0 1983 Alan R. Liss, Inc.

Cytometry 4:99-108 (1983)

REVIEW ARTICLE Somatic Cell Genetics and Flow Cytometry'

Michael E. Kamarck, James A. Barbosa, Lukas Kuhn, Pamela G. Messer Peters, Lester Shulman, and Frank H. Ruddle2
Department of Biology, Yale University, New Haven, Connecticut 06511
Received for publication February 28, 1983; accepted April 8, 1983

Human genes coding cell surface molecules can be introduced into mouse host cells using a variety of somatic cell genetic techniques. Because these human gene products can be detected using indirect immunofluorescence on viable cells, the genes themselves can be monitored and manipulated using flow cytometry and sorting. In this paper, we review ways
. -

that we have used cell sorting to develop a somatic cell genetic analysis of the human cell surface. Key terms: Somatic cell hybrids, DNA-mediated gene transfer, cell surface antigens, flow cytometry, cell sorting

In the past two decades a number of methods have been developed for the introduction of human chromosomes or DNA into mouse somatic cells. These systems include whole cell hybridization, microcell-mediated gene transfer (MMGT), chromosome-mediated gene transfer (CMGT), and DNA-mediated gene transfer (DMGT) (1,9,17,33,34,39). The method utilized in transferring human genes determines the fraction of the human genome which is introduced into the host cell. Whole cell hybridization results in the production of human x mouse hybrids which may contain up to a dozen or more intact human chromosomes, while MMGT produces somatic cell hybrids with one or several human chromosomes (9,33). When purified metaphase chromosomes are transferred into host cells by CMGT, the transfectants often contain visible chromosome fragments (17). In contrast, DNA-mediated gene transfer has been used to introduce purified, cloned human genes into mouse somatic cells (34,39). In all of the above methods, the expression of donor genes in the host cell facilitates studies on gene mapping, structure and function. This laboratory has made extensive use of whole cell hybridization and DNA-mediated gene transfer (DMGT) in the study of genes which code for human cell surface antigens. The expression of a human-specific surface antigen on a host somatic cell serves as a constitutive marker for the genetic material that codes for it (38). Since cell surface antigens can be detected on single, viable cells by the use of specific antibodies and indirect immunofluorescence, the presence of genetic material which codes for human antigens can be monitored and manipulated using fluorescence-activated cell sorting (FACS). This paper reviews the contribution of flow cytometry to somatic cell genetics in the study of genes which control molecules of the human cell surface.

WHOLE CELL HYBRIDIZATION

Human and mouse cells interspecifically fused with polyethylene glycol (PEG) or Sendai virus initially contain the entire genome of both parental cells. Shortly after hybridization, however, human chromosomes are randomly segregated from the hybrid line (33) (Fig. 1). Over the first few months after fusion this segregation occurs rapidly. Although some genomic stabilization occurs with time in tissue culture, even cloned lines are only metastable (Fig. 1) (26). Thus, the genetic constitution of such segregating lines must be monitored by isozyme and karyotype analysis to determine the human partial genome present a t any given time (18,271. These somatic cell hybrids are invaluable for the study of human genes and gene products retained by the mouse parent (23). Using standard clone panel mapping methods, it has been shown that human chromosomes 3,6,7,8,11,12,15,17,21,and X each contain at least one locus directing the synthesis of a species-specific surface antigen (for review, see [ 131). We examined the use of flow sorting as a genetic selection method for whole cell hybrids by initiating model studies on the human leukocyte antigens (HLA) and P2-microglobulin (&m) (12). The HLA-A,B,C heavy chains are polymorphic integral membrane glycoproteins of 44K daltons, which are coded within the major histocompatibility complex (MHC) on human chromosome 6 (29,37). HLA is found a t the cell surface in noncovalent association with a 12K-dalton water-soluble polypeptide, known as &-microglobulin, coded by a
'Presented at the meeting for Analytical Cytology IX, 1982, Schloss Elmau, Fcdcral Republic of Germany. 'This work was supported by NIH grant GM09966 to F.H.R. Address reprint requests to Dr. M. E. Kamarck, Yale University, 1036 KBT, P. 0. Box 6666, New Haven, CT 06511.

