The human B lymphoblastoid cell line (LCL) 721.174 sustains a large homozygous deletion in the major histocompatibility complex (MHC) class II region that results in an absence of DQ and DR molecules as well as a deficiency in the assembly and transport of class I molecules to the cell surface. The deleted genes include the transporters associated with antigen processing TAP1 and TAP2, DMA and DMB which are involved in editing class II bound peptides, and four genes whose roles in antigen processing are unclear; the low mass polypeptide genes LMP2 and LMP7, and DNA and DOB. To study this region we have integrated into 721.174 two overlapping yeast artificial chromosome (YAC) clones which cover the interval LMP2-DRA inclusive. Three clones (11.2A1.1, 4D1D10.1 and 4D1D10.2), containing complete copies of the transfected YAC, produced varying levels of mRNA from the LMP, TAP, DQ and DR genes and corresponding levels of LMP and TAP protein. Class I cell surface expression was restored in 11.2A1.1 and 4D1D10.1, as was DR expression in both 4D1D10 transfectants. These studies demonstrate the feasibility of introducing large groups of functional genes back into human lymphoblastoid cells sustaining deletions, with full restoration of biological function. The procedure could be exploited in order to restore all but one gene covered by the deletion, effectively producing a single gene defect. This could be used to introduce copies of genes engineered to contain mutations and to study cis regulatory elements at some distance from the chosen loci.
The ability to clone and manipulate large segments of DNA is pivotal to many aspects of molecular biology. Until the advent of yeast artificial chromosomes (YACs) (1 ), many problems of eukaryote molecular biology, such as the isolation of very large genes and gene clusters, analysis of large transcription units and physical mapping of ordered fragments from complex genomes, had not been possible. Nor was it possible to study the function and regulation of genes in an environment approximating their normal chromosomal context. Since then, YAC DNA has been successfully reintroduced back into, and expressed in, a variety of mammalian cell lines and transgenic animals using a number of different methods (for reviews, see 2 ,3 ). Although the majority of these studies have analysed the expression of only one gene encoded by a YAC, several have examined the combinatorial expression of multiple genes on a single cloned fragment of DNA. Using YACs carrying normal copies of the [beta]-globin locus (a cluster of five functional genes), Peterson et al. (4 ) and Gaensler et al. (5 ) found that expression in transgenic mice was both tissue- and developmental stage-specific and closely followed the pattern of expression of the endogenous mouse [beta]-globin locus. A third study (6 ) introduced two different types of mutations into this locus (a point mutation and several 3' deletions) and concluded that mutant YACs can be used successfully in the analysis of the cis control of developmentally regulated genes. Similarly Demmer and Chaplin (7 ) transferred a portion of the human major histocompatibility complex (MHC) class II region containing, amongst others, the DQA1, DQA2, DQB1, DQB2, TAP1 and TAP2 genes into Chinese hamster ovary and mouse L-cells and observed that the genes were transcriptionally active in the L-cell lines and, in the case of the DQ genes, able to express serologically detectable cell surface human leukocyte antigen (HLA)-DQ protein.
In this study, we have used a mutant human B lymphoblastoid cell line (LCL) for transfections. The LCL 721.174, which sustains a homozygous deletion in the MHC covering DPB1 through DRA inclusive (8 ), was produced by two cycles of [gamma]-ray mutagenesis on the parental LCL 721 (9 ) followed by selection for HLA antigen loss (10 ). One of the phenotypes of this mutant LCL is a total absence of all DQ and DR surface molecules. Transfection of DRA and DRB genes restored surface expression to normal levels; however, the molecules showed an abnormal conformation and impaired ability to present antigen (11 ,12 ). Defects in the assembly and subsequent transport of class I molecules to the cell surface have also been noted, resulting in total loss of B5 expression and a reduction of 70-80% of A2 on the cell surface (13 ,14 ). Expression of these molecules can be stabilised by the addition of appropriate peptide ligands (15 -17 ) or by transfecting 721.174, or its hybrid derivative T2 (18 ), with the TAP1 and TAP2 genes (19 ,20 ). This latter case, in the absence of the LMP2 and LMP7 genes, also resulted in the loading of class I molecules with endogenous peptide and re-expression at the cell surface.
