MOLECULAR REPRODUCTION AND DEVELOPMENT 30194-200 (1991)
Liposome-Mediated DNA Uptake by Sperm Cells DANIEL BACHILLER,’ KARL SCHELLANDER: JANOS PELI: AND ULRICH R ~ J T H E R ~ ‘European Molecular Biology Laboratory, Heidelberg, Germany; ‘Znstitut fur Tierzucht und Genetik, Veterinarmedizinische Universitat Wien, Wien, Austria
To investigate the potential use of ABSTRACT sperm cells as vectors to transfer exogenous DNA via the fertilization of oocytes into the germ line of mice, we have used liposomes to transfect DNA into the sperm head. Although the DNA transfer into sperm mediated by liposomes was very efficient and no obvious reduction in the fertilization frequency of oocytes could be detected, we were unable to generate transgenic mice by this method. Key Words: DNA transfer,Transgenic animals, Cationic liposomes INTRODUCTION The generation of transgenic animals has opened avenues to the analysis of many questions in oncogenesis, developmental biology, gene regulation, and even animal farming. The most established method is the microinjection of DNA into the pronucleus of fertilized eggs (for review, see Palmiter and Brinster, 1986). Although in some laboratories this is a routine technique, the generation of transgenic mice is a procedure of certain complexity. Furthermore, for certain animals the microinjection is possible only after additional treatments that are time consuming and that might influence the efficiency; e.g., pig eggs must first be centrifuged to make the pronucleus visible. Therefore, the idea of using sperm as a vector to transfer exogenous DNA into eggs is very attractive, and a recent publication reported a successful attempt at this procedure (Lavitrano et al., 1989). However, this result could not be reproduced, although more than 1,300 mice were generated by several groups following the published protocol (Brinster et al., 1989). Liposomes have been used for a long time to introduce a variety of molecules into living cells (Gregoriadis and Allison, 1980), but their utility as a carrier for nucleic acids was quite limited. Recently, a new type of liposomes made up of cationic lipids was developed (Felgner e t al., 1987; Behr et al., 1989). Cationic liposomes interact with the negatively charged nucleic acid molecules forming complexes in which the nucleic acid is coated by the lipids. The positive outer surface of the complex can then associate with the negatively charged cell membrane, allowing the internalization of the nucleic acid. This new type of liposomes turned out to be very efficient in the transfer of DNA into cells. Based on this finding, we were interested to analyze the
0 1991 WILEY-LISS, INC.
ability of the liposomes to mediate DNA transfer into sperm cells, which could subsequently be used to generate transgenic mice.
MATERIALS A N D METHODS Culture of Sperm With Radioactively Labeled DNA Sperm was collected from proven B6SJLF1 males that had abstained for 48 hr. Sperm was squeezed from one cauda epididymis from each of two males into the same drop of 500 p1 medium. The suspension was allowed to disperse for 10 min a t 37°C in 5% CO, atmosphere. After that, a volume of the suspension necessary to yield a final concentration of 1 x lo6 sperm celldm1 was placed in new drops of 500 pl final volume and incubated for 1.5 h r at 37°C. Radioactively labeled plasmid DNA (1 x lo6 Cerenkov cpm/ml final concentration) was then added, and the suspension was incubated for a further 30 min. The samples were then split into two groups. The first group was washed five times. The first supernatant containing most of the radioactivity was discarded, and the presence of counts in the others as well as in the sperm pellet and in the empty tube (background) was determined. The second group was processed in the same way, but a treatment with 100 pg/ml of DNase I (30 min at 37°C) was included. The DNA was radioactively labeled by random priming. The culture medium described by Lavitran0 et al. (1989) was used. Liposome Treatment and Labeling of DNA With Sulfone Groups The cationic liposomes we used are available under the commercial name of “Lipofectin” (BRL-Gibco). DNA was labeled with sulfone groups by a chemical reaction in which cytosines are transformed into N4methoxy-5,6 dihydrocytosine-6-sulfonate.The tagged DNA was visualized by a n immune reaction in which a specific antisulfone monoclonal antibody binds to the DNA, followed by a rhodamine labeled antimouse Ig antibody (Orgenic’s chemiprobe). Sperm was collected and kept in culture as indicated above. As the sperm
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Received April 24, 1991; accepted June 12, 1991. Address reprint requests to U. Riither, EMBL, P.O. Box 10.2209,6900 Heidelberg, Germany.
