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BOR - Papers in Press, published online ahead of print October 11, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.056952
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BIOLOGY OF REPRODUCTION 76, 336–343 (2007)
DOI: 10.1095/biolreprod.106.056952
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Effect of Transgene Concentration, Flanking Matrix Attachment Regions, and RecA-Coating on the Efficiency of Mouse Transgenesis Mediated by Intracytoplasmic Sperm Injection1

Pedro Nuno Moreira 3, Miriam Pérez-Crespo 3, Miguel Angel Ramírez 3, Julio Pozueta 4 5, Lluís Montoliu 4, and Alfonso Gutiérrez-Adán 2 3

Departamento de Reproducción Animal y Conservación de Recursos Zoogenéticos,3 Instituto National de Investigación y Technología Agranria, 28040 Madrid, Spain Departamento de Biología Molecular y Celular,4 Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Campus de Cantoblanco, 28049 Madrid, Spain Departamento de Biologia Molecular,5 Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid, 28049 Madrid, Spain

ABSTRACT

Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of ~510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.

assisted reproductive technology, early development, sperm

INTRODUCTION

Using the mouse model, intracytoplasmic sperm injection (ICSI)-mediated transgenesis has been shown to be a valuable tool for the production of transgenic animals [1], an essential instrument for basic and applied research in bioscience. This method of transgenesis consists on the comicroinjection of foreign DNA and spermatozoa into metaphase II (MII) oocytes. ICSI of DNA-loaded sperm cells has been shown to mediate mouse transgenesis at high efficiency, especially when sperm cell damage (by freeze-thaw cycles or exposure to detergents) is induced [1]. The high efficiency of ICSI-mediated transgenesis is capable of mediating complete integration and expression of DNA constructs with submegabase magnitude and foreign DNA molecules in minimal concentrations [2, and Pozueta et al., unpublished results]. The effectiveness of this procedure is much higher than that of pronuclear injection, the standard technique for transgenesis production, especially when large DNA molecules are used [2]. In addition, ICSI-mediated transgenesis leads to low frequency of embryo mosaicism, one of the major limitations of pronuclear injection for the production of transgenic livestock [3–5], and high frequency of Mendelian germ line transmission of transgenic sequences among founder animals. Although its successful application has been mainly restricted to the mouse species, transgenic pigs [6, 7], rats [8, 9], and Rhesus macaque embryos [10] were recently generated using this approach, and recent developments in bovine ICSI promise future application in this species as well.

The overall efficiency of ICSI-mediated transgenesis can be influenced by many different factors, such as sperm and oocyte donor strain, construct size and copy number, DNA vector, etc. Early work in the mouse by Brinster et al. [11] showed that the integration rate after pronuclear injection doubled when the DNA concentration increased from 0.5 to 1 ng/µl, but remained constant at about 25% when DNA was injected at 1 and 10 ng/µl. As a result, in the majority of subsequent studies on animal transgenesis production, the injected DNA concentration was never greater than 5 ng/µl. This was later challenged in domestic species, where the integration rate was reported to be lower compared with the mouse [3, 4], and, recently, higher concentrations (5–10 ng/µl) have been suggested [12]. However, we cannot assume that the DNA concentrations that have been determined as optimal for pronuclear injection will ensure optimal results in ICSI-mediated transgenesis. It is important to take into account that, in ICSI-mediated transgenesis, foreign DNA molecules are present during sperm nucleus decondensation, which involves important chromatin reorganization events, such as the replacement of paternal protamines by maternal histones. In contrast, in transgenesis procedures involving pronuclear injection, foreign DNA molecules are delivered in a completely formed male pronucleus after completion of the majority of sperm nucleus remodeling events. In such different developmental scenarios, the opportunities for transgene integration may not be exactly the same.

Within the nucleus of a eukaryotic cell, the chromatin is organized into topologically constrained loops that define functional genetic domains [13–15]. Such loops are established by matrix attachment regions (MARs); AT-rich DNA sequences that extend over several hundred base pairs contain topoisomerase II cleavage sites, flank transcriptionally active genes, and frequently map within or close to DNAse I-hypersensitive sites, enhancers, and locus control regions [16]. MARs have been shown to increase transgene expression levels in vivo and in vitro [17, 18], affect the expression of microinjected transgenes during the preimplantation developmental period [19], and have been suggested to be involved in chromatin opening via nuclear protein interaction. In addition, the fact that MARs are highly repetitive sequences within the mammalian genome suggests that transgene flanking with MARs can potentially be used to improve the transgene integration efficiency by homologous recombination. It has been recently reported that endogenous repeated sequences could be implicated in homologous recombination events in mouse embryos, involving 70% of integrated events in some experiments [20]. Also, the fact that live mouse spermatozoa can bind exogenous DNA molecules via interactions with the sperm nuclear matrix [21] further supports the importance of evaluating the impact of transgene flanking with MARs on the efficiency of ICSI-mediated transgenesis.

