Exploring Gene Function: Use of Yeast Artificial Chromosome Transgenesis

METHODS: A Companion to Methods in Enzymology 14, 199–210 (1998)

Article No. ME970578

Exploring Gene Function: Use of YeastArtificial Chromosome TransgenesisClare HuxleyDepartment of Biochemistry and Molecular Genetics, Imperial College School of Medicine at St. Mary’s,London W2 1PG, United Kingdom

Bacterial artificial chromosome (BAC) and P1-basedTransgenesis is a very powerful tool in functional analysis artificial chromosome (PAC) clones are now increas-

of proteins and control of gene expression. One of the main ingly available and they share some of the advantagesdrawbacks has been the low levels of expression, lack of tissue of YACs, including large size (up to about 300 kb) andspecificity, and inappropriate expression frequently observed for the ability to modify the DNA by homologous recombi-transgenes made with small plasmid-based constructs. The use nation. The use of BACs for transgenics is also dis-of much larger DNA fragments cloned in yeast artificial clones cussed.(YACs), bacterial artificial clones, or P1-based artificial cloneshas been found to give much better levels of expression, gener-ally very close to that of an endogenous gene, and tissue-specificexpression matching that of the endogenous gene. In addition, METHODS OF INTRODUCTION OF YAC DNAthe large DNA can easily be subtly modified by homologousrecombination. This article describes the background and meth-

The methods that have been used to transfer YACods of YAC transgenesis. q 1998 Academic Press

DNA into transgenic mice include pronuclear injectionof purified YAC DNA, lipofection of purified YAC DNAinto embryonic stem (ES) cells followed by productionof chimeric mice, and fusion of yeast spheroplasts withES cells followed by production of chimeric mice. Pro-Yeast artificial chromosomes (YACs) allow cloning ofnuclear injection of purified YAC DNA has the advan-fragments of DNA many hundreds of kilobases in sizetage of making transgenic mice directly without goingand have become central to positional cloning strate-through ES cells and is thus fairly cheap and easy.gies in both mouse and humans (1). Transfer of theHowever, the DNA must be prepared intact and is lia-YAC DNA intact into transgenic mice then allows func-ble to shearing or other breakage during preparationtional analysis of this DNA. At the end of a positionaland injection, so there is probably a limit to the size ofcloning project, transfer can be used to locate the geneDNA that can be injected intact. Shearing is not tooof interest by functional complementation if suitablemuch of a problem for molecules up to about 600 kb inmutant mice are available. Transfer of YAC DNA cansize, and for YACs up to this size pronuclear injectionalso be used to study the control of expression and func-is probably the method of choice and is described intion of a gene once the gene has been identified.detail in the Appendix at the end of this article.There are two major advantages to using YAC DNA

for the generation of transgenic mice over using otherPronuclear Injectiontypes of cloned DNA. One is the very large size of the

clones. As mammalian genes are often hundreds of kilo- To make transgenic mice by pronuclear injection, theYAC DNA is prepared intact from preparative pulsed-bases in size, and the controlling elements necessary

for full levels of expression are often located tens of field gels and injected directly into the pronuclei offertilized oocytes where it integrates into the genome,kilobases away from the gene, such large fragments of

DNA are often essential to obtain high levels of tissue- giving rise to transgenic mice. High-molecular-weighttotal yeast DNA is initially prepared in agarose blocksspecific expression of the transgene. The other useful

aspect of YACs is that the cloned DNA can be subtly to protect the DNA from shearing. The chromosomesare then separated by pulsed-field gel electrophoresismodified by homologous recombination in the yeast

host before introduction into the mice. in low-melting-point agarose and the YAC is excised

1991046-2023/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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200 CLARE HUXLEY

from the gel in a slice of agarose. The agarose is melted been used to make transgenic mice as listed in Table2. Lipofection, like microinjection, requires purificationand treated with agarase, which digests the agarose,

leaving a solution of YAC DNA. This solution is then of the YAC DNA and handling of the DNA in solution,which probably limits the size of the YACs transferred.centrifuged, concentrated, and dialyzed prior to injec-

tion. The method described in the Appendix is essen- However, two groups have successfully transferred a650-kb YAC containing the intact 400-kb human b-tially that described previously (2) and has been used

with minor modifications by several groups to generate amyloid precursor protein gene into ES cells and thenceinto transgenic mice (13, 14), and another transferredtransgenic mice (3–7). A related method of purifying

the YAC DNA that incorporates spermine and spermi- a 450-kb YAC carrying the Xist gene (15). These papersand another (16) give protocols for the handling anddine to protect the DNA from shearing has been de-

scribed (8, 9), and other variations have been used suc- transfer of such large DNA. Polyamines are usuallyused to condense the DNA and hence help protect itcessfully by a number of investigators (10–12).