LIPOSOMES AND SPERM CELLS was allowed to disperse in the concentrated suspension, 1 pg of lipofectin and 0.2-1 pg of sulfonated DNA were diluted in separated tubes containing 150 p1 of in vitro fertilization (IVF) medium each. The dilutions were then mixed, and the resulting 300 pl drop was transferred in a Petri dish and covered with paraffin oil. After dispersal, a volume necessary to yield a final concentration of 1 x lo6 sperm cells/ml was taken from the original suspension and added to the DNA-lipofectin mixture. This new suspension was incubated for 2 hr more. Once the incubation was complete, DNase at a final concentration of 100 pglml was added to half of the samples for 30 min (37°C). The sperm cells were then washed, fixed in 4% paraformaldehyde, and incubated for 16 h r at 4°C in a humid chamber with a n antisulfone antibody. After 2 h r of incubation at room temperature with a secondary antibody labeled with rhodamine, spermatozoa were washed and stained with Hoechst 33258, a heterochromatin-specific dye. The samples were then mounted for inspection.
In Vitro Fertilization Experiments CD1 females were used as egg donors and B6SJLF1 males as sperm donors. The animals and the medium were prepared according to Hogan et al. (1986). Sperm was collected as described above. At the time sperm was dispersing, 15 pg of lipofectin and 5 pg of DNA were diluted in separated tubes containing 250 p1 of IVF medium each. The dilutions were then mixed, and the resulting volume of 500 pl was added to the 500 p1 sperm suspension. The suspension was incubated at 37°C in a 5% C 0 2 atmosphere for 1.5 hr. From that point on, the normal IVF protocol was followed (Hogan et al., 1986). Embryo transfer was performed as described by Hogan et al. (1986). All DNA analysis was performed following standard protocols. Confocal Microscopy The modular confocal microscope developed and constructed at EMBL was used (for details, see CarmoFonseca et al., 1991). RESULTS Measurement of Binding and Uptake of DNA by Sperm Cells Since we were interested in measuring the efficiency of uptake of DNA by the sperm, we first had to establish a method to discriminate between the binding of DNA to the sperm surface and DNA that has been internalized. Therefore, we used radioactive label as a means to follow DNA and DNase treatment to differentiate between binding and uptake. The first experiments were performed using the conditions described by Lavitrano et al. (1989). After the incubation of sperm with radioactively labeled DNA and several washes, we treated the sperm with DNase to destroy all DNA bound at the surface of the sperm cells. The sperm was then washed again and the counts associated with the
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sperm after DNase treatment were considered as protected by internalization into the sperm head or tail. As is shown in Table 1, only about 10% of the DNA molecules seem to be internalized (fraction 4B), and about 90% are bound to the surface (fraction 4A). To determine more precisely whether the DNA is present in the sperm head, we labeled the DNA with sulfone groups. These sulfone groups can be seen using fluorescence-labeled antibodies directed against these groups. In repeating the uptake experiment with the sulfonated DNA, fluorescence-labeled sperm heads could be detected only when the DNase treatment was omitted (for details, see next section).
Liposome-Mediated Uptake of DNA by Sperm Cells It has been shown th a t tissue-culture cells and even cells of certain organs in a living organism can be successfully transfected by DNA when liposomes are used as vectors (Felgner et al., 1987; Malone, 1989). We were therefore curious to determine the efficiency of this method with sperm cells. Although the efficiency of DNA uptake correlates with the concentration of liposomes, a certain concentration is toxic for cells. The threshold, however, is different for different cell types. To reduce this toxicity to a minimum, we tested different concentrations and chose the lowest that still showed internalization of DNA as measured by the presence of fluorescence-labeled DNA (data not shown). Since we were interested in comparing the position of the fluorescence-labeled DNA and the intactness of the sperm cells, we analyzed the samples in the microscope with phase contrast and after counterstaining with Hoechst 33258 (for the position of the nucleus) and with the DNA-specific antibody for the presence of the florescence light. In the first experiment, sperm was treated (in the same media) either with or without liposomes and analyzed without any DNase treatment. As is illustrated in Figure l a and b, most of the sperm cells were positive for the labeled DNA. However, when the shape of the nucleus based on the Hoechst staining was taken into account, we could detect differences between the two experimental groups. Sperm cells that were not treated with liposomes displayed a n antibody staining that did not follow the shape of the nucleus (Fig. l c and el. In contrast, the liposome-treated sperm showed a clear overlap of the Hoechst and florescence staining (Fig. Id and 0. This finding suggests that without liposome treatment the DNA is only bound to the surface of the cells. This result was strongly supported when we incubated the sperm subsequently with DNase. The sperm cells incubated without liposomes displayed a DNAspecific staining only in broken sperm heads overlapping with the shape of the nucleus, but we could never detect any stain in or on intact sperm (Fig. 2a, c, and el. However, most of the sperm cells treated with liposomes (about 80%) showed staining for DNA that
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Fig. 1. Localization of exogenous DNA on sperm cells incubated without (a, c, e) and with (b, d, 0 liposomes. a, b: Phase contrast photograph. c, d: Hoechst 33258 nuclear staining. e, E Sulfonated DNA-specific staining.