100

KAMARCK ET AL

gene on human chromosome 15 (2,6,16). A human x mouse cell hybrid cell line, FRY 4 (Frank Ruddle Yale 4 ,was examined for the expression of both human HLA 1 (Fig. 2) and p2m (12). Figure 2a demonstrates that cell hybrids expressing HLA can be specifically identified by indirect immunofluorescence and FACS analysis. Approximately 15% of the hybrid population expresses the human surface antigen HLA and therefore has retained human chromosome 6. Expressing and nonexpressing cells were then sterilely sorted based on HLA expression and were allowed to grow up in tissue culture before reanalysis. Figure 2 demonstrates the production of somatic cell hybrid lines which were homogeneously positive (Fig. 2c) or negative (Fig. 2b) for the HLA surface antigen (12). A similar analysis of &-microglobulin expression on hybrid line FRY 4 demonstrated that 99% of the hybrid cells initially expressed human Pam. As was the case with HLA, it was possible to sort these cells based on &m expression and generate hybrid subclones homogeneously positive or negative for human (32m surface antigen expression (12). To study the interaction of HLA and human fl2m on somatic cell hybrids, homogeneous sublines were prepared for the two antigens in combination (i.e., HLAf, /32mf; HLAf, Pam-; HLA-, &m+; HLA-, &m-). This was accomplished both by sorting for the two antigens sequentially, and by sorting hybrids based on two-color immunofluorescence assays (12,221. The simultaneous detection of both antigens was accomplished by indirect immunofluorescence of HLA-A,B,C with rabbit monoclonal antibody W6/32 and fluoresceinated antimouse Ig, and &m with antihuman &m and rhodamine-conjugated antirabbit Ig. Assays of the four hybrid populations generated by these methods demonstrated that two surface antigens can be labeled and sorted simultaneously (12). A karyotype analysis of the four sublines containing the two antigens in all possible combinations is presented in Table 1(12). The original hybrid FRY 4 contained a translocation of human chromosome 15 with human chromosome 1 designated t(1;15) (lqter > lp36::Eql > 15qter). The presence of this chromosome demonstrates perfect concordance with the expression of &m in these somatic cell hybrids. Similarly, human chromosome 6 was not present in HLA-A,B,C-negative cells, but could be detected on 85% of those hybrids which expressed the antigen. This percentage is probably low because of the segregation of chromosome 6 from the hybrid during the period of study. The fact that no antigen-negative cells contained the antigen-coding chromosome demonstrates that HLA-A,B,C, and human p2m are constitutively expressed on somatic cell hybrids. The expression of human cell surface antigens on human-mouse hybrids thus allows the rapid characterization and manipulation of hybrid genotype a t the cell surface using chromosome-specific antibodies and flow sorting. In this way it is possible both to monitor the continual human chromosome loss and to produce genetically homogenous human x mouse hybrid lines. The generation of such characterized hybrids will make a

WHOLE CELL FUSION

MOUSE

HUMAN

-8

RANDOM SEGREGATION

FIG. 1. The segregation of human chromosomes in human x mouse hybrids formed as a result of whole cell fusion. Human chromosome segregation is rapid, continual, and random, so that even cloned hybrid cell lines are only metastable.

Table 1 Karvotvpe Analvsis of Sorted Linesa

FRY 4 Sublines
HLA', HLA', HLA-, HLA-,

Chromosome 6 37/42 (88.1%) 27132 (84.4%)

__

Chromosome t(1;15) 47148 (97.9%)

__

&mf Pam&mf &m-

0148 (0.0%)

54/54 (100%) 0153 (0.0%)

aKaryotype analysis of FACS-derived sublines of hybrid FRY

4. Metaphase spreads from each hybrid line were prepared by

standard methods and analyzed by viokase-Giemsa banding or Giemsa-11 staining (18) for the presence of human chromosome 6 or t(l;l5Xlqter- lp36::15ql+ 15qter). The percentage of chromosome-positive spreads of those analyzed is presented. No metaphase spreads contained multiple copies of either chromosome 6 or t(1;15). The subline designation indicate the antigen selection, and whether the selection has been for (+) or against ( - ) antigen expression.

major contribution in human DNA restriction fragment mapping to specific chromosomes using Southern blotting techniques (7,28,31). Our work on HLA and human Pam also suggested an improvement in the clone panel method for mapping human surface antigens (32). Using the FACS, a n anti-