Microinjection of YAC DNA up to nearly 700 kb has from shearing. However, the YAC DNA is transferredintact in only a minority of cell lines which have inte-been described, and published instances of YAC

transgenics made by pronuclear injection are listed in grated some of the YAC DNA. When the selectablemarker was located on the YAC, only 2 of 23 or 5 of 14Table 1. However, the proportion of transgenic mice

with intact YAC DNA is generally greater with smaller G418-resistant clones contained the intact YAC DNA(13, 15). When the YAC DNA was colipofected with theYACs and it becomes increasingly difficult to prepare

and inject larger YAC DNA intact. Thus, at relatively selectable marker, 8 of 240 clones contained some YACDNA and 3 of these contained the intact YAC (14).small sizes such as the 240-kb YAC containing the b-

globin cluster, 10 of 14 mice appeared to contain intact As it is probably possible to transfer 650 kb intact bymicroinjection, it is not clear that lipofection of the gel-YAC DNA (3). For a larger 310-kb YAC, 4 of 7 probably

contained intact DNA, while at 560 kb only 3 of 8 were purified YAC DNA has any advantages over pronuclearinjection except that lethal genes can be studied. Mak-probably intact (Huxley, unpublished data). The YAC

transgenes are often present at low copy number rang- ing transgenic mice via ES cells is, however, more labo-rious than pronuclear injection.ing from 1 to 10 and where there are multiple copies

they are usually integrated in one position in the mousegenome (6). In one case a partial inverted duplication Fusionof one copy of the YAC was found (5); in another case Fusion with yeast spheroplasts has been used a num-the 4 copies of the YAC were probably tandemly ar- ber of times to transfer YACs to ES cells (Table 2).ranged (7). Thus the YAC transgenes are integrated Fusion is the only method that does not require purifi-in a complex manner very similar to that of smaller cation of YAC DNA so there is probably no limit on thetransgenes such as plasmid fragments. size of YAC that can be transferred by this method. InLipofection the case of a 1020-kb YAC, 7 of 35 ES cell clones con-

tained the intact YAC (17). A potential drawback of theLipofection of gel-purified YAC DNA into ES cellsfollowed by germline transmission of the ES cells has method is that yeast genomic DNA is transferred into

TABLE 1

YAC Transgenics Made by Pronuclear Injection

Gene transferred Size of YAC (kb) Comments Reference

Mouse tyrosinase gene 35 (57)Human b-globin cluster 150 (10)Human b-globin cluster 248 (3)Mouse tyrosinase gene 250 (9)Human apolipoprotein (a) gene 270 (11)Human apolipoprotein B gene 108 Point mutation in open reading frame (4)Human b-globin cluster 248 Point mutation in promoter region (44)21q22.2 region °670 (12)Mouse Xist gene 460 (5)Human PMP-22 gene 560 (6)Mouse Xist gene 350 (7)Human PAX6 gene 420 (46)Human huntingtin gene 350 and 600 (58)Mouse tyrosinase gene 250 Deleted YACs (41)


201YAC TRANSGENESIS

the ES cells at the same time as the YAC during the shown in Table 3. Care should be taken to use se-lectable markers compatible with the host strain andfusion process and this is sometimes integrated into

the ES cell’s genome. However, quite large amounts of it should be noted that where mutations in the hoststrain are point mutations they can revert at an appre-integrated yeast genomic DNA do not prevent the ES

cells from going through the germline (18). Carrying ciable frequency, which can mask the desired recombi-nation events if they occur at low frequency.out fusion such that the ES cells are still germline

competent is technically quite difficult but this tech-nique is probably the only way of transferring YACs Transfer to a New Yeast Host Strainsignificantly larger than 600 kb. There are several instances where it is convenient to

transfer a YAC into a new host and the simplest wayto do this is to use karyogamy mutant (kar) strains (32,33). These strains mate and form heterokaryons withMODIFICATION OF YACSthe yeast containing the YAC but do not undergo nu-clear fusion due to the kar mutation. However, individ-ual chromosomes are sometimes transferred to the karOne of the advantages of using YACs is that they are

grown in yeast that has efficient homologous recombi- strain nucleus which can result in a haploid strain ofyeast that is the kar strain carrying the YAC. Transfernation, allowing one to modify the YAC DNA. The

methods and principles for modifying DNA in yeast into a new host using the kar strains is very simple andthe YAC generally does not rearrange as frequentlyhave been described in detail elsewhere (19). The same

methods apply for modifying YACs as long as care is happens when YAC DNA is purified and transfectedinto a new yeast strain (32, 33).taken to use suitable selectable markers (20, 21). Par-

ticularly useful modifications of YACs include introduc- kar transfer allows one to separate multiple YACs(either identical or different) present in the same hosttion of a selectable marker for selection in ES cells;

introduction of unique restriction enzyme sites to ei- cell. It also allows one to use a yeast host with nonre-verting deletion mutations in genes which can be usedther end of the YAC so that it can be analyzed by re-

striction enzyme digestion once it has been transferred as yeast selectable markers including HIS3 (note thatAB1380 is his5), TRP1, and LEU2. The genotype of theinto the mouse genome; fragmentation to reduce the

size of the YAC prior to injection; and introduction of kar strains is given in Table 3. kar transfer can be usedto move YACs from the recombination-deficient hostmodifications into the gene of interest.

Most YAC libraries have been made with the yeast into a recombination-efficient host so that modifica-tions by homologous recombination can be carried outhost AB1380 and the vector pYAC4 (22). There are

several total human libraries (23–26) and also mouse efficiently. Finally, kar strains have also been con-structed that have different-sized yeast chromosomeslibraries (27–29) which are generally available. A li-

brary of mouse DNA with large inserts has also been (called ‘‘window strains’’) so that the YAC can be intro-duced into a strain where there are no yeast chromo-made using the vector pair pRML1 and pRML2 and

the yeast host strain J57D (30) and another has been somes migrating at the same size as the YAC (34); thisallows one to gel purify the YAC DNA free of yeastmade in a recombination-deficient host (rad52) called

814/3a (31). The genotypes of various host strains are genomic DNA.