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TABLE 1. Incubation of Sperm With Radioactively promising, we next attempted to test the possibility of Labeled DNA and Consequences of generating transgenic mice with this method. To reDNase Treatment* duce toxicity of the liposome treatment to a minimum, Group we tested different concentrations of DNA and lipoFraction A somes for their efficiency in producing two-cell stages B 1 54.3 f 1.9 51.4 k 2.2 after IVF of oocytes. As is documented in Table 2 , 5 pg 2 13.7 k 2.2 41.5 f 3.0 of DNA and 15 pg of liposomes per milliliter medium 3.7 f 1.3 gave the best fertilization efficiency (about 60%). 3 3.6 k 0.6 4 26.4 3.4 2.9 k 0.4 Since i t is known from DNA microinjection experi5 1.3 0.2 0.5 f 0.2 ments into fertilized eggs that different DNA con*The data correspond to two independent experiments. In structs can differ in the frequency of integrating into each, eight samples were processed in parallel. For details, the genome, we used several DNA constructs for the see Materials and Methods. After the incubation with the radioactively labeled DNA and several washes, the samples generation of transgenic mice. In total, 5,024 oocytes were split into two groups (A and B) and counted (fraction were fertilized in vitro with DNA- and liposome-treated 1).Group B was then treated with DNaseI, both groups were sperm; 2,152 developed to the two-cell stage, and 1,984 washed twice, and the supernatant of these washes were of them were transferred into pseudopregnant females. collected and counted (fraction 2 and 3). Fraction 4 repre- Four hundred fifty-eight offspring were born and anasents the counts still bound to sperm, and fraction 5 is the lyzed for the presence of the construct used in the radioactive background of the empty tube. The counts corresponding to the five fractions were added and con- individual experiment (Table 3). However, we were sidered as loo%, subsequently the radioactivity of each of unable to detect a specific signal in the Southern blot the fractions was expressed as a percentage of the total. analysis for any of the seven DNAs tested.
**
DISCUSSION followed the shape of the nucleus even after the DNase treatment (Fig. 2b, d, and f). To test whether this result was specific for one type of DNA construct, we repeated the experiment with different DNAs varying in size from 1.8 to 14 kb. We obtained similar results with all the constructs (data not shown). To rule out a possible cross-reaction of the antisulfone antibody, the experiment was also repeated in the absence of sulfonated DNA. Under these conditions we could not detect any antibody binding to the samples (data not shown).
Localisation of the DNA in the Sperm Head To determine more precisely in which cellular compartment the exogenous DNA was accumulating, we analyzed the samples with a laser confocal microscope. One micrometer horizontal optical sections were made through liposome-treated sperm cells incubated with DNase. In a plane close to the upper surface of the sperm, the DNA-specific staining was visible (Fig. 3a). Sections through the middle of the cells displayed a n enhanced signal, which was most intense in the region of the nucleus (Fig. 3b). The staining was weaker but still detectable when a plane close to the cell-substratum attachment was analyzed (Fig. 3c). Finally, in a sagital section of the sperm head, made at the point marked by a n arrow in Fig. 3b, the fluorescent label was present all across the cell (Fig. 3d). Taken together, these analyses provide evidence that the DNA is present in or near the nucleus of the sperm head. Generation of Transgenic Mice by Liposome-Mediated DNA Transfer Since the initial analysis concerning the DNA uptake by sperm cells with the help of liposomes seemed
In this report we have provided evidence that liposomes can efficiently transfer DNA into sperm cells. About 80% of the liposome-treated sperm cells displayed a signal specific for the exogenous DNA inside the sperm head a s shown by laser confocal microscopy. The liposome-treated sperm is able to fertilize oocytes as efficiently as untreated sperm measured by the frequency of two-cell-stage embryos obtained after IVF. After transfer into pseudopregnant females, a n expected number of offspring were born. However, we were unable to generate transgenic mice by this method. Based on our initial control experiments, there is no obvious reason for this failure. Thus we have to consider that our test system is either not adequate or not precise enough. First, all analysis of the DNA internalization mediated by liposomes is performed with chemically modified DNA (coupling of sulfone groups to the cytosine residue). Although we have no evidence t h a t the sulfone groups positively influence the DNA uptake by the sperm cells, we cannot exclude that this is actually happening. Therefore, we cannot exclude t h a t the unlabeled DNA is less efficient transferred into the sperm. Second, we cannot rule out the possibility that the DNA is entering the cell only after a n extensive digestion by nuclease(s) and that these pieces are still sufficient for recognization by the antibody. Finally, since only 80% of the liposometreated sperm displayed the exogenous DNA-specific staining, we cannot rule out that the sperm without internalized DNA are more efficient in fertilizing oocytes. However, this possibility is not very likely because the fertilization efficiency of the liposometreated sperm was comparable to that of untreated sperm. It might be that the majority of the sperm with low mobility become labeled and that the more active sperm, which fertilized the oocytes, are for unknown
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Fig. 2. Localization of exogenous DNA on sperm cells incubated without (a, c, e) and with (b,d, fl liposomes, followed by DNase I treatment. a, b: Phase contrast photograph. c, d: Hoechst 33258 nuclear staining. e, E Sulfonated DNA-specific staining.
