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Reproductive Technology |
Departamento de Reproducción Animal,5 INIA, 28040 Madrid, Spain
Department of Molecular and Cellular Biology,6 Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, 28049 Madrid, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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artificial chromosomes, assisted reproductive technology, early development, gamete biology, gene regulation, genomic locus, intracytoplasmic sperm injection, in vitro fertilization, sperm-mediated gene transfer, transgene integrity, tyrosinase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100 to more than 1000 kilobases [kb], depending on its type) usually ensures faithful expression pattern of the transgene due to the inclusion of most regulatory elements responsible for the appropriate expression of the gene within these large constructs [1, 2]. Further, the availability of YAC- and BAC-integrated maps, for the mouse [3] and rat [4] genomes, along with their corresponding whole-genome sequences, makes them excellent candidates for gene function/rescue of mutation-type analyses in transgenic animals [i.e., 5, 6]. The introduction of YACs into the germline of transgenic animals by conventional, pronuclear (PN) microinjection, while feasible, remains technically difficult, even though it was first described 10 yr ago [7, 8]. Other methods have been described to generate YAC-transgenic animals, such as lipofection of YAC DNA into embryonic stem [ES] cells or fusion of yeast cells (spheroplasts) with ES cells (reviewed in [1]). However, these two ES cell-mediated methods require the more time-consuming generation of chimeras and, regarding the fusion with yeast cells, the integration of the entire yeast genome along with the YAC has to be considered. YAC transgenesis by PN microinjection typically produces founder mice with an efficiency of <5%, which is lower but comparable with that observed with plasmids [1, 2]. However, the generation of YAC transgenic mice for a given YAC by PN microinjection is often problematic (unpublished results), and it is likely to correlate with DNA batches and purity of YAC DNA samples. Therefore, the generation of new YAC transgenic animals, potentially of great scientific value, may have been hindered by currently available, inefficient procedures.
The first indication that exogenous DNA could be introduced into untreated sperm was provided in 1971 [9]. Next, it was reported that live mouse spermatozoa incubated with exogenous DNA could result in transgenic offspring [10]. This pioneer study triggered initial contradictory reports [11] that have fed an intense debate, not yet finished within the field [12–14]. Ten years later, a new technique designated metaphase II transgenesis, involving the previously described methodology of intracytoplasmic sperm injection (ICSI) [15], was shown to mediate mouse transgenesis at high efficiency [16]. Interestingly, the regulated damage of sperm heads (by freeze-thaw cycles or exposure to detergents) was found critical to produce transgenic offspring. Recently, this method was applied to bacterial and mammalian artificial chromosomes (BAC and MAC, respectively) ranging from 11.9 to 170 kb [17]. However, it remained to be demonstrated that large DNA constructs could be stably introduced in the mouse genome using sperm as a vector and that these constructs would be integrated intact. The use of large DNA constructs (harboring genomic inserts 100–1000 kb in length), such as YACs, increases the probability of mechanical shearing of DNA and, hence, partial transgene integration [18, 19]. For this reason, the efficiency of a particular method of transgenesis cannot be fully evaluated unless it is ascertained the integration of the intact construct in the genome of the host and its transmission to the offspring.
In this report, we have addressed the use of YACs, which are capable of mobilizing genomic fragments 100– 2000 kb in length [20, 21], for the efficient production of transgenic animals with large DNA constructs. Moreover, we have compared the efficiency of YAC transgenesis by PN microinjection with that of ICSI-mediated YAC transfer (IMYT) for a construct of 250 kb. This YAC transgene carried a functional tyrosinase locus, similar to previous YAC tyrosinase constructs used for the generation of transgenic mice [19]. The tyrosinase gene encodes the key enzyme for the biosynthesis of melanin, and its absence or mutation results in a syndrome known as oculocutaneous albinism type I. A number of tyrosinase constructs have been used to rescue the albino phenotype of recipient mice in transgenic experiments [22], including a standard tyrosinase construct, used as a reporter gene in animal transgenesis [23]. Indeed, the size of the mouse tyrosinase gene, including regulatory regions (a minimum of 100 kb) [19], makes it an excellent candidate for reporter purposes of large constructs because only the integration of the intact gene is likely to result in a functional transcript, and hence, pigmentation.
In this study, we describe for the first time stable incorporation and phenotypic expression of constructs with submegabase magnitude mediated by ICSI. By coinjecting spermatozoa and YACs into metaphase II (MII) oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kb.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals and media were purchased from Sigma Chemical Co. (Alcobendas, Madrid, Spain) unless otherwise stated.
Animals
Albino outbred CD1 mice (Harlan Iberica SL, Barcelona, Spain) were used as donors of oocytes and sperm for ICSI experiments. Females were 6–8 wk old at the time of the experiments, and males at least 3 mo old. CD1 females were also used for surrogate mothers for embryo-transfer experiments, mated with vasectomized CD1 males. 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 and Use Committee guidelines and in adherence with guidelines established in the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the Society for the Study of Reproduction.