Recently, it was reported that ICSI with fresh sperm cells of single-stranded DNA (ssDNA), complexed with Escherichia coli recombinase A (RecA) into mouse MII oocytes, offers significant advantages over pronuclear microinjection of RecA-complexed ssDNA and previous ICSI-based transgenesis approaches, both in terms of increased efficiency and improved embryo survival [22]. In that report, the authors suggest that the improved results were a consequence of the omission of sperm freezing or Triton X-100 treatment prior to ICSI, and an outcome of a RecA-facilitated transgene insertion mechanism, both leading to lower paternal chromosome fragmentation. RecA is a bacterial recombinase that binds and protects ssDNA from shearing and degradation [23]. This protective approach has been previously tested by pronuclear microinjection in pigs and goats with similar results [24, 25], but the recombinant effect of the prokaryote RecA in the eukaryote genome has never been demonstrated [22, 24, 25]. Moreover, in these studies, high founder animal mosaicism, a common feature of pronuclear injection-mediated transgenesis, was observed. Recently, it was also reported that the recombinase-mediated DNA transfer, by means of pronuclear microinjection and ICSI, contributed very little to the production of transgenic rats [26].

In this study, we evaluated the impact of transgene DNA concentration, transgene flanking with nuclear MARs, and the use of RecA-coated DNA on the efficiency of mouse transgenesis production by ICSI in order to optimize and understand the mechanism of ICSI-mediated transgenesis.

MATERIALS AND METHODS

Reagents and Media

Unless otherwise stated, all chemicals and media were purchased from Sigma Chemical Co. (Alcobendas, Madrid, Spain).

Animals

Pronuclear injection experiments were done in hybrid B6D2F1 mice (Harlan Iberica SL, Barcelona, Spain). This mouse strain was also used as a donor of oocytes and sperm for ICSI experiments. Females were 6- to 8-wk old at the time of the experiments, and males were at least 3-mo old. CD1 females, mated with vasectomized CD1 males, were used as surrogate mothers for embryo transfer experiments. Lactating CD1 foster mothers were occasionally used to raise pups. Mice were fed ad libitum with a standard diet and maintained in a temperature- and light-controlled room (23°C; 14L:10D). All animal experiments were performed in accordance with institutional animal care guidelines established in the Guide for Care and Use of Laboratory Animals, as adopted and promulgated by the Society for the Study of Reproduction.

Transgene Preparation

The yeast artificial chromosome (YAC; ~510 kb) used for our ICSI-mediated transgenesis experiments was a construct engineered to retain the entire human amyloid precursor protein (APP) gene, but not the surrounding loci (Pozueta et al., unpublished results). This was accomplished by homologous recombination techniques in yeast cells, using the pop-in/pop-out method, as described previously [27]. A detailed description of the human APP YAC transgene (hAPPy), and of the analysis of integrity and expression, will be published subsequently. After homologous recombination, YAC DNA was prepared and purified following reported methods [2, 28, 29], resulting in a DNA concentration of ~10 ng/µl in YAC microinjection buffer (10 mM Tris-HCl [pH 7.5], 0.1 mM EDTA, 100 mM NaCl, 30 µM spermine, and 70 µM spermidine) [30]. The integrity of the YAC DNA preparation was verified by pulsed-field gel electrophoresis. Subsequently, YAC DNA dilutions in the YAC microinjection buffer were used for ICSI-mediated transgenesis.

