Proc. Nad. Acad. Sci. USAVol. 82, pp. 6148-6152, September 1985Cell Biology

Efficient insertion of genes into the mouse germ line viaretroviral vectors

(preimplantation embryo infections/germ line integration/long terminal repeat enhancers/transgenic mice)

HERMAN VAN DER PUTTEN*, FLORENCE M. BOTTERI*, A. DUSTY MILLERt, MICHAEL G. ROSENFELDt,HUNG FAN§, RONALD M. EVANS*, AND INDER M. VERMA*

*Molecular Biology and Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, CA 92138; *Department of Molecular Biology and Biochemistry,University of California, Irvine, CA 92717; tEukaryotic Regulatory Biology Program, School of Medicine, University of California, San Diego, CA 92093; andtHutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104

Communicated by E. Peter Geiduschek, May 13, 1985

ABSTRACT We present a general strategy for the efficientinsertion of recombinant retroviral vector DNA into the mousegerm line via infection of preimplantation mouse embryos.Transgenic mice were generated that harbor a replication-competent recombinant retrovirus (AMo+Py M-MuLV) thatlacks the Moloney murine leukemia virus (M-MuLV)-typeenhancer sequence in the long terminal repeat (LTR). Instead,the LTR contains an enhancer element that permits polyomavirus F101 to grow in undifferentiated F9 embryonal carcino-ma cells. Expression studies in different tissues of animalstransgenic for AMo+Py M-MuLV indicate possibilities totarget and modulate expression of retroviral recombinants inmice via their LTR enhancer sequences. In addition, 16transgenic mice were generated that harbor proviral DNA of adefective recombinant retrovirus carrying a mutant dihydro-folate reductase gene.

Gene transfer into the mouse germ line is a crucial test forcell-specific gene expression. Transgenic animals generatedvia pronuclear DNA injection have revealed abundant infor-mation about sequences that direct tissue-specific expression(for review see ref. 1). However, such animals harbor mostlyaberrant tandem organizations of the injected gene. Thesemight affect neighboring chromatin structures, disrupt tran-scriptional units (2), and make quantitative studies on geneexpression difficult to interpret. Therefore, the developmentof techniques to insert a single copy of a gene is of greatimportance (3). Retroviral infection mediates insertion of asingle copy of a nonpermuted provirus per chromosomal site(4-14). Retroviral expression can be modulated via theenhancer sequences in the viral long terminal repeat (LTR),which determines tissue tropism and type of disease causedby a particular murine leukemia virus (MuLV) (15-19).

Until recently, only the Moloney strain of MuLV (M-MuLV), a highly leukemogenic replication-competent retro-virus, was successfully inserted into the mouse germ line(20-24). We have now developed a general strategy for theinsertion of both replication-competent and defective retro-viruses into preimplantation mouse embryos, giving rise totransgenic animals that transmit the proviral DNA to off-spring.

MATERIALS AND METHODSEmbryo Manipulations. Eight-cell stage embryos were

flushed from oviduct-uterus junctions with modified Whit-ten's medium (ref. 25; Fig. 1). The zona pellucida wasremoved by using Pronase (22 units/ml) or acidified Tyrode'ssolution (27). Embryos were cultured (16 hr) on top ofmonolayers of virus-producing cells in the presence of

Polybrene (28) at 4 gg/ml at 370C in Dulbecco's modifiedEagle's medium plus 10% fetal calf serum in 5% CO2 in air.After infection, morulae were cultured (2-4 hr) in modifiedWhitten's medium under a layer of equilibrated paraffin oil(29) before transfer into uterine horns ofpseudopregnant ICRfemales.

Cell Lines and Viruses. qi-2 cells (4) producing ecotropicLDL1 virus (6) were made as follows: Eighteen-hour super-natant harvests of cells producing amphotropic LDL1 virus(6) were used to infect i-s2 cells. On the next day medium waschanged and embryos were infected (16 hr). The titer of viruscontaining the gene for dihydrofolate reductase (DHFR),determined on NIH 3T3 thymidine kinase-deficient cells (6),was 5 x 105 colony-forming units (cfu)/ml. Several infectionswere carried out in the presence of 10 mM Ca2' to optimizeviral gp70-receptor binding (30). Viruses are described inFig. 2 and its legend.