TABLE 2

YAC Transgenics Made via ES Cells

Gene transferred Size of YAC (kb) Method Reference

Human heavy immunoglobulin locus 85 Transfectam (35)Human b-amyloid precursor protein gene 650 Lipofectin (13)Human b-amyloid precursor protein gene 650 Transfectam (14)Mouse a1(I) collagen gene 150 DOTAP (37)Human k light immunoglobulin locus 300 Fusion (59)Human HPRT gene 670 Fusion (18)Human heavy and k light immunoglobulin loci 170 and 220 Fusion (49)Human heavy and k light immunoglobulin loci 180, 225, 320 Fusion, no germline transmission (60)Mouse Xist gene 450 DOTAP (15)Human heavy and light immunoglobulin loci 800 and 1020 Fusion (17)


202 CLARE HUXLEY

Introduction of a Selectable Marker for Selection in ES ping with rare-cutting restriction enzymes. This is gen-erally time consuming and can be complicated byCellsmethylation of the restriction sites once they are inte-Introduction of YAC DNA into ES cells prior to mak-grated into the mouse genome (there is no methylationing chimeric mice requires that one can select for thein the yeast genome). If the YAC contains mouse geno-very few cells that have taken up the YAC DNA. Thismic DNA, then it is extremely difficult to determineis not necessary for making transgenic mice by pronu-whether the interior of the YAC is present, as all inter-clear injection as the frequency of transgenics is aboutnal probes will cross-hybridize to the endogenous ge-10% of potentially transgenic offspring, and these cannome, and it is virtually impossible to determinebe screened for by polymerase chain reaction (PCR). Awhether the DNA is unrearranged. Some investigatorsselectable marker can either be introduced directlyhave used interstrain or interspecies variations to showonto the YAC itself by homologous recombination or bethat the DNA of interest has been introduced (5, 7, 37)cotransfected into the ES cells along with the YACbut this does not indicate whether the whole DNA isDNA. Having the marker on the YAC ensures that allintact, and such variations are often difficult to find.the resistant ES cell colonies carry some YAC DNA

Unique restriction sites can be introduced into thewhile cotransfection results in ES cell lines of whichYAC arms by homologous recombination. These sitesabout 3% carry some YAC DNA (14, 35). One of thecan be cut with the relevant restriction enzyme oncecritical aspects seems to be having a strong enoughthe YAC has integrated into the mouse genome andpromoter driving expression of the selectable markerthe correct-sized fragment will indicate both the pres-and it has been found that having more copies of theence of the transgene and the fact that it is the rightselectable marker on the YAC increases the frequencysize (though internal rearrangements could have oc-of clones (13, 36). A number of vectors have been madecurred). A number of retrofitting vectors that introducespecifically for introduction of a selectable marker ontovery rare sites have been made and are listed in TableYACs prior to introduction into ES cells and some of4. Some of these were designed primarily for introduc-these are listed in Table 4. Resistance to G418 (neoR)tion of other sequences, such as mammalian selectableand the hypoxanthine phosphoribosyltransferasemarkers, but also introduce a NotI or other rare-cutting(HPRT) gene have both been used as selectable mark-sites. One pair has been specifically made for introduc-ers for transfer of YACs into ES cells, but there is notion of I-PpoI sites into a YAC in AB1380 (38) andreason why other mammalian selectable markersanother pair for introduction of I-SceI sites (7). I-PpoIshould not also be used in ES cells.and I-SceI are intron encoded endonucleases that have

Introduction of Unique Restriction Enzyme Sites 15- and 18-bp recognition sequences respectively andcut extremely rarely in the mouse or human genome;Once a YAC has been introduced into the mouse ge-thus YAC inserts generally do not contain these sites.nome it can be extremely difficult to determine whether

the DNA is intact. If the YAC contains human DNA itFragmentationis simple to determine whether all known regions of

the YAC have been transferred either by PCR or by Some YAC libraries have inserts up to several mega-bases in size and it is probably not possible to injectconventional Southern blotting. However, this does not

mean that the DNA is unrearranged, only that the this DNA intact. Fusion can be used to introduce verylarge YACs into ES cells but it may be easier to frag-DNA is present. To show that the DNA is unre-

arranged, it is necessary to carry out long-range map- ment the YAC to get a molecule in the right size range

TABLE 3

Selected Yeast Host Strains

Strain name Genotype Use Reference

AB1380 MATa c/ ura3-52 trp1 ade2-1OC can1-100OC lys2-1OC his5UGA Host in most YAC libraries (61)814/3a MATa c/ rad52::TRP1 ade2-1 trp1 ura3 ilv lys2-1 his3-11 Recombination-deficient host (31)

his3-15 his5-2J57D MATa ura3-52 trp1 ade2-101 can1-100 leu2-3 112 his3-6 High-transformation-efficiency YAC host (30)YPH925 MATa c-*leu2D1 trp1D63 ura3-52 ade2-101 his3D200 kar strain carrying deletion mutations (32, 33)

lys2-801 cyh2R kar1D15 of useful markersWindow strains MATa c-*leu2D1 trp1D63 ura3-52 ade2-101 his3D200 kar strains with different-sized (34)

YLBW1-9 lys2-801 cyh2R kar1D15 chromosomes


203YAC TRANSGENESIS

for easy injection and analysis. Fragmentation of chro- a 650-kb YAC (43), to make a point mutation in theapolipoprotein B gene (4), and to introduce a point mu-mosomes with constructs carrying a telomere was first

described for endogenous yeast chromosomes (39) and tation into the promoter region of the g-globin gene(44). These modified YACs can then be used to makehas since been applied to YACs. The target for fragmen-

tation can either be a known specific sequence in the transgenic mice where the function of the modifiedgene can be analyzed.YAC or a repetitive Alu, B1, or B2 element which will

give numerous different fragmentations (40), and vari-ous fragmentation vectors are listed in Table 4. Frag-mentation of a YAC has also been used to remove a ANALYSIS OF YAC DNA IN TRANSGENICDNase-hypersensitive site and construction of YAC

MICEtransgenic mice then showed that the DNase-hypersen-sitive site is important for tyrosinase expression (41).