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Fig. 3. Optical sections generated by laser confocal microscopy on liposome-treated sperm. The sulfonated DNA-specific staining is shown. a, b, c: Horizontal sections through the spermatozoa. d Sagital section of the sperm head at the point indicated by the arrow in b.
TABLE 2. Effect of Varying Liposome Concentrations on In Vitro Fertilization Frequency* 5a/Ob 126 43
DNA and liposome concentration 10/0 5/15 15/15 314 3,412c 258 167 2,055c 96
No. of oocytes No. of two-cell embryos Efficiency percent 34 53 6OC 34 *Incubation time was always 90 min (for details, see Materials and Methods). aDNA concentration in pg/ml. bLiposome concentration in pg/ml. CThisnumber includes all subsequent experiments.
reasons not susceptible to liposomes. On the other hand, we have tried to increase the efficiency of D N A uptake. For this purpose, we incubated the sperm with the liposomes for the first 30 min in the absence of BSA, which might inhibit the activity of the liposomes.
10130 681 111 16
However, this step resulted in a decrease of the fertilization efficiency (data not shown). Analysis with the laser confocal microscope indicates that the D N A has entered the nucleus but does not give any information on whether this D N A is still intact.
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Construct Tyrosinase Tyrosinase pSV2 CAT HB-c-fos LTR RSV Lac Z WAP-Dint CPR-neu MX c-fos LTR Total
+
TABLE 3. In Vitro Fertilization With Liposome-Treated Sperm and Different DNA Constructs No. of oocytes Two-cell stages Efficiency (W) Transferred pSV2 CAT 193 155 80 139 1,018 1,046 1,216 624 507 55 365 5,024
427 266 364 433 286 23 198 2,152
When DNA is directly microinjected into the nucleus of fertilized eggs, i t becomes integrated into the genome without gross alterations in most of the cases (Palmiter and Brinster, 1986). Thus the transfer of DNA through the cytoplasm of the sperm could be the site of intensive DNA digestion. These interpretations could explain why we have found on DNA Southern blot analysis of about 1%of born pups faint signals of unexpected fragment length. The intensity of the signal as well as the length of the fragments suggested less than one copy of a truncated construct per cell. Therefore, these animals were not used for breeding or expression analysis. In parallel to the mouse sperm experiments, we have initiated liposome treatment of sperm isolated from other species. Using the same protocol as for mouse sperm with the sulfonated DNA and DNase treatment, we have also found that bull and rat sperm did take up the DNA. These data suggest that the phenomena of DNA uptake mediated by liposomes is not restricted to the mouse. This finding raises the possibility that perhaps with certain species of farm animals this method may be used to generate transgenic animals. ACKNOWLEDGMENTS We thank Catherine Ovitt for critical reading of the manuscript and Hilary Davies-Ruck for typing. We also thank Carlos Dotti for help with the sulfonated DNA staining and Ernst Stelzer for confocal microscopy.
42 25 30 69 56 42 54 43
400 204 355 419 255 23 189 1,984
Born 3 102 50 87 98 73 10 35 458
Support were given by a fellowship from the Minister0 de Educacion y Ciencia, Spain, to D.B. and by a grant (Fond zur Forderung der wissenschaftlichen Forschung Wien) to K.S. REFERENCES Behr JP, Demeneix B, Loeffler JP, Perez-Mutul J (1989): Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci USA 86:6982-6986. Brinster RL, Sandgren EP, Behringer RR, Palmiter RD (1989): No simple solution for making transgenic mice. Cell 59:239-241. Carmo-Fonseca M, Tollervey D, Pepperkok R, Barabino SML, Merdes A, Brunner C, Zamore PD, Green MR, Hurt E, Lammond A1 (1991): Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. EMBO J 10:195206. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987): Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 84:7413-7417. Gregoriadis G, Allison AC (eds) (1980): “Liposomes in Biological Systems.” New York: John Wiley and Sons. Hogan B, Constantini F, Lacy E (1986): “Manipulating the Mouse Embryo: A Laboratory Manual.” Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Lavitrano ML, Camaioni A, Fazio VM, Dolci S, Farace MG, Spadafora C (1989): Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice. Cell 57:717-723. Malone RW (1989): mRNA transfection of cultured eukaryotic cells and embryos using cationic liposomes. Focus 11:61-65. Palmiter RD, Brinster RL (1986): Germ-line transformation of mice. Annu Rev Genet 20:465499.