DNA
YAC construct YRT2
LCR (unpublished) was used for IMYT and PN microinjection. DNA from the C3H mouse strain was used to prepare the original YRT2 YAC, as reported previously [7, 8]. A detailed description of YAC YRT2LCR will be published elsewhere. In brief, deletion of the LCR fragment [19] was achieved in the YRT2 parental YAC by homologous recombination techniques in yeast cells, using the pop-in/pop-out method, as described previously [24]. Thereafter, YAC DNA was prepared and purified following reported methods [8, 25], resulting in a DNA concentration of 15 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) [18]. The integrity of YAC DNA preparation was verified by pulsed-field gel electrophoresis. Subsequently, 1/10 to 1/20 YAC DNA dilutions in the YAC-microinjection buffer were used for PN microinjection and 1/4 dilutions for IMYT. The same batch of YAC DNA was used for PN microinjection and IMYT to eliminate possible differences that may occur during purification.
The efficiency of transgenesis obtained for this YAC construct was also compared with that obtained by ICSI-mediated transgenesis of a plasmid EGFP (5.4 kb, pEGFP-N1, Clontech Laboratories, Inc., Palo Alto, CA), containing the human cytomegalovirus immediate early promoter and the enhanced green fluorescent protein (GFP) gene. EGFP was linearized with Afl II prior to use.
Gamete Collection, Sperm Freezing, and Transgenic Mixing
MII oocytes were collected 14 h post-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 human chorionic gonadotropin. Cumulus cells were dispersed by a 3- to 5-min incubation in M2 medium containing 350 IU/ml hyaluronidase, and oocytes washed and maintained in K+ modified simplex optimized medium (KSOM) [26] at 37°C in a 5% CO2 air atmosphere until use.
Freeze-thawed sperm was prepared essentially as described [27], with minor differences. Briefly, 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 milliliter. The sperm extender used (M2 medium) did not contain Ca2+ chelating agents, such as EDTA or EGTA. After gentle mixing, 70- to 100-µl aliquots of the sperm suspension were transferred to cryogenic 1.5-ml vials, tightly capped, and directly placed into liquid nitrogen without complete immersion to avoid internalization of liquid nitrogen. Sperm samples 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 freshly thawed spermatozoa in M2 and 4 ng/µl YAC DNA (4–8 ng/µl plasmid EGFP) 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 in the culture dish for microinjection.
Embryo Micromanipulation, Culture, and Transfer
Mouse pronuclear injections were performed as previously described [7, 8, 19, 25]. ICSI-mediated YAC transfer with frozen-thawed spermatozoa was performed in M2 medium at room temperature within 120 min of sperm-DNA mixing. One volume of sperm-YAC solution was mixed with five of M2 medium containing 10% PVP to decrease stickiness. The ICSI dish contained a manipulation drop (M2 medium), a sperm-YAC drop (sperm-YAC solution in M2/10% PVP), and a M2/10% PVP needle-cleaning drop. Injections were performed with a PMM-150 FU piezo-impact unit (Prime Tech, Japan) using a blunt-ended mercury-containing pipette with 6–7 µm of inner diameter. Individual sperm heads decapitated by the freeze/thaw procedure were coinjected with adherent YACs into oocytes. Oocytes were injected in groups of 10. After 15 min of recovery at room temperature in M2 medium, surviving oocytes were returned to mineral oil-covered KSOM and cultured at 37°C in a 5% CO2 air atmosphere. For full-term development (of both one- and two-cell embryos, pronuclear injected plasmid, or ICSI-fertilized and pronuclear-injected YAC), embryos were transferred to oviducts of recipient females. Embryo transfer was performed as described previously [27, 28].
Analysis of Genomic DNA
Genomic DNA was prepared from tail biopsies following standard procedures [25] and used for PCR or Southern blot analysis, as described [7, 19]. Oligonucleotides used for detecting the specific 340-base pair (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.