SOMATIC CELL GENETICS AND FLOW CYTOMETRY

101

280

-4

200

1 ,

120

40

40

80

120

160

200

240

40

80

120

160

200

240

Relative Fluore8cence
FIG.2. Analysis and cell sorting of human x mouse hybrid cell line FRY 4 for the expression of HLA-A,B,C antigens. Cells were stained by indirect immunofluorescence with monoclonal antibody W6132 (29) or control myeloma supernatants ( C )and analyzed and sorted using a FACS I1 (Becton Dickinson). Photomultiplier voltages were converted
by logarithmic amplifier for display on the multichannel analyzer. The parameters for cell sorting antigen positive and antigen negative hybrid cells are indicated by the arrows in panel a. Following sterile sorting, the cells were grown up in tissue culture to large volume for reanalysis. a) FRY 4, b) FRY 4.HLA-, c ) FRY 4.HLA+.

genically heterogeneous hybrid cell population can be fractionated into two subpopulations, each of which is either homogeneously antigen positive or negative. The genomes of these sublines should differ from each other by the single human chromosome coding the surface determinant. To examine the feasibility of this method, we stained two hybrid lines which contained a large number of human chromosomes with the monoclonal antibody 4F2 (24). This antibody recognizes a human glycoprotein whose cell distribution and biochemistry have been well characterized, but for which no gene mapping data were available (11).Both hybrid lines (FRY 5 and FRY 6) examined contained a fraction of cells which stained positively for the 4F2 antigen (24). Homogeneous sublines expressing or lacking the antigen were generated by flow sorting (24). Using isozyme analysis, we then identified the human chromosomes present in the parental and sorted lines (Table 2). All human chromosomes, except 9, 14, and 22, were represented by parental hybrid lines FRY 5 and FRY 6. Table 2 demonstrates that chromosome 1 is the only human 1 chromosome which segregates concordantly with the

expression of the 4F2 determinant on the sorted sublines. The mapping of this surface antigen determinant was confirmed using a human x mouse hybrid line which 1 contained only human chromosome 1 (24). Using this combination of flow sorting and somatic cell genetics, it is thus possible to efficiently chromosomally map new human cell surface antigens. This methodology will be of increasing importance with the production of new monoclonal antibodies which recognize determinants of the human cell surface.

DNA-MEDIATED GENE TRANSFER

In the work with somatic cell hybrids, we demonstrated that HLA was constitutively expressed in association with mouse &m on the surfaces of somatic cells which lacked human chromosome 15 (12). This suggested that if isolated HLA genes could be introduced into mouse cells by DNA-mediated gene transfer, they would direct surface expression of the antigen on the mouse cell surface. In DMGT, DNA is precipitated with calcium phosphate and the precipitate applied to log phase growing

102

KAMARCK E T AL

Table 2 Isozyme Analysis of Human-Mouse Hybrids and FACS-Derived Subline9 Celllines

1 2

5 6

9 10

Human Chromosomes 1 12 13 14 15 16 1

17 18

19 20

21 22

X X

FRY 6. Unsorted FRY 6. 4F2FRY 6. 4F2+ FRY 5. Unsorted FRY 5. 4F2FRY 5. 4F2+

X X

X X

X X

X X

x x
X

x x x x x x

x x x x x x

T:
X
X

x x x x x x X X x x x x x x X x x x x x x
X X X

x x

X X X
X X

X
X X

aCell preparations, starch gel electrophoresis, and cellogel techniques were performed as described by Nichols and Ruddle (27). Cell-sorter derivation of homogeneous sublines is described in Figure 2. Lines FRY 5 and FRY 6 were examined for all human chromosomes. Presence of a chromosome is indicated by X. The chromosome containing the 4F2 locus must be present in all antigen-positive populations and absent from all antigen-negative populations. Boxes indicate that chromosome 1 is the only chromosome fitting the 1 required pattern.

xr!l:
x

x x x
X X

cells (Fig. 3) (34,39).In the work described here, DNA is transferred into mouse Ltk- cells, which lack the endogenous thymidine kinase gene and cannot survive on selective medium containing hypoxanthinelaminopteridthymidine (HAT) (21). To select for host cells which are capable of taking up DNA, the herpes simplex virus thymidine kinase (HSV-tk) gene is coprecipitated with human DNA (Fig. 3). Although the exact mechanisms of DNA uptake and processing are not clear, experimental evidence suggests that a significant percentage of cells initially take up the precipitated DNA, link it together into extrachromosomal transgenomes, and transiently express the gene products at times surrounding 60 h posttransfer (4,25) (Fig. 3). Following uptake, the donor DNA undergoes extensive degradation and segregation. By applying HAT selection pressure at this time, only those cells which have taken up HSV-tk DNA will survive. Many of these cells will also have cotransferred the human gene of interest (Fig. 3). Donor genetic material is finally stabilized in the host by integration into a chromosome, a rare event occuring in perhaps 1 x lop5of the recipient cells (34). To assess the usefulness of DMGT and flow cytometry in studying human cell surface antigens, we screened a large number of human genomic clones that hybridize to a cloned HLA cDNA probe (pDP001) for their ability to direct the synthesis of HLA-A and B surface antigens on mouse Ltk - cells following DNA-mediated gene transfer (3).Individual HLA genomic clones were mixed with the HSV-tk gene. DreciDitated with calcium nhos-