The frequency of transgenic mice by pronuclear injec-Other Modifications tion is about 10% and a proportion of these will have

the intact DNA. The quickest way to screen forHomologous recombination in yeast can be used in anumber of different ways to get the desired modifica- transgenic mice is probably by PCR on tail DNA either

for the left and right vector arms of the YAC or directlytion into a cloned gene (19–21). It can be used to insertnew sequences into a YAC along with a yeast selectable for the transgene if this is sufficiently different to the

endogenous genes.marker with concomitant duplication of the homolo-gous region, as is frequently done to introduce se- PCR and Southern blotting can then be used to detect

different sequences throughout the YAC, again assum-lectable markers or restriction sites into the YAC vec-tor arms. It can also be used for replacement of ing that there are enough differences to distinguish

the endogenous from the input gene, and this gives ansequences with new sequences along with a yeast se-lectable marker but no duplication of the homologous initial indication of whether the YAC has been trans-

ferred intact. However, PCR and conventional South-region. Finally pop-in/pop-out can be used to introduceinsertions, deletions, or point changes into the YAC in ern blotting can never indicate whether the YAC DNA

is in fact intact; it can only indicate whether short re-such a way that the yeast selectable marker is not leftin the YAC. For example, pop-in/pop-out has been used gions are present, but these may be rearranged in rela-

tion to each other.to make a 17-bp insertion into a clone of the chickenb-globin cluster (42), to introduce a pathogenic point To determine whether the YAC is intact, it is neces-

sary to carry out long-range restriction mapping withmutation into the amyloid precursor protein gene in

TABLE 4

Selected Retrofitting Vectors

Vector Selectable marker Use Reference

pRV1 LYS2 Inserts LYS2, and deletes URA3 from the right arm of (62)a YAC; also inserts a neoR gene

pLNA-1, pLNT-1, pLNB-1 LYS2 Insert NotI site and neoR into the left or right arms or (63)the insert of a YAC

pLNA, pLUNA LYS2 Insert NotI site and neoR into the left or right arm or (36)the insert of a YAC; neoR works in ES cells

pHIS3PyF101neobpA HIS3 Inserts neoR into the left arm; neoR works in ES cells (13)pLUTO LYS2 Inserts HPRT minigene, which works in ES cells into (60)

the left armpBP103, pBP108, pBP109, pBP81 HIS3 Fragment from either arm at Alu or any other cloned (40)

sequence; insert NotI sitepBCL, pB1F, pB1R LYS2 Fragment from right arm at Alu, B1, or any other (64)

cloned sequence; insert NotI sitepRFV1 Neo, pRFV2 Neo, pRFV1 Hygro, HIS5 Fragment from right arm at Alu elements; insert NotI (65)

pRFV2 Hygro and the neoR or hygro markerspUC-OK, pUC-WAN LYS2, TRP1 Insert I-PpoI sites into left and right arms, and neoR (38)

into the right arm; deletes URA3 geneyRP17his3SceI, pLUSSceIbgeo HIS3, LYS2 Insert I-SceI site into left arm and I-SceI site and bgeo (7)

under control of PGK promoter into right arm


204 CLARE HUXLEY

rare sites already present in the YAC or with rare sites expression have been achieved with almost all YACtransgenics. This is in marked contrast to smallerartificially introduced into the arms of the YACs as

described earlier. Generally this has not been carried transgenes which are often expressed at very low levelsand at very different levels in different lines ofout for YAC transgenics. However, in one case where

I-SceI sites had been introduced onto either arm of a transgenic mice due to position effects from the sur-rounding chromatin (48). The reason is probably that350-kb YAC, one of the lines was found to have the

YAC intact after digestion with I-SceI whereas another the YACs contain such a large amount of flanking DNAthat they contain all the long-range controlling ele-line had a deleted version of the YAC (7).

An alternative to introducing rare-cutting sites into ments needed for full levels of expression and to shieldthe gene from neighboring chromatin.the YAC DNA is to use RecA assisted restriction endo-

nuclease (RARE) cleavage of the genomic DNA (45). In addition to full levels of expression, the genes onYAC transgenes are usually expressed in the correctThis technique allows specific EcoRI sites in the mouse

genome to be cut while leaving the other sites uncut. tissues, are controlled by physiological signals, and areeven differentially spliced in a manner analogous toAn oligonucleotide matching the sequence around the

EcoRI site, in combination with RecA protein, makes the endogenous genes. Thus, the b-globin genes of thehuman cluster are expressed only in the erythropoietica triple-helix structure with the genomic DNA. The

triple helix then protects this site from EcoRI methyl- cells and are expressed in a developmentally controlledfashion, with the e and g genes being expressed duringase. After methylation and removal of the RecA and

the oligonucleotide, the EcoRI site is susceptible to embryogenesis and the b gene being expressed in theadult animals. However, the g gene is expressed earliercleavage while the rest of the EcoRI sites in the genome

are not cut as they are methylated. This technique has than in humans and this probably reflects the fact thatmice do not have a gene corresponding to the g genebeen used to show that a 230-kb YAC had been inte-

grated intact after microinjection into rodent cells (2); (3, 10). Furthermore, when a point mutation responsi-ble for a form of hereditary persistence of fetal hemoglo-however, it is technically quite difficult.