The enhanced green fluorescent protein (EGFP) plasmid construct (pEGFP-N1, 5.4 kb; Clontech Laboratories, Inc., Palo Alto, CA) used for our experiments contained the human CMV immediate early promoter and the enhanced GFP gene. This construct was linearized with AflII prior to use. Presented data also include assays with this plasmid EGFP construct flanked by two MAR elements (M-EGFP-M; 2.3 kb of the human ß-interferon domain boundaries) [19]. To make the plasmid M-EGFP-M, the multiple cloning site of pEGFP-N1 was eliminated, and a BclI-HindIII adapter was inserted in the AflIII and AflII sites. The MAR element was subcloned into the adapters before the CMV promoter (AflIII) and after the SV40 poly A region (AflII). The MAR element was kindly supplied by M. Kalos (Fred Hutchingson Cancer Research Center, WA). Before microinjection, plasmids were digested with BclI and HindIII to isolate the transgene cassette (6.4 kb) from the rest of the plasmid sequences. The transgene was purified using an Elu-Quit DNA Purification Kit (Schleicher & Schuell, Dassel, Germany) following the manufacturer's instructions. DNA was resuspended in TE (10 mM Tris, 0.1 mM EDTA, pH 8). Note that the concentration of EDTA was reduce from 1 mM to 0.1 mM because it has been reported that the use of ion chelators (EGTA or EDTA) reduce significantly the efficiency of the ICSI-mediated transgenesis in mouse embryos [31].

Preparation of RecA:DNA Complexes and Fluorescent Transgene Labeling

Preparation of RecA-complexed DNA was initiated by the removal of the 50% glycerol in which RecA (Epicentre, Madison, WI) is shipped, using gel filtration through a Microspin G-25 column (Amersham, Little Chalfong, UK) following the manufacture instructions. The column was washed briefly by placing 70 µl of 1x TKM buffer (25 mM Tris-HCl [pH 8.0], 150 mM KCl, 2 mM MgCl2) into the center of the column bed and centrifuging at 735 x g for 1 min at 4°C. This centrifugation step was repeated three times, with each spin lasting for 2 min, and the flow through discarded. A volume of 35 µl of RecA was mixed with an equal volume of a double-concentrated TKM buffer, placed in the center of the column bed, and centrifuged for 2 min at 4°C. The flow through free of glycerol was then mixed with denatured DNA. The denaturing was accomplished with a 5-min incubation at 95°C in TKM buffer. Denatured ssDNA was quenched on ice, and the appropriate quantity of RecA solution was added in order to satisfy a 40:1 protein:DNA (weight:weight) ratio, ensuring coating of all ssDNA and a final DNA concentration of 5 ng/µl. The reaction mixture was incubated on ice for 1 h before being mixed with freshly collected sperm cells for ICSI-mediated transgenesis.

Rhodamine labeling of the linealized EGFP plasmid was done by random, primed DNA labeling with Rhodamine-5-dUTP (Roche, Barcelona, Spain) following standard protocols. The labeled plasmid was used at a concentration of 5 ng/µl in all experiments.

Gamete Collection, Sperm Freezing, and Sperm-Transgene Mixing

Gametes for all experiments were collected from B6D2 mice. MII oocytes were collected 14 h after human chorionic gonadotropin (hCG) administration, from 6- to 8-wk-old female mice superovulated with 5 IU of equine chorionic gonadotropin, followed, 48 h later, by an equivalent dose of hCG. Cumulus cells were dispersed by 3- to 5-min incubation in M2 medium containing 350 IU/ml hyaluronidase, and oocytes were washed and maintained in potassium-modified simplex optimized medium (KSOM) medium at 37°C in a 5% CO2 atmosphere until use.

Fresh and frozen-thawed sperm was prepared essentially as described previously [32, 33], with minor differences. Briefly, fresh epididymal sperm from mature (3- to 6-mo-old) males was collected in M2 medium by excising, with a pair of fine scissors and compressing with forceps, blood and adipose tissue-free epididymal caudae. The collected sperm cells, in a minimal volume, were placed in the bottom of a 1.5-ml polypropylene centrifuge tube and overlaid with the volume of fresh medium necessary to obtain the final concentration of 2.5 million cells per ml. After gentle mixture, suspended sperm cells were immediately used for ICSI-mediated transgenesis or frozen-thawed. When the freeze-thawing of the sperm cells was required, 70- to 100-µl aliquots of this sperm suspension were transferred to cryogenic 1.5-ml vials, tightly capped, and placed directly into liquid nitrogen without complete immersion to avoid internalization of liquid nitrogen. These aliquots were stored for periods ranging from 1 day to 4 wk at –75°C. Asepsis was maintained throughout the procedure. Mixtures of equal volumes (usually 4 µl) of fresh or recently thawed spermatozoa in M2 and YAC/plasmid DNA were kept on ice for 2 min before being mixed with 40–50 µl of a 10% polyvinyl-pyrrolidone (PVP; Mr 360 000) in M2 solution, and placed on the culture dish for microinjection.