DNA-MEDIATED GENE TRANSFER

MOUSE

eHUMAN DNA
-enaMm

1
TRANSIENT E XPRE SSlON DNA UPTAKE AND PROCESSING

HSV-tk DNA

1 < -I -STABLE

DNA SEGREGATION

INTEGRATION AND

FK:.3. The uptake, segregation, and stabilization of human DNA and herpes simplex virus thymidine kinase DNA in a mouse-cell host following DNA-mediated gene transfer. Details of this process are described in the text.

SOMATIC CELL GENETICS AND FLOW CYTOMETRY

103

phate, and placed on Ltk- cells. The surface expression of human histocompatibility antigens was monitored by indirect immunofluorescence and the FACS at 60 h posttransfection to detect the transient expression of these genes immediately following transfer. After 60 h, selective medium containing HAT was applied to the cells to select for those which had taken up the HSV-tk gene and could survive HAT treatment (30). After HAT selection the resistant cells (HATR)were monitored for HLA expression using indirect immunofluorescence and the FACS, and from this HATR population, individual cells were cloned and also assayed for HLA expression. Using this protocol we screened 23 independently derived human HLA genomic clones which we obtained from Drs. A. Biro and S. Weissman of Yale University (3,5). These clones were isolated from a Charon 4A library, which was made from JY (A2, B7 homozygous) cellular DNA. Results obtained a t 60 h following transfection revealed that populations of cells transfected with

the genomic clones JY 158 and JYB3.2 showed specific staining of between 15%and 20% of their cells, respectively (3). Cells that were transfected with the pseudogene LN 1 were negative for HLA expression as were 1 cells transfected with lambda DNA. The twenty other genomic clones analyzed at the 60-h time point were also ne ative for HLA expression (3). HAT' mass populations derived from transfections of each genomic clone with an HSV-tk plasmid were also analyzed for HLA expression. Results obtained from genomic clones JY 158, JYB3.2, the pseudogene LN 11, and lambda DNA are presented in Figure 4. Thirtythree percent of the J 158 and 38% of the JYB3.2 Y cotransfected HATR mass population express the HLAA,B,C antigens. This contrasts with the total absence of expression seen with lambda DNA and LN 1 cotrans1 fected HATR mass populations (Fig. 4b,d). The twenty other HLA genomic clones which were negative for HLA surface expression a t 60 h after transfection also failed

i: k.
,.+W6/32
B . 2
2-0

Re1at ive
FIG 4. Indirect immunofluorescence and FACS analysis of HLA expression on HAT' mass populations derived from a cotransfection of L t k - cells with cloned h u m a n genomic DNA and HSV-tk. Each panel presents a population of transfected cells stained with HLA specific

FIuorescence
monoclonal antibody W6132, S1 specific monoclonal antibody W6134, and control myeloma supernatants (C). Transfection with genomic gene JY 158 (a),LN 11 pseudogene genomic HLA (b), genomic gene J Y B3.2 (c), A-DNA (d).