Fluorescence in situ hybridization (FISH) is another bin (HPFH) was introduced into the YAC, there was adelayed switch from g to b expression and continuedpowerful technique for analyzing YAC transgenics. The

position of integration into a mouse chromosome can expression of Ag chains in the adult stage of develop-ment analogous to the situation in human carriers (44).be visualized by FISH analysis of metaphase chromo-

somes prepared from spleen or from primary fibro- Differential splicing of the human amyloid precursorprotein gene was observed to mimic that of the endoge-blasts grown out from ear or tail cells (6). FISH can

also be used to determine the number of copies of the nous mouse gene (13). Finally, the apolipoprotein (a)gene, for which there is no mouse homologue, was ex-YAC that are integrated into the mouse genome; using

a cosmid located inside the YAC to probe onto in- pressed at physiological levels and with normal tissuespecificity in the liver, and its expression was de-terphase cells, it was found that the number of signals

in a cluster corresponded to the copy number of the creased by sex hormones as it is in humans (11).The situation is more complicated for the immuno-YAC transgene (46). Finally, to get a more detailed

picture of the long-range arrangement of the integrated globulin genes, where human YAC transgenes wereinitially expressed at quite low levels in comparison toYACs it is also possible to use FISH onto extended

chromatin (47). Here the relative arrangement of inter- the endogenous mouse genes (35, 49). The low overallexpression was partly due to competition between thenal and vector probes can be compared to determine

whether the YAC has been deleted and the relative transgene and the endogenous genes leading to exclu-sive expression of the mouse gene on many cells, butarrangement of multiple copies of the YAC.was also due to the small size of the YACs used. Thus,the human heavy mu chain in normal mice was ex-pressed at very low levels but the level increased by aGENE EXPRESSION IN YAC TRANSGENIC factor of about 10 in mice lacking functional endoge-

MICE nous mouse heavy chain genes (35). Similarly, on abackground that expressed no mouse heavy chains,mice produced about 500 times more human heavyThe levels of expression of transgenes on YACs haschain (350 mg/ml on the null background comparedgenerally been found to be comparable to the levelswith 0.9 mg/ml), achieving a level close to normal hu-of expression of the endogenous genes. For instance,man IgM serum levels (49). Antibody production wasquantitative reverse transcription (RT)-PCR showedalso significantly improved by using much larger YACs:that the ratio of human to mouse amyloid precursorfor the human heavy chain gene a 1020-kb YAC con-protein (APP) mRNA varied from 0.4 to 1.3 in brain,

heart, kidney, and testes (13). Similar high levels of taining an extra 61 VH genes instead of a 220-kb YAC,


205YAC TRANSGENESIS

and for the kappa light chains an 800-kb YAC instead ments. In addition, it is now possible to modify clonedBAC DNA using homologous recombination in the bac-of a 170-kb YAC (17). These large YAC transgenes were

bred onto the null mouse background and gave 700 mg/ terial host, thus allowing one to carry out modificationssimilar to those described above using the yeast hostml of human mu chain in the serum (versus 350 mg/ml

for the smaller YACs); however, the levels in the nor- (52). Furthermore they have two major advantagesover YACs. One is that they are generally very stablemal mice are not given. However, even these very large

YACs do not span the entire immunoglobulin loci. The during propagation while some YACs are quite unsta-ble and frequently rearrange. This has been strikinglyYAC transgenic mice can be compared with the chro-

mosome transgenic mice that have been generated that demonstrated by the ability to stably clone 173 kb ofpure alphoid repetitive DNA in BACs (53) while al-carry independent human chromosomes (50). Here,

mice were generated carrying the intact chromosome phoid DNA in YACs is generally deleted down to clonesof about 100 kb (54). The other is the ease of prepara-22 with the lambda chain, a deleted chromosome 2 with

the kappa chain, and a rearranged chromosome 14 with tion of intact DNA for microinjection. BACs and similarclones are supercoiled circular molecules propagated inthe mu chain. Even in chimeric mice with only a portion

of the B cells carrying the human chromosomes, and bacteria and clean DNA can be prepared in relativelywhere the endogenous mouse genes were still present, large quantities using conventional protocols for plas-the levels of expression were 7.1 mg/ml for human mu mids, including cesium chloride preparations, ready forchain and 156 mg/ml for human kappa, which is much microinjection, thus bypassing the necessity for labori-higher than achieved with the smaller YACs on a nor- ous gel purification.mal background (49). It awaits to be seen whether thechromosome transgenic mice give better expressionthan the largest YACs on a null mouse background.

The only gene for which expression was not obtainedCONCLUSIONin YAC transgenic mice is Xist, the gene responsible

for X inactivation. In one investigation, mice carryinga 460-kb YAC with 130 kb of 5* and 310 kb of 3 * DNA

YAC (and BAC) transgenesis is a powerful tool indid not show any expression of the Xist gene (5). Inthe production of genetically engineered mice whichanother, a 350-kb YAC was used and the human Xistcan be used in combination with knockouts. YAC DNAgene was expressed only when it was located in a het-can be effectively transferred into transgenic mice by aerochromatic region of the Y chromosome (7). However,number of methods, of which microinjection of smallera similar 450-kb YAC has also been transferred intoYACs or fusion with ES cells for larger YACs are partic-ES cells, and here there was good expression and Xularly useful. Using the published techniques it is rela-inactivation (15), indicating that this 450 kb of DNAtively straightforward to obtain the YAC DNA inte-was adequate to drive good levels of controlled expres-grated intact. As the inserts of YACs are so large, theysion. The reason that the Xist gene was not expressedoften contain mammalian genes intact including all thefrom the YACs in transgenic mice is therefore probablylong-range controlling elements needed to give full lev-due to expression of the Xist gene in euchromatic re-els of position-independent expression. In addition, thegions being lethal. Hence the only mice that were recov-genes are generally expressed in a tissue-specific man-ered were ones where the gene was inactivated by somener, are differentially spliced, and are modulated bymechanism or where the expression was on the Y chro-physiological mechanisms.mosome and did not matter.