ICSI-mediated transgenesis was performed using two DNA concentrations (1 and 15 ng/µl) of the M-EGFP-M transgene, three DNA concentrations (1, 6, and 15 ng/µl) of the pEGFP transgene, and three DNA concentrations (2, 3.6, and 5 ng/µl) of the YAC construct.

Embryo Micromanipulation, Culture, and Transfer

ICSI-mediated transgenesis was done in the B6D2 hybrid mouse strain, as previously described [2, 34]. Briefly, ICSI was performed in MII oocytes with fresh or frozen-thawed spermatozoa at room temperature within 120 min of sperm-DNA mixing. One volume of sperm-DNA solution was mixed with five of M2 medium containing 10% PVP to decrease stickiness. The ICSI dish contained an injection drop (M2 medium), a sperm-DNA drop (sperm-DNA solution in M2/10% PVP), and an M2/10% PVP needle-cleaning drop. Injections were performed in an inverted microscope equipped with Eppendorf micromanipulators (Hamburg, Germany) and a PMM-150 FU piezo-impact unit (Prime Tech, Japan) using a blunt-ended mercury-containing pipette with an inside diameter of 6–7 µm. Individual sperm heads, decapitated either by the freeze/thaw procedure or mechanically with the piezo unit, were co-injected with adherent YAC/plasmid DNA into oocytes. Oocytes were injected in groups of 10. After an injection recovery period of 15 min at room temperature in M2 medium, surviving oocytes were returned to mineral oil-covered KSOM and cultured at 37°C in a 5% CO2 atmosphere. For evaluation of full-term development, embryos developing to two-cell stage were transferred to oviducts of synchronized recipient females. Embryo transfer was performed as described previously [32].

Analysis of Genomic DNA and EGFP Expression

Genomic DNA was prepared from tail biopsies following standard procedures [29], and was used for PCR or Southern blot analysis for the detection of the integrated transgene, as previously described [35, 36]. Oligonucleotides used for detecting the specific 340-bp PCR product of EGFP were: GFP1F 5'-tgaaccgcatcgagctgaagg-3'; GFP2R 5'-tccagcaggaccatgtgatcg-3'. PCR conditions were as follows: Taq polymerase (Promega, Madison, WI), 2 min at 93°C, 30 cycles of 30 sec at 93°C, 45 sec at 60°C, and 35 sec at 72°C, followed by a final extension step of 10 min at 72°C.

PCR analysis was used for the detection of yeast markers (TRP1 and LYS2) present in both vector arms of the YAC transgene. The oligonucleotides used for the detection of the specific 488-bp TRP1 and 592-bp LYS2 PCR products of the YAC hAPPy, respectively, were: trp1 5'-gcccaatagaaagagaacaattgacc-3'; trp2 5'-acacctccgcttacatcaacacc-3'; lys1 5'-accaagccagcatctgtatcacc-3'; and, lys2 5'-gccaaatccatccacttctcatc-3'. PCR conditions were as follows: AmpliTaq (Roche, France), 2 min at 94°C, 35 cycles of 30 sec at 94°C, 30 sec at 65°C, and 2 min at 72°C, followed by a final extension step of 10 min at 72°C. Genomic DNAs from unrelated YAC transgenic mice [36] were systematically included as positive controls for PCR analyses.

Analysis of EGFP expression in transgenic animals was realized by immunocytochemistry, as described by Villuendas et al. [35]. The EGFP expression was analyzed in samples from testis, kidney, heart, lung, liver, spleen, striated muscle, endothelium, and pancreas.

Statistical Analysis

Differences in the efficiency of ICSI-mediated transgenesis were evaluated by a Z-test analysis. Chi-square analysis was used for all other comparisons. SigmaStat statistical software version 3.11 (Jandel Scientific, San Rafael, CA) was used for the statistical analysis. Differences of P < 0.05 were considered significant.

RESULTS

Effect of DNA Concentration

In order to assess the impact of transgene concentration on the efficiency of mouse transgenesis production by ICSI, ICSI-mediated transgenesis was performed in the B6D2 hybrid mouse strain, with frozen-thawed sperm cells, using three DNA concentrations of a 5.4-kb pEGFP transgene, and three DNA concentrations of the 510-kb YAC (hAPPy) construct. Selection of pEGFP concentration range was done after a preliminary analysis with 25 ng/µl. With this concentration, the majority of the fetuses were resorbed, and live offspring were not obtained (Table 1). As shown in Supplementary Figure 1 (available online at www.biolreprod.org), the majority of the resorptions (80%) expressed the transgene. For this reason, in the subsequent experiments, we decided to use our standard concentration for this procedure (6 ng/µl) [2], a lower concentration, (1 ng/µl [usually used in pronuclear injection experiments]), and a higher concentration (15 ng/µl). Selection of YAC transgene concentrations were within our standard concentration previously tested with success (~4 ng/µl) [2], considering that concentrations higher than 6 ng/µl, used in preliminary experiments, resulted in embryo developmental arrest (Moreira and Gutiérrez-Adán, unpublished results).


