype

factor

karyotype

for yeast.

Sci. USA 82 (1985) 3756-3760. A. and

Robertson,

in heterozygous

II gene disrupted

E.J.:

mice carrying

by targeting.

A growth-

an insulin-like

Nature

345 (1990)

78-80. Deininger,

P.L.: SINES:

short

interspersed

repeated

higher eukaryotes.

In: Berg, D.E. and Howe,

DNA.

Society

American

pp. 61Y-637. Doetschman, T., Maeda,

for

Microbiology,

N. and Smithies,

Hprt gene in mouse embryonic

DNA element

in

M.M., (Eds.), Mobile Washington,

0.: Targeted

1989,

mutation

of the

stem cells, Proc. Natl. Acad. Sci. USA

85 (1988) 8583-8587. Dorm, J.R., Inglis, J.D. and Porteous, somal targeting Science D’Urso,

of a dominant

D.J.: Selection

for precise chromo-

marker by homologous

recombination.

243 (1989) 1357-1360.

M., Zucchi,

I., Ciccodicola,

A., Palmieri,

G., Abidi, F.E. and

Schlessinger, D.: Human glucose-6-phosphate dehydrogenase gene carried on a yeast artificial chromosome encodes active enzyme in monkey

cells. Genomics

Eibel, H. and Philippsen,

7 (1990) 531-534. P.: Identification

LYS2 gene by an integrative Genet.

of the cloned

transformation

approach.

S. cerevisiae Mol. Gen.

191 (1983) 66-73.

NmR would favor replacement of part or all of a gene rather than inactivation, with the cloned gene tending to retain its function. Such a YAC could be used for corrective repair of a defective gene by a normal copy (as discussed in Schlessinger, 1990). It remains to be seen if mutant allele replacement might also be favored by the large size of the homologous region in a YAC, in line with a trend previously noted with lambda clones (Capecchi, 1989).

Feinberg,

ACKNOWLEDGMENTS

1990, p. 9. Hutchison, C.A. III, Hardies, S.C., Loeb, D.D., Shehee, W.R. and Edgell, M.H.: LINES and related retroposons: long interspersed repeated

The work was supported by NSF grant 8406949 to D.S. and by the WU-Monsanto Research Agreement.

PMS PCM Biomedical

Eliceiri, B., Labella,

T., Hagino,

Pilia, G., Palmieri,

Y., Srivastava,

sion in mouse cells of yeast artificial genes, Proc. Natl. Acad. endonuclease

chromosomes

B.: A technique fragments

Biochem. 132 (1983) 6-13. Gnirke, A., Barnes, T., Gardiner, M.V.: Cloning and expression

sequences

in the eukaryotic

Mobile

DNA.

ington,

1989, pp. 593-619.

harboring

for radiolabeling

to higher specific activity.

K., Patterson, of the human

artificial chromosomes. Genome Mapping Abstract. Cold Spring Harbor Laboratory,

(Eds.),

D.,

and expreshuman

Sci. USA 88 (1991) 2179-2183.

A, and Vogelstein,

restriction

A.K., Schlessinger,

G. and D’Urso, M.: Stable integration

DNA Anal.

D., Schild, D. and Olson, GART locus using yeast and Sequencing Meeting Cold Spring Harbor, NY,

genome. In: Berg, DE. and Howe, M.M.,

American

Society

for Microbiology,

Wash-

59 Huxley,

C., Hagino,

HPRT

transferred Jasin,

Y., Schlessinger,

gene on a yeast

D. and Olson,

artificial

chromosome

to mouse cells by cell fusion. Genomics

M. and Berg, P.: Homologous

without Labella,

target

gene selection.

T. and Schlessinger,

integration

Genes

that expresses

rDNA

Genomics

cells units

5 (1989) 752-760.

J. and Varmus,

tumor

virus

regulation

Laboratory

Scott,A.F.,

Harbor,

trait. Mol. Cell.

repeat

of the mouse

confers

mammary

glucocorticoid

Singer,

hormone

S.L., Thomas,

proto-oncogene strategy

gene. Proc. Natl. Acad. Sci. USA

Sambrook,

Cold Spring

Harbor

J.: Molecular Laboratory,

Cloning.

A

Cold Spring

M.R.: Disruption

of the

stem cells: a general

to non-selectable

genes. Nature

336

(1988) 348-352. Pachnis,

artificial

P., Rothstein, chromosome

R. and Costantini, carrying

myces cerevisiae into mammalian

human

DNA

F.: Transfer

of a

from Saccharo-

cells. Proc. Nat]. Acad. Sci. USA 87

(1990) 5109-5113. Pavan,

carcinoma

yeast artificial

chromosome.

cell line of a 360-kilobase

and transfer

into

human-derived

Mol. Cell. Biol. 10 (1990) 4163-4169.

Sci. USA 87

M., Comey, C.T., O’Hara,

B.,

P., Smith, K.D. and Margolet,

L.:

proposed

DNA sequence.

repeat

progenitor

Genomics

J.: Making

sequence

and

6 (1990) 248-258.

in

genes deduced

1 (1987) 113-125.

sense out of LINES:

mammalian

genome.

long Trends

Sci. 10 (1985) 119-122.

DNA-mediated Natl. Acad.

heritable

W.: Genetics

transformation

of human

cell lines, IV.

of a biochemical

trait. Proc.

Sci. USA 48 (1962) 2026-2034.

K.R. and Capecchi,

targeting

in mouse

M.R.: Site-directed

embryo-derived

mutagenesis

stem cells. Nature

by gene 346 (1990)

847-850. Traver,

C.N.,

Klapholz,

S., Hyman,

R.W.

and

Davis,

R.W.:

Rapid

of a human genomic library in yeast artificial chromosomes

for single copy sequences.

Proc.

Natl. Acad.

Sci. USA

86 (1989)

5898-5902. Yanisch-Perron, cloning

W.J., Hieter, P. and Reeves, R.H.: Modification

an embryonal

elefor its

tools for mapping

Genetics

B.J., Abdelrazik,

E.H. and Szybalski,

screening

V., Larysa,

yeast

Biochem.

Thomas, K.R. and Cappecchi, mutations

Trends

J.P., Cooley, T., Heath,

M.F. and Skowronski,

Szybalska,

int-2 in mouse embryo-derived

for targeting

chromosomes:

genomes.

Schmeckeper,

interspersed

NY, 1982.

Mansour,

of complex

from a consensus

E.F. and

Manual.

D.: Yeast artificial

analysis

Origin ofthe human LZ elements:

selective

B.H.: Regulatory

gene are necessary

stem cells. Proc. Natl. Acad.

a dominant

on a linked heterologous

T., Fritsch,

in embryonic

HPRT

(1990) 4299-4303.

Rossiter,

H.E.: A small region

long terminal

0. and Keller,

of the human

P.M.: A stable bovine papillomavirus

80 (1983) 5866-5870. Maniatis,

R.G., Smithies,

ments in the introns

Schlessinger,

repeat

Biol. 3 (1983) 2110-2115. Majors,

Reid, L.H., Gregg, expression

9 (1991) 742-750.

in mammalian

human

isolated in yeast artificial chromosomes. plasmid

when

Dev. 2 (1988) 1353-1366.

D.: Complete

Law, M., Byrne, J.C. and Howley, hybrid

M.: The human

is functional

M13mp18

C., Vieira,

vectors

and

and pUC19

J. and Messing, host

strains:

vectors.

J.: Improved

nucleotide

Ml3

sequences

Gene 33 (1985) 103-119.

phage of the