Using YACs from the human MHC class II region, we aimed here to produce new stable cell lines where part of the MHC class II region is reintroduced back into 721.174. We specifically chose YACs that contained neither the DM or DO genes with the view to examining the effect of these genes on DR/DQ antigen processing and presentation in future studies. The four YACs used cover the regions from LMP2-DRA, LMP2-DRB1, DQB1-DRA and DQB3-DRB1. We also wanted to assess whether the genes contained in the introduced YAC DNA would be expressed successfully both internally and on the cell surface.
As far as we can determine, this is the first time that a large deletion has been restored in a human LCL. As such, this work is potentially useful not only in generating a variety of mutant cell lines on an identical genomic background but also in the identification of disease genes in YACs where cell lines established from patients express the phenotype (e.g. lymphoblastoid cells from Fanconi anaemia patients).
The 4D1 (21 ,22 ) and 11.2 (23 ) YACs were first retrofitted with the amplification vector pCGS990 (24 ) and then with the fragmentation/selectable marker vector, pBP109hyg. Eightyseven 4D1, and 94 11.2 derived fragmentation clones were analysed by pulsed-field gel electrophoresis (PFGE). The 4D1 YAC yielded six different product sizes (145, 265, 390, 435, 485 and 535 kb) and the 11.2 YAC eight different product sizes (145, 180, 250, 290, 310, 335, 360 and 385 kb). From these, two products were chosen from each YAC fragmentation for subsequent transfection [4D1D11: 265 kb (DQB1-DRA) and 4D1D10: 535 kb (LMP2-DRA) derived from YAC 4D1; and 11.2C10: 180 kb (DQB3-DRB1) and 11.2A1: 385 kb (LMP2-DRB1) derived from YAC 11.2] (see Fig. 1 ).
Twelve transfections each of 4D1D11, 4D1D10, 11.2C10 and 11.2A1 into 721.174 resulted in a total of 12 independent hygromycin BR cell lines (five from 4D1D10, four from 4D1D11, and three from 11.2A1). As an initial screen, all positive cell lines were tested by PCR for the presence of the genes TAP1, TAP2, DQA and DRB. Nine of the 12 clones were negative for all genes and presumably contained only the hygromycin BR marker. The remaining three cell lines (11.2A1.1, 4D1D10.1 and 4D1D10.2) were positive for the four genes and were subsequently used for further analysis.
The karyotype of the 721.174 cell line was established as 45,XX,-6,der(16)t(6;16)(q11.2;q11.2). This cell line is therefore monosomic for 6p and 16q. The 11.2A1.1, 4D1D10.1 and 4D1D10.2 cell lines were also karyotyped. All had an additional deletion of the long arm of chromosome 1 with breakpoints at q23 and q31.2.
To determine that the YACs had integrated stably into the 721.174 genome, the cosmid probes U15 (25 ,26 ), LC10 (27 ) and LH1 (23 ) (outlined in Fig. 1 ) initially were mapped to metaphase spreads from this cell line, and to a normal LCL (CB72) by fluorescence in situ hybridisation (FISH) (Fig. 2 ). With the exception of a hybridisation signal on the p-terminus of chromosome 1 seen with LC10 (also noted in CB72), no other signal was observed in 721.174. Conversely, in CB72, a single hybridisation signal at 6p21.2/21.3 was seen with these probes. In the transfectants, the YACs were observed to have integrated into one site per cell in 11.2A1.1 and 4D1D10.2, at the q-arm of an anomalous acrocentric chromosome and at 18q respectively. Similarly, one YAC integration site per cell was observed in 4D1D10.1 but a variety of sites were observed; integration was also seen at some G-group satellites and in at least two chromosomes of the C- and E-groups (further characterisation of these chromosomes was not possible due to their poor morphology).
To determine whether each integrated YAC was complete, the three transfected clones and the 11.2A1 and 4D1D10 YACs were digested with BssHII and MluI, electrophoresed on a pulsed-field gel, blotted and probed with two widely separated genomic probes and a probe derived from the retrofitting vector pCGS990. The fragment sizes detected from digests of the two YACs using these probes are shown in Figure 1 .
Finer analysis of the integrated DNA was ascertained by probing Southern blots of HindIII- and EcoRI-digested LCLs and YACs with LMP2 and DRB. The results from LMP2 suggest that this end is intact in at least one copy in all transfected LCLs. In the case of 4D1D10.2, however, two extra smaller bands were found in both digests with this probe. Hybridising these blots with a probe to detect the other end (DRB) revealed a consistency in banding pattern between the two 4D1D10 clones, L0081785 and the 4D1D10 YAC, and between 11.2A1.1 and the 11.2A1 YAC, suggesting that all integrated YACs are complete at this end.