Nucleic Acids. Extraction of tail DNA, nick-translations,hybridizations, and final stringent washes were as describedpreviously (31, 32, 36). Total RNA was isolated by extractionwith guanidinium thiocyanate (37).

RESULTSInsertion of Replication-Competent Retroviruses. Embryos

were exposed (Fig. 1) to cells producing a mixture of 107plaque-forming units (pfu) of M-MuLV and 106 cfu of therecombinant LPHGL retrovirus (Fig. 2) per ml. To monitorthe infection, 50 progeny were analyzed for the presence ofproviruses in tail DNA and infectious M-MuLV in the blood.The infected eight-cell stage mouse embryos could be mo-saic, since all cells of the morula may not be infected.Furthermore, if some cells are multiply infected, they wouldcarry more than one provirus but each in a different chro-mosomal site. Integration of proviral DNA can be monitoredby digesting tail DNA with the restriction endonuclease SstI and hybridization to the U3 LTR probe (Fig. 2). Provirusesin different chromosomal positions should each contribute inthe detection of the respective internal Sst I fragments.M-MuLV proviral DNA yields a specific 5.6-kb fragment,whereas the LPHGL provirus yields a 5.3-kb fragment (Fig.2). Furthermore, if many cells in the tail carry a provirus,another Sst I fragment should be detected, which representsa 5'-LTR-host DNA fusion fragment. Sixteen animals out of50 analyzed carried M-MuLV proviral DNA, of which 13showed fragments representing a 5'-LTR-host DNA junc-tion. Eleven out of 16 animals were viremic at 6 weeks of ageas detected by XC plaque assay (Table 1). None of the mice

Abbreviations: LTR, long terminal repeat; DHFR, dihydrofolatereductase; MuLV, murine leukemia virus; M-MuLV, Moloney strainof MuLV; PyF101, polyoma virus F101; rGH, rat growth hormone;HPRT, hypoxanthine phosphoribosyltransferase; kb, kilobase(s);cfu, colony-forming units; pfu, plaque-forming units.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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ovary oviduct ampulla uterus zonapellucida

PMS

-2

Implantation

FIG. 1. Infection of preimplantation mouse embryos. Schematic representation of preimplantation mouse embryo stages at various timespost coitum and their location in the reproductive tract. Female mice were induced to superovulate with injections of 5 international units ofpregnant mare serum (PMS) and 2.5 international units of human chorionic gonadotropin (HCG) given 42-52 hr apart (26). Embryos wererecovered in the morning ofday 3 post coitum, and after zona removal they were infected. C, compaction (60-72 hr post coitum); I, implantationof blastocysts into uterine wall; 1CM, inner cell mass.

analyzed were positive for DNA from the recombinant virusLPHGL.

Since the germ line probably originates from ectoderm, thetail DNA may not yield a representative picture ofprovirusesin the germ line of mosaic animals. Therefore, 11 out of 16mice were bred and tail DNAs of offspring were tested forproviral DNA. Seven out of 11 mice yielded offspring thatcarried proviruses. The transmission frequencies varied(5-80%), reflecting mosaicism in the germ line ofthe parentalmice (not shown). All offspring had 5'-LTR-host DNAjunctions in their tail DNA also found in the tail DNA of theirfounder (Fig. 3). Offspring T235 and T237 of animal T3,however, have 5'-LTR-host DNA junctions only weaklyrepresented in the parent (T3) tail DNA. The pattern ofinheritance ofthe three proviral copies detected in DNA fromanimal T48 suggest that a single cell of the embryo wasmultiply infected. The segregation of proviruses in the F1generation is expected from the chromosomal segregationduring meiotic division which leads to germ cell formation. Inconclusion, genetically transmittable proviruses in transgen-

ic mosaic animals can reliably be identified in the tail DNAof the parental mosaic mouse.

Insertion of a Replication-Competent Recombinant Provi-rus. AMo+Py M-MuLV is a replication-competent, ecotro-pic recombinant virus that lacks the M-MuLV-type enhancerin its LTR (Fig. 2). Instead it contains an enhancer elementof the polyoma virus mutant PyF101 (38). This elementconfers replication competence to PyF101 in undifferentiatedF9 embryonal carcinoma (EC) cells and allows gene expres-sion in F9 EC cells of the AMo+Py M-MuLV LTR (35). Incontrast, M-MuLV expression does not occur in EC cells andpreimplantation embryos (39, 40-43). Furthermore,AMo+Py M-MuLV, in contrast to M-MuLV, is nonleuke-mogenic (34).Three positive male mice (T96, T99, and T126) yielded F1

progeny carrying AMo+Py M-MuLV DNA in their germ line(Fig. 4 A and B). None of the animals had detectable levelsof virus in the blood at 10 weeks of age (Table 1), even thoughexpression of the two characteristic viral RNAs of 35S and21S was detected in spleen. No viral RNA was detected in