104

KAMARCK ET AL

60 hours

zo

10

(10

Sort 1

to produce HLA on HATR populations. The presence of this large number of defective genes suggests that they may have a role in the genetic polymorphism seen in this region. HLA-positive cells from both cotransfer experiments were then sorted for antigen expression to produce mouse cell lines expressing a single HLA gene. Using allospecific antibodies, it was possible to identify the product of the JYB3.2 genomic clone a s HLA-A2, and the product of genomic clone JY 158 as HLA-B7 (3). These mouse cell lines express a single human polypeptide and have been particularly useful in the generation of serum and monoclonal antibodies against these gene products (14). In addition to the study of isolated HLA genes in a mouse cell background, it is also useful to study the expression of each human gene in the context of other human antigens which might effect its expression. To develop a methodology for sequentially placing human genes into a recipient, we transferred cloned HLA-B7 genes into Ltk- cells without cotransfer with the HSVtk DNA. This DNA was transiently expressed 60 h r after gene transfer, and approximately 23% of the cells produced detectable HLA (Fig. 5). These antigen-positive cells were sorted using the FACS (Fig. 5 ) . Among these cells which briefly express HLA are a very small number that will eventually integrate the donor DNA and stabilize the gene. This initial enrichment of approximately ten-fold reduces the difficulty of separating the permanent transfectants. To isolate these cells, we applied selection pressure by sorting for antigen positive cells a t ten day intervals, followed by expansion of the positive, sorted population in tissue culture (Fig. 5). In this way we continued to recover those transfectants that retained HLA DNA, but may have not yet stably integrated the gene. After the fourth sort for HLA expression, the transfectants were cloned and characterized (Fig. 5). Most of the cloned lines displayed stable HLA expression after two months of tissue culture. These cell lines express HLA-B7, but remain thymidine kinase negative, so that we can easily introduce additional human genes into these cells using the HSV-tk cotransfer methodology discussed earlier. In this way it should be possible to rebuild the human cell surface one polypeptide a t a time to monitor the effects of each gene on each other and on the biology of the transfected cell.
FIG.5. The isolation of a J Y 158/HLA-B7 transfectant using selection by cell sorting alone. In each panel transfectants have been stained by indirect imrnunofluorescence with monoclonal antibody W6132 compared to control staining with myeloma supernatant (C). The first panel (top) presents the expression of HLA-A,B,C antigens 60 h after transfection. Antigen-positive cells were sorted sterilely and expanded in tissue culture for 10 days before each subsequent reanalysis and sorting. The second, third, and fourth panels present HLA expression on the selected line following one, two, and four sorts, respectively.

SOMATIC CELL GENETICS AND FLOW CYTOMETRY

105

SURFACE ANTIGEN GENE REGULATION

We also have been interested in studying the regulation of genes which control antigen expression. In a n effort to identify cells with unusual HLA expression patterns, we cloned the HATR mass population of cells produced from the JY 158/HLA-B7 gene transfer. We then used flow cytometry to monitor and compare the level of HLA expression on clones derived from the HATRpopulation. Sixty HATRclones were isolated from cotransfection experiments performed with the genomic Y clone J 158/HLA-B7.The FACS analysis of these clones demonstrated that significant variation could be observed in their level of HLA surface expression (3). Southern blot analysis of the DNA from each of these cloned lines allowed us to conclude that this level of HLA expression correlated very well with the number of intact HLA gene copies (3). This indicated that, a s was the case in somatic cell hybrids, the HLA gene sequences were constitutively expressed. As such, this system was not useful for the study of gene regulation of the transferred gene sequences. It has been observed that histocompatibility antigens of both mouse and man are increased over normal levels when the cells are treated with interferon (8). To examine the possibility that the transferred HLA gene sequences might be under similar regulation, we treated Ltk- parental cells, a transfectant expressing HLA-A2, and a transfectant expressing HLA-B7 with mouse /?interferon. In all three lines, the mouse histocompatibility antigens H-2Kk and H-2Dk were induced, although H-2Kk was consistently induced a t a higher level (Table 3) (36). The two human alloantigens were also shown to be induced on the mouse cell surface in response to mouse /?-interferon. As was the case with the endogeneous histocompatibility antigens, these antigens were disproportionately induced: HLA-A2 increased 2.7-fold over 36 h in response to mouse interferon, while HLAB7 increased only 2.2-fold (Table 3). We have also observed disproportionate increases in these antigens on human cells in response to human interferon (36).Thus, flanking gene sequences that determine not only the constitutive expression of the human HLA antigens, but also their differential response to interferon are present within the human DNA introduced into the mouse host. By altering these genes before transfection, it should be possible to determine exactly which sequences are crucial in gene regulation.

Table 3 Induction of Histocompatibility Antigens With Interferon Treatmenta Surface Antinen H-2Kk H-2Dk HLA-B7 HLA-A2 Ltk2.7 2.2
-

JY 158B7
2.7 2.2 2.7
-

JYB 3.21A2
2.7 2.2
-

2.2

aIndirect immunofluorescence assays with monoclonal antibodies 11-4.1 (H-2Kk), 15-5-5s (H-2Dk), or W6132 (HLAA,B,C) were used to specifically quantitate the induction of mouse and human histocompatibility antigens on Ltk- and transfected cell lines (3,12) following interferon treatment. The cell lines were incubated with mouse 0-interferon (1,000 units/ml) or control medium for 36 h, and the difference in antigen expression was quantitated using the fluorescence signal preamplifier (12).