In this work, we examined the feasibility of restoring lost class I and II functions by introducing modified YAC clones from the human MHC class II region back into a LCL sustaining a homozygous deletion for part of this region. Of the 181 fragmentation clones analysed from the two parental YACs, four were selected for subsequent transfection. A total of 12 hygromycin BR cell lines were isolated from 48 transfections. Of these, using a combination of PCR, FISH and Southern blotting, three were found to contain either complete copies or large fragments of the modified YAC. Metaphase and interphase FISH analysis of these three clones revealed a single integration site for both 11.2A1.1 and 4D1D10.2, with at least two copies of the YAC present in the former clone and three in the latter, and at least three integration sites for 4D1D10.1 with one copy at each position.
Because there are BssHII and MluI sites within both YAC end vectors, if the YAC has been incorporated totally intact then the fragments generated by these two restriction enzymes should be identical between transfectants and the original YAC. Of the three clones, 4D1D10.1 was the only one that showed the same banding pattern on a pulsed-field gel (PFG) with the LMP2, DRB and pCGS990 vector probes as found in the 4D1D10 YAC, indicating that all copies of the transfected YAC at the three different chromosomal sites had been incorporated complete. By comparison, the banding patterns observed in 11.2A1.1 and 4D1D10.2 with the same probes were inconsistent. With the exception of an MluI and a BssHII fragment in the LMP2 hybridisation, all band sizes in 11.2A1.1 were different from the 11.2A1 YAC. The most likely explanation for this pattern is that there is one virtually complete and one truncated YAC in this clone. Since the TAP1 and DQA probes are present in 88% of 11.2A1.1 nuclei as doublet signals, both integrants are complete from TAP1 up to at least DQA2. FISH on 4D1D10.2 interphase nuclei using U15 and LH1 suggests that there are three integrated copies of this YAC in chromosome 18q. The additional bands observed after hybridising both the PFG and conventional Southern blots with the LMP2 probe suggest that at this end some integrated YACs are incomplete. The size of the two extra bands found in the EcoRI and HindIII digests indicates that two of the three integrated YACs sustain a deletion covering at least the EcoRI site 430 bp 3' to the LMP2 gene through to the HindIII site within the gene (29 ). At the other end, when the DRB and pCGS990 probes were hybridised to the PFG blots a number of bands were detected in this clone (none of which corresponded to the 4D1D10 YAC), consistent with the explanation that all integrated copies are incomplete. No differences between the 4D1D10 YAC, 4D1D10 clones and L0081785 cell line were detected when the DRB probe was hybridised to the EcoRI and HindIII digests, indicating that the break points of these integrated products are outside these restriction sites.
Expression of the YAC genes in the transfected cell lines was determined by Northern and Western blotting. With the exception of TAP2 in 4D1D10.2 (which was not detected at either the mRNA or protein level), all other genes tested for in the three transfected clones were expressed. It is surprising that TAP2 is not expressed in 4D1D10.2, since the expressed LMP2 gene is closer to the site of integration and as a result would be more likely to be influenced by the surrounding chromosomal sequences than TAP2. Given that several fragmented copies of the integrated YAC have been found in this clone, it is not implausible to suggest that there may be additional small deletions or rearrangements which specifically affect TAP2 expression that have not been detected by PCR or Southern analysis. It is also possible that a locus control region (LCR) involved in TAP2 expression, like that found upstream from the mouse MHC class II Ea gene (30 ), is absent or disrupted in this cell line. (Likewise the regulation of DR[alpha] expression in the 4D1D10 clones may be due to a similar LCR existing between the human DRA and DRB genes.)
The above findings were supported by the flow cytometry data where the levels of class I cell surface expression were restored to normal levels in 11.2A1.1 and 4D1D10.1 as was the level of DR expression in the two 4D1D10 transfectants. In 4D1D10.2, however, the expression of HLA-A2 was reduced (~3-fold) and HLA-B5 was totally absent. Expression of the TAP and LMP proteins correlated with the levels of mRNA produced in the three clones.