Liposome-Mediated DNA Uptake by Sperm Cells DANIEL BACHILLER,’ KARL SCHELLANDER: JANOS PELI: AND ULRICH R ~ J T H E R ~ ‘European Molecular Biology Laboratory, Heidelberg, Germany; ‘Znstitut fur Tierzucht und Genetik, Veterinarmedizinische Universitat Wien, Wien, Austria
To investigate the potential use of ABSTRACT sperm cells as vectors to transfer exogenous DNA via the fertilization of oocytes into the germ line of mice, we have used liposomes to transfect DNA into the sperm head. Although the DNA transfer into sperm mediated by liposomes was very efficient and no obvious reduction in the fertilization frequency of oocytes could be detected, we were unable to generate transgenic mice by this method. Key Words: DNA transfer,Transgenic animals, Cationic liposomes INTRODUCTION The generation of transgenic animals has opened avenues to the analysis of many questions in oncogenesis, developmental biology, gene regulation, and even animal farming. The most established method is the microinjection of DNA into the pronucleus of fertilized eggs (for review, see Palmiter and Brinster, 1986). Although in some laboratories this is a routine technique, the generation of transgenic mice is a procedure of certain complexity. Furthermore, for certain animals the microinjection is possible only after additional treatments that are time consuming and that might influence the efficiency; e.g., pig eggs must first be centrifuged to make the pronucleus visible. Therefore, the idea of using sperm as a vector to transfer exogenous DNA into eggs is very attractive, and a recent publication reported a successful attempt at this procedure (Lavitrano et al., 1989). However, this result could not be reproduced, although more than 1,300 mice were generated by several groups following the published protocol (Brinster et al., 1989). Liposomes have been used for a long time to introduce a variety of molecules into living cells (Gregoriadis and Allison, 1980), but their utility as a carrier for nucleic acids was quite limited. Recently, a new type of liposomes made up of cationic lipids was developed (Felgner e t al., 1987; Behr et al., 1989). Cationic liposomes interact with the negatively charged nucleic acid molecules forming complexes in which the nucleic acid is coated by the lipids. The positive outer surface of the complex can then associate with the negatively charged cell membrane, allowing the internalization of the nucleic acid. This new type of liposomes turned out to be very efficient in the transfer of DNA into cells. Based on this finding, we were interested to analyze the
0 1991 WILEY-LISS, INC.
ability of the liposomes to mediate DNA transfer into sperm cells, which could subsequently be used to generate transgenic mice.
MATERIALS A N D METHODS Culture of Sperm With Radioactively Labeled DNA Sperm was collected from proven B6SJLF1 males that had abstained for 48 hr. Sperm was squeezed from one cauda epididymis from each of two males into the same drop of 500 p1 medium. The suspension was allowed to disperse for 10 min a t 37°C in 5% CO, atmosphere. After that, a volume of the suspension necessary to yield a final concentration of 1 x lo6 sperm celldm1 was placed in new drops of 500 pl final volume and incubated for 1.5 h r at 37°C. Radioactively labeled plasmid DNA (1 x lo6 Cerenkov cpm/ml final concentration) was then added, and the suspension was incubated for a further 30 min. The samples were then split into two groups. The first group was washed five times. The first supernatant containing most of the radioactivity was discarded, and the presence of counts in the others as well as in the sperm pellet and in the empty tube (background) was determined. The second group was processed in the same way, but a treatment with 100 pg/ml of DNase I (30 min at 37°C) was included. The DNA was radioactively labeled by random priming. The culture medium described by Lavitran0 et al. (1989) was used. Liposome Treatment and Labeling of DNA With Sulfone Groups The cationic liposomes we used are available under the commercial name of “Lipofectin” (BRL-Gibco). DNA was labeled with sulfone groups by a chemical reaction in which cytosines are transformed into N4methoxy-5,6 dihydrocytosine-6-sulfonate.The tagged DNA was visualized by a n immune reaction in which a specific antisulfone monoclonal antibody binds to the DNA, followed by a rhodamine labeled antimouse Ig antibody (Orgenic’s chemiprobe). Sperm was collected and kept in culture as indicated above. As the sperm
~~~~~
~
Received April 24, 1991; accepted June 12, 1991. Address reprint requests to U. Riither, EMBL, P.O. Box 10.2209,6900 Heidelberg, Germany.