Table 1. Embryo infections

Cell line Probe and no. positive No. positive(ref.) Viruses and vectors Embryos Manipulations Born/transferred in tail DNA in XC test

A (14) M-MuLV, 107 pfu/ml El Pronase/-Ca2+ 56/292 (19o) U3 LTR 16/50 (32%) 11/50LPHGL, 106 cfu/ml rGH or HPRT 0/50 (0%o)

B (34) AMo+Py M-MuLV, E2 Acidic Tyrode's/+Ca2+ 62/228 (-30%) Pvu II-4 4/62 (6%) 0/621i5 pfu/ml

MS-4 4/62 (6%)C (6) LDL1, 6 x 107 cfu/ml E2 Acidic Tyrode's/+Ca2+ 76/193 (-40o) U3 LTR 32/74 (43%)

M-MuLV, 1 x 107 pfu/ml DHFR 15/74 (20%)D (see LDL1, 5 x 10' cfu/ml El Acidic Tyrode's/+Ca2+ 58/197 (-30%) U3 LTR 1/58 (2%)

text) DHFR 1/58 (2%)All viruses are ecotropic. Pronase or acidic Tyrode's solution was used to remove the zona pellucida. Some infections were performed in the

presence of 10 mM Ca2+ (30). El indicates that embryos were C57BL/6J x SJL/J; E2 were (C57BL/6J x SJL/J) x SJL/J. Tail DNAs wereanalyzed as described in the text. Four of 62 mice were positive for the AMo+Py M-MuLV provirus. Only three were further analyzed. XCtests were performed with 20 ,ul of blood (for AMo+Py M-MuLV, see ref. 34). Virus at concentrations <50 pfu/ml would have remainedundetected.

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M-MuLV :1 2 3 4 5 6

2.5kb .0 5.6kL.-

-445 -335 -175 1 70 145

AMo+PYM-MuLV PyF101 U3 R U5LTR

F

RNA3' g45'

LPHGL

5.3kb / - -

Hpa I

LDL 1

L-0.8- 2.OkbJ

1= Sst I

FIG. 2. Viruses, vectors, and probes. MS-2, MS-3, and MS-4 (31) as well as the U3 LTR (32) probe are M-MuLV-specific probes. The0.8-kilobase (kb) HindIII/Nco I fragment of pFR400 (33) was used as a DHFR-specific probe. The 0.8-kb Xho 1-HindI11 fragment of the ratgrowth hormone (rGH) minigene was used as a growth hormone probe (14). The combined 0.6-kb Pst 1/HindI1 fragments of p4aA8 were usedas a probe for the hypoxanthine phosphoribosyltransferase (HPRT) gene (13). AMo+Py M-MuLV proviruses were identified by using theM-MuLV-specific probes MS-4, MS-3, and U3 LTR. The Pvu 11-4 fragment of polyoma virus F101 (PyF101) (34, 35) was used to identify theenhancer region. The LPHGL and LDL1 viruses have been described before (6, 14) as well as AMo+Py M-MuLV (34). The position of thePyF101 enhancer element within the AMo+Py M-MuLV LTR is indicated.

liver, while brain contained significant levels of 35S viralRNA but no 21S viral RNA (= spliced env mRNA) (Fig. 4C).Brain, however, did contain a 30S RNA that, like the 35Sviral RNA, hybridized both to the PyF101-Pvu II enhancerand to the U3 LTR probes (Fig. 4C). The 30S RNA probablyrepresents an aberrantly spliced viral mRNA. Furthermore,since the other two positive mice (T99 and T126) also showedRNA expression in the brain, it is unlikely to be the result ofsome specific chromosomal integration site (data not shown).

F10C N LO cc M

co r- r- N- N- N- N-

kb H H H H H H H

None of the F2 mice carrying AMo+Py M-MuLV showednew proviral insertions. Therefore, the PyF101-Pvu II-4fragment does not seem to promote virus production duringearly embryogenesis resulting in reinfection of cells in theearly embryo.