Yeast. The Saccharomyces cerevisiae yeast strains YPH274 (42 ) and AB1380 (1 ) containing the 11.2 and 4D1 YAC clones respectively were routinely grown in synthetic dextrose minimal medium (SD) (43 ) containing histidine and tryptophan (20 [mu]g/ml); lysine and leucine (30 [mu]g/ml) and adenine (50 [mu]g/ml). The Gal+ amplification strain CGY2570 (44 ) was grown in YPD medium (43 ). All strains were grown at 30oC.Mammalian. The LCLs 721.174, 721 (HLA class I control), L0081785 (HLA class II control) and CB72 (PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK) were all routinely grown in RPMI-1640 medium (Sigma, Poole, Dorset, UK) containing 10% fetal calf serum (FCS) and 4 mM l-glutamine (Gibco-BRL, Paisley, UK). LCL 721 was also grown with 100 U/ml penicillin and streptomycin (Sigma). Established transfected cell lines were grown in the same media containing 200 [mu]g/ml hygromycin B (Boehringer Mannheim, Lewes, East Sussex, UK). Expansion of transfected 721.174 cells was carried out as previously described (45 ). Growth of all lines was at 37oC in a humidified atmosphere of 95% air, 5% CO2.
YAC transfer to strain CGY2570. To maximise the level of amplification, both the 11.2 and 4D1 YACs were transferred from their respective strains into the CGY2570 strain using an adaptation of the method of Burgers and Percival (46 ). An agarose block containing the YAC was equilibrated overnight at 4oC in 10 mM Bis-Tris (pH 6.5) (Sigma), 1 mM EDTA, 0.7 mM spermidine, 0.3 mM spermine (equilibration buffer). The block was melted at 67oC for 10 min, cooled to 40oC for 10 min then 2 U of [beta]-agarase (New England Biolabs, Inc., Hitchin, Herts, UK) was added and the sample incubated at 40oC for 3 h. Fifty [mu]l of this solution was added to 150 [mu]l of 1 M sorbitol, 10 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 0.7 mM spermidine and 0.3 mM spermine and used to transform 1*108 CGY2570 spheroplasts. Selection for colonies taking up the YAC was carried out using SD medium (as above) containing 1 M sorbitol and 2% agar.YAC modification with pCGS990 and pBP109hyg. One [mu]g of the amplification vector pCGS990 was linearised with SalI, gel purified and used to transform yeast containing the 11.2 and 4D1 YACs according to the method of Gietz et al. (47 ) with the modifications of Smith et al. (48 ). Once the colonies had grown, they were replica plated onto selective media without tryptophan. Those colonies that failed to grow on these plates were deemed to have been retrofitted correctly and subsequently were amplified (see below).
To facilitate both fragmentation and the addition of a mammalian selectable marker to the YACs, the vector pBP109 (49 ) was modified by ligating in a resistance cassette from pREP4 (Invitrogen, Abingdon, UK). The hygromycin BR gene, TK promoter and TK polyadenylation signal and transcription terminator were excised from pREP4 as a single fragment using SalI and NruI, and gel purified. pBP109 was linearised at the NotI site and both vector and fragment were blunt ended with T7 polymerase (50 ), and gel purified prior to ligation. The modified plasmid (pBP109hyg) was transformed into the Escherichia coli strain DH5[alpha]tm (Gibco-BRL) and selected for on plates containing ampicillin (50 [mu]g/ml) and hygromycin B (100 [mu]g/ml). pBP109hyg was linearised with SalI and gel purified. Transformation was exactly as described for pCGS990. Correctly retrofitted clones were those that failed to grow on plates minus uracil.
DNA for Southern analysis (and PCR) was prepared in blocks essentially as previously described (50 ). [For PCR, blocks were melted at 65oC for 10 min and then diluted 1/8 with TE (pH 8.0); 4 [mu]l of this was used per PCR reaction.)] Restriction digestion of yeast/YAC and mammalian plugs was as described by Anand (53 ). PFGE and regular electrophoresis were carried out in 0.5* TBE, 1% agarose (the PFGE conditions were as described above). Preparation of gels and subsequent alkaline transfer to Hybondtm-N+ membranes (Amersham, Little Chalfont, Bucks, UK) was performed using standard protocols. Hybridisation was overnight at 65oC, and membranes were washed at high stringency.
Total cellular RNA was extracted using the Purescripttm RNA Isolation Kit (Gentra Systems, Inc., Minneapolis, MN, USA) and poly(A)+ RNA using the PolyATract® mRNA Isolation System IV Kit (Promega, Southampton, UK). Samples were electrophoresed in 1% agarose/formaldehyde gels and transferred to Hybondtm-N+ membranes according to the supplied protocol. Hybridisation was at 42oC overnight in formamide hybridisation solution, and membranes were washed at high stringency (0.1* SSC/0.1% SDS for 2*15 min at 68oC).