LIPOSOMES AND SPERM CELLS was allowed to disperse in the concentrated suspension, 1 pg of lipofectin and 0.2-1 pg of sulfonated DNA were diluted in separated tubes containing 150 p1 of in vitro fertilization (IVF) medium each. The dilutions were then mixed, and the resulting 300 pl drop was transferred in a Petri dish and covered with paraffin oil. After dispersal, a volume necessary to yield a final concentration of 1 x lo6 sperm cells/ml was taken from the original suspension and added to the DNA-lipofectin mixture. This new suspension was incubated for 2 hr more. Once the incubation was complete, DNase at a final concentration of 100 pglml was added to half of the samples for 30 min (37°C). The sperm cells were then washed, fixed in 4% paraformaldehyde, and incubated for 16 h r at 4°C in a humid chamber with a n antisulfone antibody. After 2 h r of incubation at room temperature with a secondary antibody labeled with rhodamine, spermatozoa were washed and stained with Hoechst 33258, a heterochromatin-specific dye. The samples were then mounted for inspection.
In Vitro Fertilization Experiments CD1 females were used as egg donors and B6SJLF1 males as sperm donors. The animals and the medium were prepared according to Hogan et al. (1986). Sperm was collected as described above. At the time sperm was dispersing, 15 pg of lipofectin and 5 pg of DNA were diluted in separated tubes containing 250 p1 of IVF medium each. The dilutions were then mixed, and the resulting volume of 500 pl was added to the 500 p1 sperm suspension. The suspension was incubated at 37°C in a 5% C 0 2 atmosphere for 1.5 hr. From that point on, the normal IVF protocol was followed (Hogan et al., 1986). Embryo transfer was performed as described by Hogan et al. (1986). All DNA analysis was performed following standard protocols. Confocal Microscopy The modular confocal microscope developed and constructed at EMBL was used (for details, see CarmoFonseca et al., 1991). RESULTS Measurement of Binding and Uptake of DNA by Sperm Cells Since we were interested in measuring the efficiency of uptake of DNA by the sperm, we first had to establish a method to discriminate between the binding of DNA to the sperm surface and DNA that has been internalized. Therefore, we used radioactive label as a means to follow DNA and DNase treatment to differentiate between binding and uptake. The first experiments were performed using the conditions described by Lavitrano et al. (1989). After the incubation of sperm with radioactively labeled DNA and several washes, we treated the sperm with DNase to destroy all DNA bound at the surface of the sperm cells. The sperm was then washed again and the counts associated with the
195
sperm after DNase treatment were considered as protected by internalization into the sperm head or tail. As is shown in Table 1, only about 10% of the DNA molecules seem to be internalized (fraction 4B), and about 90% are bound to the surface (fraction 4A). To determine more precisely whether the DNA is present in the sperm head, we labeled the DNA with sulfone groups. These sulfone groups can be seen using fluorescence-labeled antibodies directed against these groups. In repeating the uptake experiment with the sulfonated DNA, fluorescence-labeled sperm heads could be detected only when the DNase treatment was omitted (for details, see next section).
Liposome-Mediated Uptake of DNA by Sperm Cells It has been shown th a t tissue-culture cells and even cells of certain organs in a living organism can be successfully transfected by DNA when liposomes are used as vectors (Felgner et al., 1987; Malone, 1989). We were therefore curious to determine the efficiency of this method with sperm cells. Although the efficiency of DNA uptake correlates with the concentration of liposomes, a certain concentration is toxic for cells. The threshold, however, is different for different cell types. To reduce this toxicity to a minimum, we tested different concentrations and chose the lowest that still showed internalization of DNA as measured by the presence of fluorescence-labeled DNA (data not shown). Since we were interested in comparing the position of the fluorescence-labeled DNA and the intactness of the sperm cells, we analyzed the samples in the microscope with phase contrast and after counterstaining with Hoechst 33258 (for the position of the nucleus) and with the DNA-specific antibody for the presence of the florescence light. In the first experiment, sperm was treated (in the same media) either with or without liposomes and analyzed without any DNase treatment. As is illustrated in Figure l a and b, most of the sperm cells were positive for the labeled DNA. However, when the shape of the nucleus based on the Hoechst staining was taken into account, we could detect differences between the two experimental groups. Sperm cells that were not treated with liposomes displayed a n antibody staining that did not follow the shape of the nucleus (Fig. l c and el. In contrast, the liposome-treated sperm showed a clear overlap of the Hoechst and florescence staining (Fig. Id and 0. This finding suggests that without liposome treatment the DNA is only bound to the surface of the cells. This result was strongly supported when we incubated the sperm subsequently with DNase. The sperm cells incubated without liposomes displayed a DNAspecific staining only in broken sperm heads overlapping with the shape of the nucleus, but we could never detect any stain in or on intact sperm (Fig. 2a, c, and el. However, most of the sperm cells treated with liposomes (about 80%) showed staining for DNA that
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Fig. 1. Localization of exogenous DNA on sperm cells incubated without (a, c, e) and with (b, d, 0 liposomes. a, b: Phase contrast photograph. c, d: Hoechst 33258 nuclear staining. e, E Sulfonated DNA-specific staining.