YAC transgenics thus allows complementation ofmouse mutants as a means of identifying functionalgenes. In combination with knockout technology to in-

BAC AND PAC TRANSGENICS activate the endogenous mouse genes, YAC trans-genesis also allows the production of humanized mice.This has been demonstrated with the mice expressingBAC and PAC clones are becoming increasingly usedhuman antibodies which is allowing the production ofin physical cloning and can also be used to makehuman monoclonal antibodies for the first time. Thetransgenic animals by pronuclear injection (51, 52) andtechnology can be extended to obtain humanized miceprobably by introduction into ES cells as well. Althoughwith high-fidelity expression of subtly modified trans-they are smaller than many YACs, having an uppergenes. Such mice can be used to study the structure–size limit around 300 kb, and are thus not appropriatefunction relationship of genes and can also be used tofor very large genes such as the immunoglobulin loci,assess drug or other therapies aimed at specific humanBACs are large enough to contain many mammalian

genes intact with their full repertoire of controlling ele- mutations.


206 CLARE HUXLEY

ing the ability to synthesize. Thus a Lys0 medium isAPPENDIX: PROTOCOLS FOR PREPARATION lacking lysine but contains the other compounds thatAND MICROINJECTION OF YAC DNA the yeast host cannot synthesize. The compounds listed

below are mixed very well in the proportions given (un-der g in mix), leaving out the relevant compound (orGrowth of YAC-Containing Yeast Strainscompounds if one wishes to select for more than oneMost YAC libraries have been made in the pYAC4 marker at once). A certain amount of this dropout mixvector which has the yeast TRP1 selectable maker on (given in the final column of the table as mg of mix/the left arm and URA3 on the right arm. Such YACs liter) is then used to make 1 liter of medium.are standardly cultured in AHC medium at 307C with

agitation (23). AHC lacks both tryptophan and uracilFinal Sigma g in mg ofand hence selects for retention of the YAC in the yeast

mg/liter Ingredient Catalog No. mix mix/literhost which cannot synthesize either of these com-pounds. AHC is normally made with low adenine (10 40 Adenine hemisulfate A-9126 2 610

40 L-Arginine hydrochloride A-5131 2 610mg/liter) which gives the yeast a red color due to the20 L-Histidine hydrochloride H-8125 1 630ade2 mutation in the yeast host. However, this limits60 L-Isoleucine I-2752 3 590the growth of the yeast; the aAHC medium described60 L-Leucine L-8000 3 590

here contains 40 mg/liter adenine and does not limit 50 L-Lysine hydrochloride L-5626 2.5 600the growth of the yeast so much, though the yeast are 20 L-Methionine M-9625 1 630

50 L-Phenylalanine P-2126 2.5 600still pink. YACs that have other combinations of se-200 L-Threonine T-8625 10 450lectable markers are grown in the appropriate dropout40 L-Tryptophan T-0254 2 610medium while YPD is a rich medium for growth of the 50 L-Tyrosine T-3754 2.5 600

yeast host strain or for growth of the YAC-containing 20 Uracil U-0750 1 630strains for short periods.

As with bacteria, the yeast is first grown on agarAlternatively, Bio 101 sells dropout mixtures.plates to obtain single colonies and then single colonies

To make the dropout medium, combine 6.8 g yeastare grown up in liquid medium. Yeast have a doublingnitrogen base without amino acids (Difco No. 0919-15),time of about 90 min and thus take longer to reachthe amount of dropout mix indicated above (under mgsaturation than bacteria. Yeast strains are stored atof mix/liter), and 20 g glucose. Dissolve in H2O; bring0707C after adding glycerol to 15%.the pH to 5.8 with 1 M NaOH and bring to 1 liter.Add 17 g agar (Difco No. 0140-01) per liter for plates.aAHC MediumAutoclave. For a richer medium add 50 ml of filter-

YAC-containing strains grow to nearly the same den- sterilized 40% glucose per liter after autoclaving rathersity in aAHC medium as in YPD medium, Ç108/ml. than before.

Per 1 liter, combine 6.7 g yeast nitrogen base withoutamino acids (Difco No. 0919-15), 10 g casein hydroly- YPD Mediumzate-acid low salt (U.S. Biochem No. 12852), 40 mg

To prepare this complete medium for growing alladenine hemisulfate (Sigma No. A-9126), and 20 g glu-yeast strains, combine 20 g glucose, 10 g yeast extract,cose. Add water; bring to pH 5.8 with 4 M NaOH ifand 20 g peptone per liter. Autoclave. For a richer me-necessary. Add 17 g agar (Difco No. 0140-01) for agardium add 50 ml of filter-sterilized 40% glucose per literplates. Autoclave. For a richer medium, add 50 ml ofafter autoclaving rather than before.40% glucose (filter-sterilized) per liter of aAHC after

autoclaving rather than adding the glucose before auto-Preparation of High-Molecular-Weight Yeast DNA inclaving.Agarose Plugs

Dropout Medium To protect the very long DNA molecules from shear-ing in solution, the yeast are embedded in agaroseWhen one is selecting for a specific yeast markerplugs prior to spheroplasting and preparation of thesuch as LYS2, one grows the yeast in a medium thatDNA. The plugs described here are at high concentra-is lacking lysine, so that only those yeast that have thetion so that the preparative pulsed-field gels will con-LYS2 gene, and can synthesize lysine, are able to grow.tain as much YAC DNA as possible. The yeast are sphe-A dropout medium is a basic minimal medium withroplasted in the agarose plugs. The plugs are thenadded ingredients that include all the amino acids andtreated with lithium dodecyl sulfate solution (no pro-other molecules that laboratory yeast strains are com-teinase K is used in this protocol). The protocol is basedmonly unable to synthesize. However, it lacks the spe-

cific compound, such as lysine, for which one is select- on that described by Southern et al. (55).