HUMAN WHOLE CELL DNA

HSV-tk GENE

DNA-MEDIATED GENE TRANSFER

MOUSE Ltk-CELLS

I
I
b
0
0 0 0 0

HAT SELECTION

I - m l
POOL OF HATR COLONIES

C E L L SORTING FOR HUMAN SURFACE ANTIGEN

0

:I

M O U S E CELL E X P R E S S I N G H U M A N ANTIGEN
FIG. Isolation of mouse transfectants expressing human cell sur6. face antigens following DMGT with whole cell DNA. Details of this process are described in the text.

SURFACE ANTIGEN GENE CLONING

To complete a genetic analysis of the human cell surface, it is necessary to develop a general method for cloning surface antigen genes. As a first step in such a cloning strategy, a method has been developed for isolating small fragments of the human genome in a mouse cell background (Fig. 6) (15,19,20). Whole cell human DNA is cotransferred together with HSV-tk into mouse Ltk- cells, and HATR mass populations are recovered as already described. Because the size of donor DNA that is taken up and stabilized by integration in a host

chromosome is between 1,000 and 10,000 kilobases (30), and the human genome contains 3 x lo6 kb, the isolation of approximately 3,000 independently derived cellular clones should comprise a complete library of the human genome present in a mouse cell genetic background. To recover transfectants which contain the gene

106
'000~

KAMARCK ET AL

9ooy
600;

Sort 2

700;

5001
4001

a
E
2
3

L Q)

20

40

60

80

100

120

140

160

160

200

220

240

260

Sort 4

20

40

so

80

100

120

140

180

160

200

220

240

280

Relative Fluorescence
FIG. 7. Indirect immunofluorescence and FACS analysis of sorted mouse transfectants derived from cotransfection of L t k - cells with HSV-tk and human whole cell DNA. The panels present the second
and fourth sort populations stained with monoclonal antibody W6132 compared to control staining with myeloma supernatant (C).

coding for any specific antigen, a representative population of the entire HATR library is stained by indirect immunofluorescence and the antigen-positive cells isolated by cell sorting (Fig. 6). Because the cells of interest are represented in approximately 0.03% of the population, several enrichment sorts are performed before antigen-positive transfectants are cloned by single cell cloning. Figure 7 demonstrates the recovery of a transfectant population containing the gene(s) for a n HLAA,B,C determinant. After two enrichment sorts a n antigen-positive position can be detected. This transfectant

population is then sorted to homogeneity and cloned on the fourth sort (Fig. 7). The transfectants produced in this way contain a fragment of the human genome surrounding the HLA genes and will be useful for genetic dissection of this region a t a n intermediate level of resolution (31).In addition, these cell lines represent valuable starting material for cloning of the surface antigen genes (15J9). The human genome can be further fragmented by using the transfectant cell DNA as starting material for a secondary transformation experiment. The DNA from a secondary transformant can then be cloned

SOMATIC CELL GENETICS AND FLOW CYTOMETRY

107

Table 4 Flow Cytometry and Sorting in Somatic Cell Genetics" Flow Cytometry Methodology Sorting somatic cell hybrids for expression of chromosome-specific surface antigens (Fig. 2, Table 1) Sorting somatic cell hybrids for the expression of genetically uncharacterized human surface antigens (Table 2) Analysis and sorting of mouse cells which have been cotransfected with cloned human surface antigen genes and HSV-tk (Fig. 4) Isolation of mouse cells expressing human surface antigens which have been transfected with cloned surface antigen genes alone (Fig. 5) Quantitation of human surface antigen expression on transfected mouse cells (Table 3 ) Isolation of mouse cells expressing human surface antigens which have been cotransfected with whole cell human DNA and HSV-tk (Fig. 6. 7) Somatic Cell Genetic Application Manipulation of human chromosome segregation in hybrids. Generation of genetically defined lines for chromosome mapping. Rapid and efficient chromosomal mapping of surface antigen genes Bioassay of gene function in mouse cells. Production of mouse lines containing a single human gene for biological and immunological studies Starting material for placing multiple human genes in mouse cells. Studies of gene and gene product interaction Studies of gene structure and regulation

Starting material for cloning human surface antigen genes

aThe interaction of flow cytometry with somatic cell genetics in the study of human surface antigen genes. The methods outlined are illustrated i n the text.

