Proc. Natd. Acad. Sci. USA Vol. 91, pp. 1138-1142, February 1994 Immunology Exogenous Mtv-7 superantigen transgene expression in major histocompatibility complex class II I-E- mice reconstituted with embryonic stem cell-derived hematopoietic stem cells (minor lymphocyte stmulator antigen 1) CYNTHIA A. CHAMBERS*, JOONsoo KANG*t, JUDY PAWLING*, BRIGITTE HUBERt, NOBUMICHI HOZUMI*t, AND ANDRAS NAGY§f *Division of Neurobiology and Molecular Immunology and §Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario MSG DX5, Canada; tDepartment of Pathology, Tufts University, Boston, MA 02111; and tDepartment of Medical and Molecular Genetics, University of Toronto, Toronto, Ontario, Canada Communicated by Robert P. Perry, September 17, 1993 (receivedfor review July 19, 1993) ABSTRACT Direct genetic manipulation of hematopoietic cells was performed in our initial report. Further, the ability cells is limited by the lack ofan established hematopoietic stem of genetically manipulated ES cells to generate these em- cell line. It has been demonstrated that embryonic stem (ES) bryos and the resultant phenotypic changes in animals re- cell + tetraplold embryos are completely ES cell-derived and constituted with ES cell-derived FL cells has not been that fetal liver (FL) cells from these embryos support he- demonstrated. In this report we show that a gene transferred matopolesis in lethally irradiated recipients. In this report, we to the ES cells can be expressed in hematopoietic cells ofthe d onstrate that FL cells from ES cell 4- tetraploid embryos animals reconstituted with the ES cell-derived FL cells. The support normal lymphopolesis and T-cell repertoire develop- transgene chosen for this study encodes a minor lymphocyte ment. Moreover, the introduction of the Mtv-7 superantigen stimulatory antigen (Mls) that is critically involved in in- transgene coding for minor lymphocyte stimulatory] antigen 1 trathymic selection of developing thymocytes (7, 8) and in into murine hematopoletic cells via reconstitution with ES cell conferring resistance to exogenous mouse mammary tumor *+ tetraplold FL cells demonstrates that this method can virus (MMTV) infections in mice (9). effectively confer stable genetic changes into the hematopoietic Mls activate a large proportion of T cells via specific V, tises without going togh the germ line. Long-term and chains of the T-cell receptor (TCR) and are classified as secondary reconstitution with ES cell + tetraploid FL cells superantigens (sag). Recently, it has been demonstrated that expressng the Mtv-7 superantigen transgene clonally deleted Mls are encoded by the open reading frames of the long minor lymphocyte stimulatory antigen 1-reactive T-cell recep- terminal repeat of MMTV (7, 8). Specifically, Mis-i is a tor Vp6+, -8.1+, and -9+ T cells, but not Vp7+ T cells, in H-2b 45-kDa type II transmembrane glycoprotein encoded by (I-E-) mice. This model system will be extremely important for endogenous MMTV provirus Mtv-7 sag gene (10-13). M4s-i analyzing structure-ftcon reationss of molecules in- expression in association with major histocompatibility com- volved in proliferation, differentiation, and selection of hema- plex (MHC) class II I-E molecules results in the intrathymic topoletic cells in Wvo and for examining hematopolesis-specific clonal deletion of Mls-1-reactive V,06+, -7+, -8.1+, and -9+ T effects of mutations that are lethal during embryogenesis. cells in vivo (14-18). The ability of Mis antigens to be presented by I-A remains controversial. Strong Mis stimu- Hematopoietic stem cells (HSCs) differentiate and give rise lators such as Mls-i can be presented by MHC I-A molecules to all myeloid and lymphoid cells throughout the entire life of in vitro but much less efficiently than by I-E molecules (8). the animal (1). Genetic alteration ofthe HSC thereby has the Studies with backcross inbred, recombinant animals (19) and potential to alter all of the cells in these lineages. Although bone marrow (BM) chimeras (20) indicate that there are retroviral vectors have been used to introduce genes into varying degrees ofpartial deletion in V.6+ and V,8.1+ T cells HSCs, there are a number oflimitations, including low levels when Mls-1 is presented by H-2b (I-E-) MHC molecules in of gene expression and poor infection efficiency (2). More- vivo. Mtv-7 sag transgene expression in H-2b mice would over, null mutations cannot be directly generated using directly address this issue and clarify the role of I-A in HSCs. Thus, the ability to genetically alter HSCs without tolerance generating transgenic wice is limited by the lack of an Mls-mediated induction. established HSC cell line. To this end, ES cell lines constitutively expressingMtv-7sag We have that stem cells transgene