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TABLE 1. Incubation of Sperm With Radioactively promising, we next attempted to test the possibility of Labeled DNA and Consequences of generating transgenic mice with this method. To reDNase Treatment* duce toxicity of the liposome treatment to a minimum, Group we tested different concentrations of DNA and lipoFraction A somes for their efficiency in producing two-cell stages B 1 54.3 f 1.9 51.4 k 2.2 after IVF of oocytes. As is documented in Table 2 , 5 pg 2 13.7 k 2.2 41.5 f 3.0 of DNA and 15 pg of liposomes per milliliter medium 3.7 f 1.3 gave the best fertilization efficiency (about 60%). 3 3.6 k 0.6 4 26.4 3.4 2.9 k 0.4 Since i t is known from DNA microinjection experi5 1.3 0.2 0.5 f 0.2 ments into fertilized eggs that different DNA con*The data correspond to two independent experiments. In structs can differ in the frequency of integrating into each, eight samples were processed in parallel. For details, the genome, we used several DNA constructs for the see Materials and Methods. After the incubation with the radioactively labeled DNA and several washes, the samples generation of transgenic mice. In total, 5,024 oocytes were split into two groups (A and B) and counted (fraction were fertilized in vitro with DNA- and liposome-treated 1).Group B was then treated with DNaseI, both groups were sperm; 2,152 developed to the two-cell stage, and 1,984 washed twice, and the supernatant of these washes were of them were transferred into pseudopregnant females. collected and counted (fraction 2 and 3). Fraction 4 repre- Four hundred fifty-eight offspring were born and anasents the counts still bound to sperm, and fraction 5 is the lyzed for the presence of the construct used in the radioactive background of the empty tube. The counts corresponding to the five fractions were added and con- individual experiment (Table 3). However, we were sidered as loo%, subsequently the radioactivity of each of unable to detect a specific signal in the Southern blot the fractions was expressed as a percentage of the total. analysis for any of the seven DNAs tested.
**
DISCUSSION followed the shape of the nucleus even after the DNase treatment (Fig. 2b, d, and f). To test whether this result was specific for one type of DNA construct, we repeated the experiment with different DNAs varying in size from 1.8 to 14 kb. We obtained similar results with all the constructs (data not shown). To rule out a possible cross-reaction of the antisulfone antibody, the experiment was also repeated in the absence of sulfonated DNA. Under these conditions we could not detect any antibody binding to the samples (data not shown).
Localisation of the DNA in the Sperm Head To determine more precisely in which cellular compartment the exogenous DNA was accumulating, we analyzed the samples with a laser confocal microscope. One micrometer horizontal optical sections were made through liposome-treated sperm cells incubated with DNase. In a plane close to the upper surface of the sperm, the DNA-specific staining was visible (Fig. 3a). Sections through the middle of the cells displayed a n enhanced signal, which was most intense in the region of the nucleus (Fig. 3b). The staining was weaker but still detectable when a plane close to the cell-substratum attachment was analyzed (Fig. 3c). Finally, in a sagital section of the sperm head, made at the point marked by a n arrow in Fig. 3b, the fluorescent label was present all across the cell (Fig. 3d). Taken together, these analyses provide evidence that the DNA is present in or near the nucleus of the sperm head. Generation of Transgenic Mice by Liposome-Mediated DNA Transfer Since the initial analysis concerning the DNA uptake by sperm cells with the help of liposomes seemed
In this report we have provided evidence that liposomes can efficiently transfer DNA into sperm cells. About 80% of the liposome-treated sperm cells displayed a signal specific for the exogenous DNA inside the sperm head a s shown by laser confocal microscopy. The liposome-treated sperm is able to fertilize oocytes as efficiently as untreated sperm measured by the frequency of two-cell-stage embryos obtained after IVF. After transfer into pseudopregnant females, a n expected number of offspring were born. However, we were unable to generate transgenic mice by this method. Based on our initial control experiments, there is no obvious reason for this failure. Thus we have to consider that our test system is either not adequate or not precise enough. First, all analysis of the DNA internalization mediated by liposomes is performed with chemically modified DNA (coupling of sulfone groups to the cytosine residue). Although we have no evidence t h a t the sulfone groups positively influence the DNA uptake by the sperm cells, we cannot exclude that this is actually happening. Therefore, we cannot exclude t h a t the unlabeled DNA is less efficient transferred into the sperm. Second, we cannot rule out the possibility that the DNA is entering the cell only after a n extensive digestion by nuclease(s) and that these pieces are still sufficient for recognization by the antibody. Finally, since only 80% of the liposometreated sperm displayed the exogenous DNA-specific staining, we cannot rule out that the sperm without internalized DNA are more efficient in fertilizing oocytes. However, this possibility is not very likely because the fertilization efficiency of the liposometreated sperm was comparable to that of untreated sperm. It might be that the majority of the sperm with low mobility become labeled and that the more active sperm, which fertilized the oocytes, are for unknown
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Fig. 2. Localization of exogenous DNA on sperm cells incubated without (a, c, e) and with (b,d, fl liposomes, followed by DNase I treatment. a, b: Phase contrast photograph. c, d: Hoechst 33258 nuclear staining. e, E Sulfonated DNA-specific staining.