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  		TABLE 1. ICSI-mediated transgenesis efficiency obtained for a 510-kb YAC (hAPPy) construct and a 5.4-kb plasmid EGFP transgene tested at different concentrations.


Figure 76021001
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  		FIG. 1. This figure illustrates the difference in the binding of exogenous DNA to frozen-thawed and fresh mouse spermatozoa after a 2 min co-incubation period. Representative fluorescent images of frozen-thawed mouse spermatozoa co-incubated with rhodamine-labeled DNA (A); fresh spermatozoa co-incubated with rhodamine-labeled DNA (B); and fresh spermatozoa co-incubated with RecA-complexed, rhodamine-labeled DNA (C). When frozen-thawed sperm cells are co-incubated with DNA, most sperm cell structures become labeled, including the tail (A). When fresh sperm preparations are co-incubated with DNA without RecA, just nonmotile sperm cells become fluorescently labeled, specifically in their postacrosomal and mid-piece regions (B). When fresh sperm preparations are co-incubated with RecA-complexed DNA, the majority of the cells become fluorescently labeled in their acrosomal, postacrosomal and mid-piece structures (C). Original magnification x400.

For each concentration (2, 3.6, and 5 ng/µl) of YAC DNA, 346, 350, and 224 oocytes, respectively, were used, 244 (71%), 311 (89%), and 180 (80%) survived injection, and 215, 228, and 175 reached the 2-cell stage and were transferred into pseudopregnant recipient females (Table 1). Out of these, 20 (9 %), 52 (23 %), and 30 (17 %) developed to term. When sperm microinjection was performed with 1, 6, and 15 ng/µl pEGFP DNA, 309, 219, and 313 oocytes, respectively, were used for each concentration, 233 (75%), 195 (78%), and 255 (81%) survived injection, and 194, 163, and 230 reached the 4-cell stage and were transferred into pseudopregnant recipient females. Out of these, 23 (12%), 22 (13%) and 30 (13%) developed to term. For both constructs, the three concentrations tested did not generate significant differences in oocyte survival, in vitro embryo development and proportion of live offspring. As shown in Table 1, the proportion of transgenics obtained from live offspring with 2, 3.6 and 5 ng/µl of the YAC construct was 10, 21 and 13 %, respectively. The data generated with this large construct indicated that the number of transgenic offspring, when expressed as a proportion of injected ova (efficiency rate of the procedure), increased with DNA concentration up to a threshold of 3.6 ng/µl, and indicated that higher concentrations of this transgene decreased the efficiency rate of the procedure. Interestingly, although much higher transgene concentrations were tested for the pEGFP construct, this threshold was not reached. With an increase in transgene concentration (from 1 to 15 ng/µl), the proportion of transgenics significantly increased (from 39% to 70%), as did the efficiency rate of the procedure (from 3% to 7%). As expected, the proportion of transgenic animals among live offspring and the efficiency rate of the procedure obtained with the smaller pEGFP construct were always significantly higher than the one obtained with the hAPPy transgene, even when a lower transgene concentration was used. In addition, in this study, two founders exhibiting germ line transmission of an intact and functional hAPPy transgene of 510 kb were produced (Pozueta et al., unpublished results), supporting our previous observation [2] that incorporation in the host genome and phenotypic expression of large DNA constructs of submegabase magnitude can be mediated by ICSI.

The expression of EGFP was analyzed in three transgenic lines generated by ICSI using a transgene concentration of 6 ng/µl. As shown in the Supplementary Table 1 (available online at www.biolreprod.org), the pattern of transgene expression in the organs analyzed was similar in the transgenic lines generated by ICSI and pronuclear injection [35], except that, in two of the transgenic lines generated by ICSI, EGFP expression was also detected in heart and striated muscle.