in a gene library using a lambda or cosmid vector, and ACKNOWLEDGMENTS human repetitive sequences are used to identify human The authors thank John Hart, Suzy Pafka, Gloria Schoolgenomic material. Finally, the antigen coding gene can field, and Marie Siniscalchi for their expert technical be isolated by testing the cloned DNA in a final set of assistance. transformation experiments. Except for the transfectant selection procedure, this cloning strategy is identical to LITERATURE CITED that used for isolating the onc gene sequences (10,35). 1. Anderson WF, Killos L, Sanders-Haigh L, Kretschmer PJ, DiacuWe are interested in applying this technique to cloning makos EG: Replication and expression of thymidine kinase and genes that code for human cell surface receptors (20). human globin genes microinjected into mouse fibroblasts. Proc

CONCLUSIONS
We have illustrated ways in which flow cytometry and sorting can be used to monitor and manipulate human genes which have been introduced into mouse cells by somatic cell genetic methods. A summary of these methods and their applications is presented in Table 4.It is clear that flow cytometry and sorting facilitates studies on the human cell surface ranging from the initial chromosomal mapping of a surface antigen gene to its isolation and cloning. The manipulation of the human partial genome present in somatic cell hybrids also provides valuable material for mapping other human genes. Finally, by introducing cloned surface genes sequentially into mouse cells, it will be possible not only to study the biological regulation of the gene product, but also to examine the effects of multiple surface antigen genes on ) each other (Table 4 .

Natl Acad Sci USA 77:5399, 1980. 2. Arce-Gomez B, Jones EA, Barnstable CJ, Solomon E, Bodmer WF: The genetic control of HLA-A and B antigens in somatic cell hybrids: Requirement for &microglobulin. Tissue Antigens 11:96, 1978. 3. Barbosa JA, Kamarck ME, Biro PA, Weissman SM, Ruddle FH: Identification of human genomic clones coding the major histocompatibility antigens HLA-A2 and HLA-B7 by DNA-mediated gene transfer. Proc Natl Acad Sci USA 79:6327, 1982. 4. Barbosa JA, Kamarck ME, Ruddle FH: unpublished data. 5. Biro PA, Pereira D, Sood AK, DeMartinville B, Francke U, Weissman SM: The structure of the human major histocompatibility locus. In Immunoglobulin Idiotypes, ICN-UCLA Symposium on Molecular and Cell Biology, Janeway C, Setcare CA, Wigzell H (eds) Academic Press, New York, Vol 20, p 315, 1981. 6 . Cresswell P, Springer TA, Strominger J ,Turner MJ, Grey HM, L Kubo RT: Immunological identify of the small subunit of HL-A antigens and &microglobulin and its turnover on the cell mem~ brane. Proc Natl Acad Sci USA 71:2123, 1974. 7. D'Eustachio P, Pravtcheva D, Marcu K, Ruddle FH: Chromosomal localization of the structural gene cluster coding murine immunoglobulin heavy chains. J Exp Med 151:1545, 1980.

108

KAMARCK ET AL 24. Messer Peters PG, Kamarck ME, Hemler ME, Strominger JL, Ruddle FH: Genetic and biochemical characterization of a human surface determinant on somatic cell hybrids: The 4F2 antigen, Somatic Cell Genet 8(6):825, 1982. 25. Milman G, Herzberg M: Efficient DNA transfection and rapid assay for thymidine kinase activity and viral antigenic determinants. Somatic Cell Genet 7:161, 1981. 26. Nabholz M, Miggiano V, Bodmer WF: Genetic analysis with human-mouse somatic cell hybrids. Nature 223:358, 1969. 27. Nichols EA, Ruddle FH: A review of enzyme polymorphism, linkage and electrophoretic conditions for mouse and somatic cell hybrids in starch gels. J Histochem Cytochem 21:1066, 1973. 28. Owerbach D, Rutter WJ, Cooke NE, Martial JA, Shows TB: The prolactin gene is located on chromosome 6 in humans. Science 2122315, 1981. 29. Parham P, Barnstable CJ, Bodmer WF: Use of a monoclonal antibody (W6132) in structural studies of HLA-A,B,C antigens. J Immunol 125:342, 1979. 30. Perucho M, Hanahan D, Wigler M: Genetic and physical linkage of exogeneous sequences in transformed cells. Cell 22:308, 1980. 31. Ruddle FH: A new era in mammalian gene mapping: somatic cell genetics and recombinant DNA methodologies. Nature 294:115, 1982. 32. Ruddle FH, Creagan RP: Parasexual approaches to the genetics of man. Ann Rev Genet 9:407, 1975. 33. Ruddle FH, Kucherlapati RS: Hybrid cells and human genes. Sci Am 231:36, 1974. 34. Scangos G, Ruddle FH: Mechanisms and applications of DNAmediated gene transfer in mammalian cells-A review. Gene 14:1, 1981. 35. Shih C, Weinberg RA: Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell 29:161, 1982. 36. Shulman LM, Kamarck ME, Barbusd JA, Ruddle FH: Interferon induced surface expression of HLA in LTK-cells transfected with HLA genomic clones (Abstract). Tn: The Third Annual Interna tional Congress for Interferon Research, 1982, p 86. 37. Someren HV, Westerveld A, Hagemeijer A, Mees JR,Meera Khan P, Zaalberg OB: Human antigen and enzyme markers in manChinese hamster somatic cell hybrids: Evidence for synteny between the HL-A, PGM3, ME1 and IPO-B loci. Proc Natl Acad Sci USA 71:962, 1974. 38. Weiss MC, Green H: Human-mouse hybrid lines containing partial complements of human chromosomes and functioning human genes. Proc Natl Acad Sci USA 76:1104, 1967. 39. Wigler M, Silverstein S, Lee L-S, Pellicer A, Cheng T, Axel R Transfer of purified Herpes virus thymidine kinase gene to cultured mouse cells. Cell 11:223, 1977.