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Fig. 3. Optical sections generated by laser confocal microscopy on liposome-treated sperm. The sulfonated DNA-specific staining is shown. a, b, c: Horizontal sections through the spermatozoa. d Sagital section of the sperm head at the point indicated by the arrow in b.
TABLE 2. Effect of Varying Liposome Concentrations on In Vitro Fertilization Frequency* 5a/Ob 126 43
DNA and liposome concentration 10/0 5/15 15/15 314 3,412c 258 167 2,055c 96
No. of oocytes No. of two-cell embryos Efficiency percent 34 53 6OC 34 *Incubation time was always 90 min (for details, see Materials and Methods). aDNA concentration in pg/ml. bLiposome concentration in pg/ml. CThisnumber includes all subsequent experiments.
reasons not susceptible to liposomes. On the other hand, we have tried to increase the efficiency of D N A uptake. For this purpose, we incubated the sperm with the liposomes for the first 30 min in the absence of BSA, which might inhibit the activity of the liposomes.
10130 681 111 16
However, this step resulted in a decrease of the fertilization efficiency (data not shown). Analysis with the laser confocal microscope indicates that the D N A has entered the nucleus but does not give any information on whether this D N A is still intact.
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Construct Tyrosinase Tyrosinase pSV2 CAT HB-c-fos LTR RSV Lac Z WAP-Dint CPR-neu MX c-fos LTR Total
+
TABLE 3. In Vitro Fertilization With Liposome-Treated Sperm and Different DNA Constructs No. of oocytes Two-cell stages Efficiency (W) Transferred pSV2 CAT 193 155 80 139 1,018 1,046 1,216 624 507 55 365 5,024
427 266 364 433 286 23 198 2,152
When DNA is directly microinjected into the nucleus of fertilized eggs, i t becomes integrated into the genome without gross alterations in most of the cases (Palmiter and Brinster, 1986). Thus the transfer of DNA through the cytoplasm of the sperm could be the site of intensive DNA digestion. These interpretations could explain why we have found on DNA Southern blot analysis of about 1%of born pups faint signals of unexpected fragment length. The intensity of the signal as well as the length of the fragments suggested less than one copy of a truncated construct per cell. Therefore, these animals were not used for breeding or expression analysis. In parallel to the mouse sperm experiments, we have initiated liposome treatment of sperm isolated from other species. Using the same protocol as for mouse sperm with the sulfonated DNA and DNase treatment, we have also found that bull and rat sperm did take up the DNA. These data suggest that the phenomena of DNA uptake mediated by liposomes is not restricted to the mouse. This finding raises the possibility that perhaps with certain species of farm animals this method may be used to generate transgenic animals. ACKNOWLEDGMENTS We thank Catherine Ovitt for critical reading of the manuscript and Hilary Davies-Ruck for typing. We also thank Carlos Dotti for help with the sulfonated DNA staining and Ernst Stelzer for confocal microscopy.
42 25 30 69 56 42 54 43
400 204 355 419 255 23 189 1,984
Born 3 102 50 87 98 73 10 35 458
Support were given by a fellowship from the Minister0 de Educacion y Ciencia, Spain, to D.B. and by a grant (Fond zur Forderung der wissenschaftlichen Forschung Wien) to K.S. REFERENCES Behr JP, Demeneix B, Loeffler JP, Perez-Mutul J (1989): Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci USA 86:6982-6986. Brinster RL, Sandgren EP, Behringer RR, Palmiter RD (1989): No simple solution for making transgenic mice. Cell 59:239-241. Carmo-Fonseca M, Tollervey D, Pepperkok R, Barabino SML, Merdes A, Brunner C, Zamore PD, Green MR, Hurt E, Lammond A1 (1991): Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. EMBO J 10:195206. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987): Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 84:7413-7417. Gregoriadis G, Allison AC (eds) (1980): “Liposomes in Biological Systems.” New York: John Wiley and Sons. Hogan B, Constantini F, Lacy E (1986): “Manipulating the Mouse Embryo: A Laboratory Manual.” Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Lavitrano ML, Camaioni A, Fazio VM, Dolci S, Farace MG, Spadafora C (1989): Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice. Cell 57:717-723. Malone RW (1989): mRNA transfection of cultured eukaryotic cells and embryos using cationic liposomes. Focus 11:61-65. Palmiter RD, Brinster RL (1986): Germ-line transformation of mice. Annu Rev Genet 20:465499.