Effect of Transgene Flanking with Nuclear MARs

In our second experiment, with the objective of evaluating the impact of transgene flanking MARs on the efficiency of ICSI-mediated transgenesis, ICSI of frozen-thawed sperm cells was done with pEGFP and pEGFP flanked by two MAR elements (M-EGFP-M; 2.3 kb of the human ß-interferon domain boundaries). This comparison was made for transgene concentrations of 1 and 15 ng/µl. For each pEGFP concentration, 170 and 148 oocytes were used, respectively; 132 (78%) and 117 (79%) survived injection, and 114 and 108 reached the 4-cell stage and were transferred into pseudopregnant recipient females. When sperm microinjection was performed with 1 and 15 ng/µl M-EGFP-M DNA, 139 and 165 oocytes were used; 101(73%) and 138 (84%) survived injection, and 80 and 122 reached the 4-cell stage and were transferred into pseudopregnant recipient females. For both constructs, neither of the two concentrations tested generated significant differences in oocyte survival, in vitro embryo development, or proportion of live offspring (Table 2). PCR analysis of fetal tissue for the detection of the genomic presence of the integrated transgene at Day 14 indicated that, independently of the transgene DNA concentration tested, there was no significant difference between ICSI with pEGFP and ICSI with M-EGFP-M in the proportion of transgenics obtained and efficiency rate of the procedure (Table 2).


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  		TABLE 2. ICSI-mediated transgenesis efficiency obtained for EGFP and M-EGFP-M.a

Effect of the Use of Live or Dead Spermatozoa and RecA-Coated and Uncoated DNA

In our third and last experiment, we evaluated the effect of fresh sperm cells co-incubated with ssDNA complexed with E. coli recombinase RecA on the efficiency of mouse transgenesis production by ICSI. For this evaluation, three replicates were performed of mouse ICSI with fresh sperm cells of EGFP DNA complexed with E. coli recombinase RecA, used at a concentration of 5 ng/µl. In total, 104 oocytes were microinjected; 73 (70%) survived the micromanipulation, and 72 were transferred, giving rise to 16 (22%) live animals. Out of these, six (five males and one female) were transgenic for EGFP. The proportion of transgenic animals among live offspring obtained with this procedure was similar to that obtained for the uncoated transgene with the common ICSI-based transgenesis approach with frozen-thawed sperm cells (38% vs. 45%, respectively); no difference in the efficiency rate of the two procedures was observed either (6% vs. 5%, respectively). However, as shown in Table 3, a significant difference was observed when the germ line EGFP transmission of the six founder animals produced with fresh sperm cells and RecA-complexed DNA was compared with that of five EGFP-positive controls generated with the common ICSI-based transgenesis approach with frozen-thawed sperm cells and uncoated DNA. From the proportion of EGFP-positive animals in the F1 generation, it was clear that, while all transgenic control animals displayed a Mendelian EGFP transmission, just one parent (named Rec-9) generated by ICSI with a fresh sperm cell and EGFP DNA complexed with RecA, displayed a similar behavior. Four founders had lower than 50% transmission; two of them were significantly different from control animals. Using the additive properties of the chi-square analysis, we observed that many of the transgenic founders generated with RecA-complexed DNA displayed a significant deviation from a Mendelian transgene transmission, indicating the possibility that the integration of the transgene took place after the first embryo cleavage.


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  		TABLE 3. Summary of the meiotic EGFP transmission to the F1 generation of transgenic mice produced by standard ICSI-mediated transgenesis or by ICSI with RecA.

Concomitantly, we could observe that RecA-complexed and non-complexed rhodamine-labeled EGFP DNA attaches differently to fresh sperm cells, and that this attachment is also different from the one that occurs between RecA-free rhodamine-labeled EGFP DNA and frozen-thawed sperm cells. As shown in Figure 1, when a frozen-thawed sperm sample was co-incubated for 2 min with RecA-free rhodamine-labeled EGFP DNA, fluorescent transgene attachment was observed in most sperm cell structures (Fig. 1A). However, when a fresh sperm sample was exposed to rhodamine-labeled EGFP for the same period, a fluorescent transgene attachment was only detected in the postacrosomal region of nonmotile sperm cells (Fig. 1B). Finally, when a fresh sperm sample was co-incubated with RecA-complexed rhodamine-labeled EGFP DNA, a generalized fluorescent transgene attachment was observed in the head and mid-piece of motile and nonmotile sperm cells (Fig. 1C). In some nonmotile sperm cells, an exclusive transgene attachment to the postacrosomal region was also observed, probably due to poor RecA-coating of labeled DNA. The same pattern of rhodamine-DNA-labeled sperm was also observed after 120 min of sperm and rhodamine-DNA co-incubation.