Insertion of a Defective Recombinant Provirus. Fig. 2 showsthe proviral DNA structure of the recombinant retrovirusLDL1. To favor insertion of the defective recombinant virusa cell line (Table 1, cell line C) was used that produces a 6-fold

Ft

C NO co cnHY H~ H H

F1 F1lcc 0 MI 1Fc -1

LO Cc Cc O 0CU CxJ Cj Ctj N LOcc cc cc ccF cc c

6.7-

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_ _

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4

FIG. 3. M-MuLV proviral DNA in mosaic animals. Each lane contains 10 ,g of tail DNA, digested with Sst I, which, after Southern blotting,was hybridized with the U3 LTR probe (Fig. 2). Control (C) DNA was from a tail of an unmanipulated animal. Proviral DNA copies in tail DNAfrom four different parental mosaic animals (T48, T3, T15, and T5) and some of their offspring (F1) are shown. The internal 5.6-kb Sst I fragmentofM-MuLV is indicated by an arrow. All other fragments represent 5'-LTR-host DNA junctions. The new 5'-LTR-host DNAjunctions in T235and T237 were visible in parental T3 DNA only after very long exposure (not shown). T15 DNA displays a proviral copy that was not transmittedto any of six F1 animals tested.

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_k )

A cL 2

m al

kb

5.6 - A

4:00 w

= -.4.1 u)

(D (D0o CDCD CO0

-_.

1-1k

(0 (DCM CUj

(.C) F- C

6:.0.-

4*

-*0

*:k

a)&- I'l CD LO ragA = a c ,r- r- r- Co

0 0Hkb t) H H23.7-

B HD

VW9.4-

6.7 -

_w 4.3u-U w

_R 4*2.3-

- bag.mw *.

*

B | F1 || F lcCO N CM (D

C) LI) LI) 0 (0 01kb

23.7 -

4.3 -

2.3 -

F1

ccCoM A-d FIG. 5. DHFR-vector insertion into mice. (A) Ten micrograms of- [,- Nlco cO co co DNA of the cell line used for mixed infection with the recombinantH H H H DHFR virus and M-MuLV (Table 1, cell line C) and various mouse

tail DNAs are shown positive for the recombinant DHFR provirus.The cell line carries multiple DHFR proviruses and was generated via

1W- *X; ~ selection in methotrexate (6). After digestion with Sst I, Southernblots were probed with a DHFR probe (Fig. 2). The two character-

* 111*19 istic internal Sst I DNA fragments of the DHFR vector (LDL1) areindicated by arrows. The three upper fragments present in each lanerepresent the endogenous mouse DHFR gene. The asterisk indicatesthe position of a DHFR-proviral DNA fragment derived from arearranged provirus in cell line C. One of the 15 mice positive for theDHFR-recombinant provirus harbored this rearranged provirus (notshown). (B) Mouse T554 was generated via infection with ecotropicLDL1 virus without helper M-MuLV (Table 1, cell line D).

C

Cc

m

Pvu 11-4

cna

CL)

0J + 0.) + 0D +

28S - t

18S-

U3 LTR

a)1 .ia-5-

/) + O

- J* 35S

- * -21S

FIG. 4. Mice carrying AMo+Py M-MuLV proviruses. (A) Screen-ing of tail DNA (Sst I), using MS-4 and PyF101-Pvu H-4 probes (Fig.2). Three positives (T99, T96, and T126) and control (C) DNAs areshown. The 5.6-kb band represents the internal Sst I DNA fragment(Fig. 3). (B) EcoRl analysis (A&Mo+Py M-MuLV has no EcoRI site) ofF1 tail DNAs (hybridized with PyF101-Pvu 11-4 probe). Control mouseDNA contains endogenous cross-hybridizing sequences. Proviruses areindicated by dots. Male T126 gave rise to two types of F1. One type(T378 and T372) carrying two, the other type (T374 and T372) eachcarrying one provi-al copy. (C) Analysis of 10 ,ug of total RNA from theindicated tissues of a positive F1 mouse of T99 and a control animal,electrophoresed through a formaldehyde gel, and after blotting probedwith PyF101-Pvu H-4 probe and the U3 LTR probe.

excess of LDL1 virus over M-MuLV helper. Of the 74 mice,32 were found positive for M-MuLV proviral DNA, whereas15 were found to harbor both a DHFR recombinant provirus(Table 1; Fig. 5A) and one or more M-MuLV proviruses (notshown). Unexpectedly, the helper seems to have an advan-tage and/or to interfere with recombinant virus infection in away that is disproportionate to their ratio.