8. Fellous M, Kamoun M, Gresser I, Bono R: Enhanced expression of HLA antigens and Pz-microglobulin on interferon-treated human lymphoid cells. Eur J Immunol 9:446, 1979. 9. Fournier REK, Ruddle FIT: Stable association of the human transgenome and host murine chromosomes demonstrated using trispecific microcell hybrids. Proc Natl Acad Sci USA 74:319, 1977. 10. Goldfarb M, Shimizu K, Perucho M, Wigler M: Isolation and preliminary characterization of a human transforming gene from T24 bladder carcinoma cells. Nature 296:404, 1982. 11. Hemler ME, Strominger JL:Characterization of the antigen recognized by the monoclonal antibody 4F2: Different molecular forms on human T and B lymphoblastoid cell lines. J Immunol 129:623, 1982. 12. Kamarck ME, Barbosa JA, Ruddle FH: Somatic cell genetic analysis of HLA-A,B,C and human Pz-microglobulin expression. Somatic Cell Genet 8(3):385,1982. 13. Kamarck ME, Ruddle FH: Somatic cell genetic analysis of human cell surface antigens. In: Membranes and Genetic Disease, Alan R. Liss, Inc., New York p 219, 1982. 14. Kamarck ME, Barbosa JA, Ruddle FH: manuscript in preparation. 15. Kavathas P, Herzenberg LA: Stable transformation of mouse L cells for human membrane T-cell differentiation antigens, HLA and &microglobulin: Selection by fluorescence-activated cell sorting. Proc Natl Acad Sci USA 80524, 1983. 16. Klein G, Terasaki P, Billing R, Honig R, Jondal M, Rosen A, Zeuthen P, Clements G: Somatic cell hybrids between lymphoma lines. 111. Surface markers. Int J Cancer 19:66, 1977. 17. Klobutcher LA, Ruddle FH: Chromosome mediated gene transfer. Annu Rev Biochem 50533, 1981. 18. Kozak CA, Lawrence JB,Ruddle FH: A sequential staining technique for the chromosomal analysis of interspecific mousehamster and mousehuman somatic cell hybrids. Exp Cell Res 105:109, 1977. 19. Kuhn LC, Barbosa JA, Kamarck ME, Ruddle FH: Isolation of mouse L-cells expressing human transferrin rerceptor, 4F2-antigen and HLA after gene transfer with total human DNA. J Cell Biochem, Supplement 7A:130,1983. 20. Kuhn, LC, Ruddle FH: Gene transfer with total human DNA as a step in the molecular cloning of human transferrin receptor and 4F2 antigen. Fed Proc 429133,1983. 21. Littlefield JW: Selection of hybrids from matings of fibroblasts in uitro and their presumed recombinants. Science 145:709, 1964. 22. Loken MR, Parks DR, Herzenberg LA: Two-color immunofluorescence using a fluorescence-activatedcell sorter. J Histochem Cytochem 25:899, 1977. 23. McKusick VA, Ruddle FH: The status of the gene map of human chromosomes. Science 196:390,1977.