DISCUSSION

The results of our ICSI-mediated transgenesis assays with different concentrations of plasmid DNA suggest that ICSI allow higher transgene concentrations than traditional pronuclear microinjection. However, even with ICSI, when a certain threshold is bypassed, the viability of the embryos/fetuses decrease dramatically. This effect might result from the high efficiency of transgene integration obtained by ICSI-mediated transgenesis [1, 2, 34], which may lead to chromosomic alterations in microinjected embryos. It has been reported that incubation of spermatozoa with exogenous DNA leads to paternal chromosomal breaks [31, 37]. The decrease in the frequency of normal karyoplates at blastocyst stage was reported to be 2-fold when ICSI-mediated transgenesis was executed with frozen-thawed spermatozoa incubated with 5 ng/µl of pEGFP [31]. These results suggest that incorporation of transgenic DNA into the embryonic genome by ICSI-mediated transgenesis may be directly correlated with paternal chromosome breaks. The relationship between transgene concentration and transgenesis efficiency was similar for the YAC constructs tested. When the YAC construct was used, the efficiency rate of the procedure increased with DNA concentration up to a threshold of 3.6 ng/µl, decreasing when a higher transgene concentration was used. Interestingly, this threshold for the plasmid construct was observed with much higher DNA concentrations. Considering that the kilobase magnitude of pEGFP is about 100 times smaller that of the YAC tested, these observations suggest that, for smaller constructs, higher transgene concentrations may be used in order to ensure optimal ICSI-mediated transgenesis results. Moreover, because small plasmids and large YACs require different preparation methods, our results might also reflect a difference in the concentration of copurified contaminants in YAC and plasmid DNA samples.

The results of our experiment also indicate that the integration rate after ICSI-mediated transgenesis is different from what was described by Brinster et al. for pronuclear injection [11], which seems to remain constant when DNA is injected at concentrations above 1 ng/µl. According to our results with pEGFP, the efficiency of the ICSI-mediated transgenesis procedure with plasmid constructs may be significantly improved by increasing the transgene concentration up to values significantly higher than those commonly used for pronuclear injection. In this study, the efficiency of the ICSI-mediated transgenesis procedure with pEGFP was significantly enhanced by increasing the transgene presence up to concentrations as high as 15 ng/µl. These results suggest that the opportunities for transgene integration might be far superior when the transgene is delivered by ICSI rather than by pronuclear injection. These observations can be explained by considering that, in ICSI-mediated transgenesis, foreign DNA molecules are present during several steps of sperm nucleus transformation into a functional pronucleus, which is concomitant with important chromatin reorganization events, such as phosphorylation and replacement of sperm-specific histones by maternal ones [38]. In contrast, in transgenesis procedures involving pronuclear injection, foreign DNA molecules are delivered in a completely formed pronucleus after completion of the majority of sperm nucleus remodeling events. This may also explain the much higher frequency of founder animal mosaicism obtained as the result of late transgene integration (after first mitosis), when the transgene is introduced by pronuclear injection, than when it is delivered by ICSI. In our experience, most ICSI-generated founders do not display mosaicism, indicating that the integration of the transgene took place before first embryo division. In addition, and based on our experience with these techniques, our results also seem to indicate that the toxicity of the transgene is lower when the transgene is delivered by ICSI than when it is delivered by pronuclear injection. These observations can be explained by considering that, in ICSI-mediated transgenesis, the sperm cell-transgene mixture is delivered intracytoplasmically, while, during pronuclear injection, a volume of transgenic DNA is directly delivered in one of the pronuclei.

In our second experiment, when the efficiency of ICSI-mediated transgenesis with pEGFP and pEGFP flanked with MAR elements was compared, significant differences were not found independently of the transgene concentration used (either 1 or 15 ng/µl). These results indicate that, regardless of the findings of a previous report [21] demonstrating interaction between exogenous DNA molecules and the nuclear matrix of mouse spermatozoa, the impact of transgene flanking with MARs on the efficiency of this procedure is not significant. Our results also suggest that the mechanism of transgene integration occurring after ICSI might not involve homologous recombination events mediated by MAR sequences. It will be worth evaluating, however, if transgene flanking with MARs can lead to enhanced and persistent transgene expression after ICSI, as it does in transgenesis procedures involving pronuclear injection [39].