were used to generate ES cell + tetraploid embryo demonstrated embryonic (ES) aggregates. The resultant embryos were 100% ES cell-derived. aggregated with tetraploid embryos (ES cells + tetraploid embryo aggregates) develop into viable embryos in which the FL cells from embryonic day (E) 15.5 embryos were then used embryos are entirely ES cell-derived and most of the ex- to reconstitute lethally irradiated adult recipients. E15.5 FL traembryonic cell lineages are tetraploid-derived (3). Al- cells from unmanipulated ES cells supported normnal lym- though the offspring die perinatally, the organs of the viable phopoiesis, specifically the generation of the normal TCR embryos can be used as a source of completely ES cell- repertoire in vivo. Importantly, Mtv-7 sag gene expression in derived tissue. The fetal liver (FL) is the primary site of H-2b (I-E-) animals modified the TCR repertoire and demon- hematopoiesis during the second half of gestation (4, 5). Long-term reconstitution of hematopoiesis in lethally irradi- Abbreviations: BM, bone marrow; ES, embryonic stem; FL, fetal ated recipients by ES cell-derived FL cells has been dem- liver; HSC, hematopoietic stem cell; Mls, minor lymphocyte stim- the ulatory antigen(s); RAG-2, recombination-activating gene 2; sag, onstrated (6). However, no detailed analysis of lymphoid superantigen(s); TCR, T-cell receptor; SN, spleen; LN, lymph node; THY, thymus; E, embryonic day; MMTV, mouse mammary tumor The publication costs ofthis article were defrayed in part by page charge virus; MHC, major histocompatibility complex; GPI, glucose phos- payment. This article must therefore be hereby marked "advertisement" phate isomerase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom reprint requests should be addressed. 1138 Downloaded by guest on September 30, 2021 Immunology: Chambers et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1139 -J IL -J N LL .0 .0 Z I E z z E c U, I Z z z z Cl) Cl) (1ut) F-I- e >- CI I- -, -3 o o a: E z : Gn U .0 2 ( 0 0 w 0E m w 0 0 0 0 0) 0 QllJ CDU n o 0 IT""P 0o 0o cI: c: 'r NV C 23 - i.... 9.4- *:: sI.I 6.6- 4.4- d_ AN FIG. 1. Detection of the transgene in the hematopoietic cells of ES cell-derived FL-reconstituted mice. Genomic DNA was prepared from the ES cell lines, FL, and lymphoid organs from the reconstituted mice. Southern blot analysis was performed as described (24), and the blots were probed with the Bal I-BamHI fragment of the neomycin phosphotransferase cDNA. 129 (Ri-FL) were mice reconstituted with FL cells from tetraploid embryos generated with parental cell line R1. The sizes of the DNA bands (in kb) are indicated on the left. Morf 1, 4-1, 4-2, 8, and 10 are group A; Morf 16 is group B; and 129 and R1-FL reconstituted mice are group C. One integrant in R1i(orf 13 was never detected in the recipients, suggesting that there was a second clone, which did not contribute to hematopoiesis. strated that the Mls-specific V+ T cells have varying avidities overnight at 370C in M16 medium to reach the 4-cell stage. ES for Mls-i associated with I-A molecules. cells were gently trypsinized, and 5-10 loosely connected cells were placed between two tetraploid embryos and cul- METHODS AND MATERIALS tured overnight. The aggregates were transferred into 2.5 day pseudopregnant females with the day of transfer considered Generation of ES Cell Lines. R1 ES cells [129/Sv; H-2b E2.5. (I-E-), Mls-l-; ref. 21] were maintained and electroporated FL and BM Reconstitution. 129/SvJ mice were irradiated as described (22) with the pHf3A-ORF vector (13). The open (10 Gy) and injected i.v. with E15.5 FL (2-3 x 106 cells per reading frame region of the Mtv-7 sag gene was inserted into mouse) or BM (4 x 106 cells per mouse). Animals were the pHPAPr-l-neo expression vector (23), which contains the analyzed 3-9 months postreconstitution. human f-actin promoter/enhancer and neomycin phospho- Southern Blot Analysis. Genomic DNA was isolated from transferase cDNA driven by the simian virus 40 early pro- the indicated organs and ES cell lines and analyzed for the moter and has been shown to give high expression in vitro presence of the transgene as described (24, 25). After the (13). The cells were selected for neomycin phosphotransfer- DNA was transferred, the filters were hybridized with an ase expression with G418 (200 pug/ml, dry weight; GIBCO). [a-32P]dCTP (Amersham)-labeled neomycin phosphotrans- Colonies were picked and expanded, and expression of the ferase probe (Bal I/BamHI fragment of the PGKneo vector). Mtv-7 sag gene was tested by RNA dot blot analysis. Clones Some of the blots were stripped and rehybridized with a showing high expression, R1i3orf 13 and 4, were used for the (3-actin probe to normalize for the amount of DNA as in vivo studies.