The TAP1 and TAP2 genes were amplified using the PCR primers TAP1/ARMS1 and TAP1/ARMS4, and TAP2/ARMS1 and TAP2/ARMS4 respectively (54 ), the DQA and DRB genes were amplified using primers GH26 and GH27 (55 ), and GH46 and GH50 (56 ) respectively. In all instances, the conditions were as previously described. The probes used for Southern and Northern analysis were as follows: DRA (1.2 kb cDNA, 57 ), DRB (pRTV-II, 58 ) DQA [1.3 kb insert from probe #57390, American Type Culture Collection (ATCC), Rockville, MD, USA], DQB (176 bp PCR product using primers GH28 and GH29, 59 ), TAP1 (2.8 kb cDNA, 60 ), TAP2 (427 bp PCR product using primers TAP2/ARMS1 and TAP2/ARMS4, 54 ), LMP2 (0.7 kb cDNA, 61 ) and LMP7 (1.1 kb cDNA, 62 ), [beta]-actin (2.0 kb cDNA, Clontech, Cambridge, UK). The cosmid clones used for FISH analysis were: U15 which contains the TAP1 gene (25 ,26 ), LC10 which contains the DQA locus (27 ) and LH1 which is at the telomeric end of the class II region (23 ).
YAC and cosmid clones were labelled with biotin-14-dATP (Gibco-BRL) and/or digoxigenin-11-dUTP (Boehringer Mannheim) and used for FISH as previously described (63 ). FISH was carried out on metaphase chromosomes which were prepared from the various LCLs by standard cytogenetic techniques (64 ). Karyotyping of the various cell lines was carried out using a combination of standard G-banded chromosome analysis and FISH with whole chromosome paints (Cambio Ltd, Cambridge, UK) and chromosome-specific centromere probes (Appligene, Oncor, Gaithersburg, MD, USA). Signals were visualised using a Zeiss Axioplan microscope equipped with a cooled charge-coupled device camera (Photometrics, Tucson, AZ, USA) and Smartcapture image analysis system [Vysis (UK) Ltd, Richmond, Surrey, UK]. Chromosomes were counterstained with DAPI (Sigma) and G-banding was enhanced during image analysis.
The interphase nuclei present in the chromosome preparations were also investigated in order to establish how many copies of the transfected YAC were present per cell. One cosmid from one end of each YAC was labelled with biotin and developed with fluorescein isothiocyanate (FITC; green) and one from the other was labelled with digoxigenin and developed with tetrametylrhodamine isothiocyanate (TRITC; red) (see Fig. 2 ). Nuclei were then examined for the presence of doublet signals composed of one of each of the cosmids and therefore indicating the presence of one copy of the YAC. A nucleus was considered informative when one or more doublet signals were observed and at least 50 informative nuclei were examined per sample.
Antibodies. The antibodies W6/32, 4D12, BB7.2 and L243 are directed against HLA-A,B,C, HLA-B5, HLA-A2 and HLA-DR respectively (65 -68 ) and were obtained from the ATCC. Rabbit antisera against the C-terminus of the proteasome subunits LMP2, LMP7 and delta and against the C-terminus of TAP1 have been described (28 ,69 ). Rabbit antisera against TAP2A and TAP2B were generated using the following C-terminus peptides: TAP2A, HQILVLQEGKLQLAQL; TAP2B, QEGQDLYSRLVQQRLMD. FITC-conjugated F(ab')2 rabbit anti-mouse immunoglobulins and horseradish peroxidase (HRP)-conjugated swine anti-rabbit antibody were purchased from Dako Ltd. (High Wycombe, Bucks, UK).Flow cytometric analysis. Cells were stained indirectly with antibodies specific for HLA-A,B,C, HLA-A2, HLA-B5 and HLA-DR molecules using standard methods. Briefly, 3*105 cells were incubated with the corresponding hybridoma supernatants for 20 min at 4oC. Cells were then washed and incubated with FITC-conjugated F(ab')2 rabbit anti-mouse immunoglobulins, for 20 min at 4oC. After washing, the cells were analysed using a FACScan® flow cytometer with CellQest® software (Beckton Dickinson, San Jose, CA, USA). Dead cells were excluded on the basis of forward and side light scatter.