Helper-Virus-Free Insertion of a Defective RecombinantProvirus. Recombinant retroviruses were prepared in celllines that permit their helper-free propagation (4, 6). Theecotropic helper-MuLV-free DHFR virus (LDL1) estab-lished its proviral DNA into preimplantation embryos (Table1, cell line D). Mouse T554 (Fig. 5B) was generated byinfection with a helper-free defective recombinant DHFRvirus, indicating that a defective recombinant retrovirus canbe inserted into the early mouse embryo in the absence ofhelper virus.

DISCUSSIONInfection of Embryos. Our findings demonstrate that

preimplantation embryos can be infected with recombinantretroviruses. Efficient and precise integration of proviralsequences is feasible. Screening for positive animals via tailDNA analysis is fast and reliable even though all animals areessentially mosaic. Mosaic animals do transmit proviralcopies efficiently to subsequent generations. Often, animalshaving several proviral DNA copies initially integrated in oneor more blastomeres of the early embryo give rise to mice inwhich these copies have segregated (mouse T48; see Fig. 3).

Expression of a Recombinant Vector. A recombinant ret-rovirus (A&Mo+Py M-MuLV) containing an altered LTRenhancer sequence can be inserted into the germ line andstably transmitted to offspring (Fig. 4A and B). The AMo+PyM-MuLV LTR can be expressed in EC cells and thus offers

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the possibility of expression in preimplantation mouse em-bryos. Second, it would show that altering enhancer elementsin the LTR, which confers tissue tropism, does not affectMuLV integration in preimplantation embryos. Finally,A&Mo+Py M-MuLV is not leukemogenic (34) and therefore isideal as a vector for gene transfer into animals. Transgenicanimals containing AMo+Py M-MuLV expressed specific35S and 21S viral RNA in spleen but not liver (Fig. 4C), asshown previously for mice containing genetically transmittedM-MuLV (22-24). Surprisingly, however, mice containingAMo+Py M-MuLV (Fig. 4C) showed expression of 35S and30S viral RNA in brain not observed in mice carrying agenetically transmitted M-MuLV (22-24). However, whenthe germ line copy is transcriptionally activated duringembryogenesis, tissues in the developing embryo are infectedand newly integrated proviruses do express viral RNA(22-24). Several findings argue against reinfection of tissuesin mice carrying aAMo+Py M-MuLV. First, no amplificationofthe internal 5.6-kb Sst I fragment was detected in any tissueDNA tested (not shown). Second, no infectious virus wasdetected in the serum (Table 1). Third, no 21S spliced envmRNA was detected in the brain (Fig. 4C), making thepresence of infectious virus within the brain unlikely. It thusappears that the presence of the PyF101 enhancer in the LTRimparts specificity for expression in brain tissue and thatrecombinant retroviruses introduced into preimplantationmouse embryos can be expressed. More importantly, thisenlarges the scope of recombinant retroviral vectors contain-ing a wide variety of tissue-specific enhancers for generatingtransgenic mice.

Transgenic Mice with a Mutant DHFR Gene. From ourstudies (Table 1) we conclude that the recombinant DHFRvirus can be efficiently inserted into preimplantation mouseembryos. Also, germ line transmission has been detected (notshown). The presence of replicating helper virus is notrequired, though it could play a promoting role. In conclu-sion, retrovirus-mediated gene insertion in preimplantationmouse embryos allows us now to study and modulate (e.g.,via the LTR) expression of single-copy genes in transgenicmice. Preliminary studies on DHFR proviral gene expressionfurther suggest that recombinant proviruses can be ex-pressed.

We thank Marguerite Vogt and Wiebe Kruijer for sharing theirexpertise concerning the virus and RNA work, respectively. Wethank Richard Mulligan for providing us the qi-2 cell line. We thankMarguerite Vogt, Wiebe Kruijer, and Bart Sefton for discussion andcritical reading of the manuscript, and we thank Marijke terHorst forsecretarial assistance. This work was supported in part by grantsfrom the National Institutes of Health. A.D.M. is a fellow of theLeukemia Society of America. R.M.E. acknowledges support fromthe Mathers Foundation.

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