In our last experiment, we found that, although the transgenesis efficiency of the common ICSI-based transgenesis procedure with frozen-thawed sperm cells is similar to that of ICSI with fresh sperm cells and RecA-complexed DNA, the traditional ICSI-mediated transgenesis procedure with frozen-thawed spermatozoa is much more efficient in maintaining a low frequency of founder animal mosaicism. These results suggest that transgene integration takes longer when RecA and fresh sperm cells are used for ICSI-mediated transgenesis, resulting more frequently in delayed transgene integration (after completion of first mitosis). It is known that the regulated damage of sperm heads, by freeze-thaw cycles or exposure to detergents, is critical to improving the production of transgenic offspring with this transgenesis procedure [1]. This improvement comes, possibly, through the facilitation of the sperm cell-transgene interaction after membrane fracture and/or by induction of small sperm DNA breaks that, when combined with the oocyte's DNA repair mechanisms, result in higher transgene integration rates. E. coli RecA is a bacterial recombinase that, by binding and coating the ssDNA, has been shown to protect it from shearing and degradation [23]. During previous pig and goat pronuclear microinjection transgenesis attempts, this protective mechanism of RecA has also been suggested to influence the transgene DNA stability [24, 25]. When ICSI-mediated transgenesis is performed with live sperm cells, RecA may facilitate transgene integration, first by protecting the DNA from nucleases present in the sperm solution before sperm injection, and second by protecting it from the intracellular oocyte degradation that usually occurs in its absence (Table 4). In contrast, or concomitantly, RecA may also be interfering with the structural opening of DNA strands that is necessary for the insertion of foreign DNA molecules. However, at present, we cannot discriminate whether the delayed transgene integration observed after ICSI with fresh sperm cells of DNA complexed with RecA is a consequence of the use of the bacterial recombinase, or of the use of intact sperm cells, or both. Future experiments are presently being planed in order to clarify this issue.


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  		TABLE 4. Comparative of tested mouse ICSI-mediated transgenesis procedures with live and frozen-thawed sperm cells.

A concern about the use of ICSI with frozen-thawed DNA-coated sperm to generate transgenic mice is whether transgene expression is similar to that obtained by pronuclear microinjection. We have done this comparison using three of the transgenic EGFP lines generated by ICSI with frozen-thawed DNA-coated sperm and three transgenic lines for the same construct generated by pronuclear microinjection [35]. In this study, we demonstrate that the transgenic lines generated by ICSI with frozen-thawed DNA-coated sperm follow the same pattern of expression observed in the transgenic lines produced by pronuclear microinjection. In relation to the YAC expression in the transgenic offspring generated by ICSI, we have reported correct phenotypic expression of large YAC DNA constructs using this procedure [2]. Our results demonstrate that ICSI with frozen-thawed DNA-coated sperm is at least as efficient as pronuclear microinjection in mediating transgene expression in mice.

The general purpose of this study was to optimize and understand the mechanism of transgenesis production by ICSI in mice, specifically to evaluate the impact of transgene DNA concentration, transgene flanking with MARs, and the use of RecA-coated DNA on the efficiency of this procedure. Collected data suggested that:

1) ICSI allows the use of higher plasmid DNA concentrations than those used for pronuclear microinjection; however, when a certain threshold is exceeded, the viability of the embryos/fetus decreases dramatically. Moreover, for genomic-type transgene preparations, such as YAC DNA (which usually still carry traces of contaminants after purification), concentrations higher than 4 ng/µl may reduce the transgenesis efficiency.

2) Regardless of previous reports demonstrating interaction between exogenous DNA molecules and the nuclear matrix of mouse spermatozoa, the impact of transgene flanking with MARs on the integration efficiency of this procedure is not significant.

3) ICSI with fresh sperm cells of RecA-complexed DNA is not as efficient as the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa in maintaining a low frequency of founder animal mosaicism.

4) The expression pattern of the transgenic offspring generated by ICSI with frozen-thawed DNA-coated sperm displays the same pattern observed in the transgenic offspring generated by pronuclear microinjection.

The information gathered here is particularly relevant for the application of this procedure in the mouse species, but it will be important to determine if it is also valid for other mammalian species.

ACKNOWLEDGMENTS

We would like thank to Dr. Belen Pintado for her help with the scientific revision of the final manuscript.

FOOTNOTES

1Supported by grants AGL2003-05783 from the Ministerio de Educación y Ciencia and CARM BIO2005-01-6463 from the Comunidad Autónoma de Murcia to A.G.-A., and by grant BIO2003-08196 from the Ministerio de Educación y Ciencia to L.M. Back

Correspondence: 2FAX: 34 91 347 4014; e-mail: agutierr{at}inia.es

Received: 1 September 2006.

First decision: 1 October 2006.

Accepted: 6 October 2006.

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