207YAC TRANSGENESIS

Solution I 1 M sorbitol 40 ml (1 M) 8. Mix and place in a plug mold that has been chilled20 mM EDTA, pH 8.0 1.6 ml (0.5 M) on ice.14 mM 2-mercaptoethanol 40 ml (14 M)Make up fresh. We use molds that are cut out of a 0.8-cm-thick block

Enzyme 6 mg/ml yeast lytic enzyme of Perspex. Slots 2.5 cm 1 1.5 mm are drilled throughsolution (70,000 U/g, ICN No. 36- the Perspex. One side of the Perspex is sealed with

094-2) in solution Itape. When the agarose has set, the tape is removedMake up fresh.and the agarose pushed out with a Pasteur pipet. TheseAgarose Solution I without 2- 10 ml

solution mercaptoethanol large plugs are preferable to small 100-ml plugs for2% SeaPlaque GTG 0.2 g preparative gels.

agarose (FMC No.9. Chill on ice for 10 min.50112)

Melt in a microwave, then 10 ml (14 M) 10. Place plugs in enough solution II (with yeastadd 2-mercaptoethanol lytic enzyme at 2 mg/ml) for the plugs to be able toto 14 mM. move around.

Make up fresh and hold at11. Incubate at 377C for 2 h with agitation.407C.12. Replace solution II with at least 10 ml of LDS/Solution II 1 M sorbitol 40 ml (1 M)

20 mM EDTA, pH 8.0 1.6 ml (0.5 M) ml of plug.10 mM Tris, pH 7.5 400 ml (1 M) 13. Incubate at 377C for 1 h with agitation, then14 mM 2-mercaptoethanol 40 ml (14 M) replace solution with same and incubate at 377C over-2 mg/ml yeast lytic enzyme 80 mg

night with agitation. It is important that the plugsMake up fresh.move around and that there is plenty of LDS solution.LDS 1% Lithium dodecyl sulfate 5 g

(Sigma No. L-4632) The LDS is the active ingredient in this protocol; no100 mM EDTA, pH 8.0 100 ml (0.5 M) proteinase K is needed.10 mM Tris, pH 8.0 5 ml (1 M) 14. Wash plugs in 10 ml of 11 NDS/ml of plug for 2H2O to 500 ml

h at room temperature with agitation.Filter-sterilize and store at15. Repeat wash.room temperature.

51 NDS Mix 350 ml H2O, 93 g EDTA, and 0.6 g Trizma Plugs can now be loaded directly onto gels or stored atbase.

47C in 11 NDS. It is very important that the plugs areBring pH to ú8.0 with Ç100–200 pellets of solidwashed thoroughly to remove the LDS.NaOH.

Add N-laurylsarcosine (5 g predissolved in 50 mlPreparation of YAC DNA for Pronuclear InjectionH2O).

The plugs of high-molecular-weight DNA are loadedBring pH to 9.5 with concentrated NaOH.Bring volume to 500 ml with H2O. on a pulsed-field gel and the chromosomes separated.Filter-sterilize and store at room temperature. A slice containing the YAC is excised and agarased.

Figure 1 shows what a preparative pulsed-field gelInoculate 200 ml of selective medium with 2 ml ofa saturated o/n culture. Grow overnight at 307C withshaking. The nearly saturated culture should containbetween 5 1 107 and 1 1 108 cells/ml.

1. Spin down cells at 1000g for 5 min and discardmedium.

2. Resuspend cells in 100 ml of 50 mM EDTA. Re-peat step 1.

3. Using a preweighed tube, resuspend cells in 40ml of solution I. Repeat step 1. Remove all supernatantat this point.

4. Weigh the tube and determine the weight of thepellet of yeast. Assume a density of 1 g/ml and calculate

FIG. 1. Preparative pulsed-field gel of YAC DNA. High-concentra-the volume of the pellet. tion plugs of high-molecular-weight yeast DNA were separated on a

5. Let yeast pellet volume Å x. Add half x volume of 1% low-melting-point agarose gel with 23-s switching, 200 V, and117C, for 36 h on a Chef-DRII apparatus (Bio-Rad). The edge slicesenzyme solution to give a concentration of 2 mg/ml ofwere stained and marked with a nick at the position of the YAC.enzyme. Resuspend yeast cells.The YAC was cut out of the center portion prior to staining this part6. Warm briefly to 407C. of the gel. The figure shows the gel after it has been completely

7. Immediately add an equal volume of Agarose So- stained and reassembled, and it can be seen that the YAC has beencorrectly excised.lution (i.e., a volume of 1.5x).


208 CLARE HUXLEY

should look like after the YAC DNA has been excised This clears the solution so that it will go through amicroinjection needle.and the whole gel stained with ethidium bromide. The

very wide bands of the yeast chromosomes indicate that 12. Keep 40 ml of the DNA solution for checking ona pulsed-field gel.a large amount of DNA has been loaded onto the gel.