Cells were lysed at 4*107 cells/ml in 50 mM Tris-HCl (pH 7.5) buffer containing 0.5% NP-40, 0.5% Mega 9, 150 mM NaCl, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM iodoacetamide. After removing nuclei and aggregates, the protein concentration in the lysates was determined using the BCA reagent (Pierce, Rockford, IL, USA). Samples (25 [mu]g) were separated by SDS-PAGE (70 ) using gels of 10 and 12.5% acrylamide for the TAP and LMP proteins, respectively. After electrophoresis, proteins were electrotransferred onto PVDF membranes (Immobilon Millipore, Bedford, MA, USA). Membranes were blocked in phosphate-buffered saline containing 0.01% Tween-20 and 5% skimmed milk powder. Membranes were then probed with antisera specific for TAP1, TAP2A, TAP2B, LMP2, LMP7 and delta proteins diluted in the above blocking solution. Antibody binding was detected by incubation with HRP-conjugated swine anti-rabbit antibody, followed by enhanced chemiluminescence (Amersham).
We thank Dr R. DeMars for the 721 and derivative cell lines. This work was supported by grants from Guy's and St. Thomas's special trustees and by MRC grant no. 69212533MA.
Human Molecular Genetics
Pages
Introduction
Results
Generation of MHC class II YAC variants for transfection
Introduction of YAC variants into the LCL 721.174
Karyotype determination, analysis of integrated YAC DNA and estimation of copy number
Determination of YAC integrity
Expression analysis of YAC genes in transfected cells
Discussion
Materials And Methods
Cell culture and strains
YAC manipulation
YAC amplification, concentration and lipofection
DNA extraction, Southern analysis and PCR
RNA extraction and Northern analysis
PCR primers and DNA probes
FISH
Flow cytometry
Western blotting
Acknowledgements
References
Transcriptional analysis. Northern blot hybridisation of mRNA samples from the three transfected clones was carried out with DRA/B, DQA/B, TAP1/2, LMP2/7 and [beta]-actin probes (Fig. 3 ). Expression of all MHC genes was positive in the L0081785 cell line and negative in the 721.174 cell line. In 11.2A1.1, the expression of LMP2, LMP7 and DQA and DRB appeared close to normal whereas TAP1, TAP2 and DQB expression was elevated. 4D1D10.1 expressed increased levels of DRA, DRB and DQB and approximately normal levels of the remaining genes, whereas 4D1D10.2 showed decreased levels of TAP1, LMP2 and LMP7 expression and no discernible TAP2 expression.
YAC amplification. Amplification of the yeast strain CGY2570 containing the 11.2 and 4D1 YACs was carried out essentially as previously described (44 ), except that in order to apply selection at all stages the various media were deficient in lysine. To achieve this, the 1% casamino acids were omitted from the S-gal, MST-gal and S-glc media and replaced with an amino acid mix consisting of arginine, isoleucine, uracil (all 20 [mu]g/ml), phenylalanine (50 [mu]g/ml), valine (150 [mu]g/ml) and tyrosine (30 [mu]g/ml) and the final solution adjusted to pH 5.8. For convenience, confluent amplified cultures were centrifuged, resuspended in YPD media containing 20% glycerol and frozen at -70oC. A 1/1000 dilution of the amplified cultures subsequently was grown under selection until confluent in S-glc(lys-) medium.YAC concentration. The method used to prepare yeast chromosomes in agarose plugs was as previously described (51 ). To prevent too much distortion after PFGE, yeast cells were usually concentrated to no more than 1/40th of the original culture volume. PFGE was carried out on a CHEF-DR® II apparatus (Bio-Rad, Hemel Hempstead, Herts, UK) using the conditions described by Maule et al. (52 ) at 6 V/cm with 10-60 s switch times for 18-20 h. Further concentration was also as described by Maule et al. (52 ), except that the concentrated DNA was equilibrated three times at 4oC in 25 vol of equilibration buffer containing 100 mM NaCl. The samples were then weighed, melted at 67oC for 10 min and cooled to 40oC for 10 min before adding 3 U of [beta]-agarase I per 100 mg of molten agarose. Incubation was at 40oC for 2 h. The concentration and integrity of the DNA was determined by PFGE using the same conditions as described above. This DNA was used without further manipulation in transfections.Lipofection. The lipofection complex consisted of 150 [mu]l of agarased YAC DNA (containing 0.5-1 [mu]g of purified DNA) added to 50 [mu]l of equilibration buffer containing 100 mM NaCl and 4 [mu]l of Lipofectin® reagent (Gibco-BRL). The lipofection conditions and subsequent cloning were as previously described (45 ).
REFERENCES
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