If the gel is overloaded, then the bands smear into eachother. Concentration with a Filter Unit

1. Place 400 ml oocyte-grade water into a Millipore1. Load the plugs right across a 1% low-melting- Ultrafree-MC 30,000 NMWL Filter Unit (Millipore No.

point agarose (SeaPlaque from FMC) gel. If you are UFC3 TTK 00).using the large plugs described above, cut the plugs in 2. Spin at 6000 rpm in a microcentrifuge for 5 minhalf longways and load right across the width of the or until all the water has gone through the filter. Dis-gel. Seal the plugs in with agarose. card the water. This washes the filter.

2. Run the pulsed-field gel. For low-melting-point 3. Transfer 400 ml of the agarased and spun DNAagarose, run the gel at the same switching time as solution into the filter unit with a cutoff blue tip.normal but you may need to run the gel for 36 h instead 4. Spin at 6000 rpm in a microcentrifuge for 5 min.of 24 h to obtain good resolution. Set the switching so 5. Measure the volume of liquid that has passedthat the YAC is near the top of the gel to obtain good through the filter.resolution. 6. Continue spins until 320 ml has passed through

3. Cut 2-cm strips off the left and right sides of the the filter. This would give a 51 concentration.gel and stain these with ethidium bromide. Store the 7. Close filter unit and let it sit on the bench for 4center portion of the gel in 0.51 TBE. Visualize the h or at 47C overnight.DNA in the edge strips on a UV light box and mark 8. Carefully remove the DNA solution with a cutoffthe strips at the position of the YAC (cut a nick in the yellow tip. Pipet up and down a couple of times slowlyside of the gel strip). Reassemble the gel on a glass to dislodge the DNA from the surface of the filter.sheet and cut out a slice about 2 mm wide that contains 9. Keep 20 ml of the DNA solution for checking on athe YAC; the narrower the slice, the higher the concen- pulsed-field gel.tration of DNA. Cut the strip into 1-cm pieces and putinto a 50-ml tube with agarase buffer (below). Stain

Dialysisthe central portion of the gel and reassemble the whole1. Preequilibrate a Millipore filter Catalog No. VMWgel on a UV box to check that the YAC was cut out.

02500, type VM, pore size 0.05 mm), for several hours4. Equilibrate the pieces of agarose two times for 2on the surface of microinjection buffer (10 mM Tris, pHh with 20 ml of 11 agarase buffer (10 mM BisTris–HCl,7.4, 0.2 mM EDTA, 100 mM NaCl) made with embryo-pH 6.5, 0.2 mM EDTA, 100 mM NaCl) with agitation atgrade water. Use 30 ml of buffer in a Petri dish androom temperature. This agarasing buffer is very im-place the filter shiny side up.portant; make up 100 ml of 101 stock and store 10-ml

2. Transfer the filter to new buffer and place 200 mlaliquots at 0207C. Make the buffer up with embryo-of agarased, spun, and concentrated DNA solution ontoquality water (Sigma No. W-1503) and use this qualitythe filter using a cutoff yellow tip. Dialyze for severalwater for all the following steps.hours.5. Place pieces of agarose into preweighed 1.5-ml

3. Carefully take up the DNA solution with a cutofftubes and determine the weight of agarose; insertyellow tip.about 500 mg per tube.

4. Keep 20 ml of the DNA solution for checking on a6. Spin the agarose to the bottom of the 1.5-ml tubepulsed-field gel.by pulsing for about 6 s in a microcentrifuge.

7. Place in a 687C water bath for 10 min. It must beChecking the DNAhot enough to completely melt the agarose.

8. Place in a 407C water bath for 5 min. Figure 2 shows a 320-kb YAC after various stages of9. Add 1 ml of agarase (NEB No. 392L, 1 U/ml) per preparation and purification run out on a pulsed-field

100 mg of agarose. Mix by pipetting once slowly with gel. The purified YAC DNA can easily be seen on thea cutoff blue tip (cut the end off a blue pipet tip with ethidium bromide-stained gel.a razor blade to give an approximately 2-mm aperture).

10. Leave at 407C for 2 h. 1. Add DNA loading buffer to the samples of agar-11. Centrifuge the solution at 13,000 rpm in a micro- ased DNA with a yellow tip but do not mix. Leave the

centrifuge at room temperature for 20 min and care- tubes on the bench for about 30 min, by which time thefully transfer the supernatant to a new tube (if the loading buffer will have diffused into the DNA solution.

2. Prepare a pulsed-field gel with size markers andcentrifuge gets too hot this step will shear the DNA).


209YAC TRANSGENESIS

empty wells for the agarased DNA samples. Cool the injection needle and allowed to run up to the tip bycapillary action (the needles must have an internal cap-gel in the pulsed-field gel apparatus for about 30 min.

3. Switch off the pump in the pulsed-field gel appa- illary). To do this, put about 10 ml of DNA in a 1.5-mltube. Place the needle in the tube back end down; youratus and load the liquid DNA samples into the wells

with cutoff yellow tips (the samples should be pipetted will see the needle tip fill in a minute or two. Tenmicroliters is enough DNA for about five needles.up and down in the tubes once to mix in the loading

buffer). The frequency of transgenics is approximately thesame as that achieved with plasmid DNA. However,4. Switch on the voltage of the pulsed-field gel appa-

ratus and let it run for about 30 min with the pump some difficulty may be encountered with injection ofthe YAC DNA solution, which tends to be rather sticky.still off. When the dye has migrated about 1 cm into

the gel, set the pump going and let the gel run so thatthe YAC DNA is well resolved.

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Exploring Gene Function: Use of Yeast Artificial Chromosome Transgenesis