For IMYT experiments, PCR analysis was used for the detection of yeast markers (TRP1 and LYS2) present in both vector arms of the YAC transgene. Oligonucleotides used for detecting the specific 488-bp or 592-bp TRP1 and LYS2 PCR products of YAC YRT2LCR, 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), 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 [19, 22] were systematically included as positive controls for PCR analyses. For Southern blot analysis of YAC transgenes. the internal 3'LCR probe has been reported before (probe E5; [29]) and results in a Hind III polymorphic pattern that allows clear distinction between the transgenic YAC DNA (6.5 kb) and the endogenous tyrosinase locus (1.2 kb).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LCR, with a size of 250 kb, was used for PN microinjection and IMYT transgenic experiments (Fig. 1a). In IMYT experiments, 367 MII oocytes were microinjected, 252 (69%) survived injection, and 201 (80%) cleaved to the two-cell stage. In PN microinjection experiments, 1733 pronuclear-stage oocytes were microinjected, 1219 (70%) survived injection, and 959 (79%) cleaved to the two-cell stage. Similar values were obtained for ICSI assays with a 5.4-kb EGFP standard transgene (Table 1). The absence of significant differences in embryo survival and progression to the two-cell stage suggests that embryo manipulation and the method and timing of transgene administration do not significantly impact early embryo viability. Transfer of 163 (EGFP), 218 (IMYT), and 959 (PN microinjection) embryos produced 149 offspring. The integrity of genomic integrants was evaluated by PCR and Southern blot analysis for YRT2LCR and by PCR analysis for EGFP. Of 34 offspring generated by IMYT, 12 (35%) were shown to carry transgene DNA (Table 1). This proportion was significantly different from that obtained by PN microinjection of YRT2LCR, in which only one (1%) of the 93 offspring produced by this method carried transgene DNA. Of 22 live offspring in the EGFP series, 10 (45%) carried the transgene. This efficiency is comparable with that of IMYT. Considering that EGFP is a transgenic construct about 50 times smaller than YRT2LCR, the transgenesis obtained by IMYT becomes even more impressive. It is important to note that it is the mass (or length) of the DNA, not its molarity, that is important in ICSI-mediated transgenesis and predicts the feasibility of introducing megabase-size transgene constructs [1, 2, 21]. To assess germline transmission, three IMYT-produced founders were crossed with nontransgenic mice. All transmitted transgene DNA to their progeny, approximating Mendelian genetics, assuming single or closely linked integration sites: founder 1, 12 out of 26; founder 3, 5 out of 15; and founder 6, 11 out of 17 offspring, received the YAC transgene from their respective transgenic progenitor. Besides the huge difference in size, this rate of YRT2LCR germline transmission is comparable with that previously reported for EGFP [16] by an analogous method, attesting the stability of YAC transgene integrants. Most of the IMYT-derived transgenic animals contained integrations of only part of the YAC transgene construct (Table 2). However, the presence of terminal or internal markers (alone or in combination) was, in any case, considerably higher than that in the offspring produced by PN microinjection, where only a single terminal yeast marker was detected.
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Of the IMYT-derived transgenic offspring, 1/12 (8%), corresponding to founder 1 (Table 2), apparently harbored an intact copy of the 250 kb YAC, thus supporting the value of our improved ICSI-derived methodology for the generation of transgenic animals with large DNA constructs. This was confirmed, physically, with three well-separated transgene markers (Fig. 1a and Table 2) and, functionally, by a coinherited coat color characteristic of tyrosine YAC transgene expression (Fig. 1b). All three YAC transgene-specific DNA markers and the characteristic pigmented phenotype were observed in the offspring of founder 1. The YAC YRT2LCR construct carries a tyrosinase gene in which its locus control region (LCR) has been experimentally deleted, resulting in variegated expression in skin, coat, and eyes, as reported previously with analogous YAC constructs in mice [28], generated by PN microinjection [8, 19]. This phenotype is recapitulated here as heavily pigmented eyes, with clear but low expression in skin (pigmented patches can be found in ears) and hair (very light gray coat color) (Fig. 1b).
Although we are consistently and efficiently able to generate transgenic offspring via PN microinjection [reviewed in 22; unpublished results], we were unable to use the PN microinjection method to produce a YRT2LCR transgene-expressing mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10 times narrower than that of IMYT pipettes (7 µm). Larger pipettes ease handling of, and likely reduce damage to, very large DNA fragments. We do not have sufficient information, however, to explain why PN microinjection did not produce the higher levels of partial YRT2LCR integration observed with IMYT. It is possible that remodeling within an MII oocyte of a sperm nucleus delivered by ICSI generates open chromatin and thereby opportunities for large DNA fragment integration, which do not occur at the pronuclear stage. It is also possible that our modified sperm-freezing procedure, executed in the absence of calcium-chelating agents, such as EDTA or EGTA, which inhibit chromosome damage during freezing [30] and are commonly used in conventional ICSI-mediated transgenesis, results in a higher level of sperm DNA exposure [30] and/or breakage, which might facilitate transgene DNA integration (as observed in this study after ICSI-mediated transfer of YAC YRT2LCR and plasmid EGFP transgenic constructs).
We have here described the high-efficiency introduction by IMYT of a 250-kb YAC transgene, YRT2LCR. Expression of the transgene product, tyrosinase, resulted in the rescue of the albino background of the host. The pigmented phenotype was associated with the presence of an intact copy of the transgene, which was expressed and stably maintained in the offspring. To our knowledge, this is the first report that shows intact integration, correct phenotypic expression, and germline transmission of a construct of submegabase magnitude, such as a YAC, mediated by ICSI.
These data clearly demonstrate the YAC transgenesis by IMYT. Moreover, they indicate that the ability to generate functional transgenic animals with large YAC constructs via ICSI is greater than that of PN microinjection. Most emphasis should be placed on the fact that only one half as many sessions and a fifth as many oocytes were needed to generate 12 IMYT transgenics than the ones used to generate a single transgenic animal by PN microinjection. IMYT is poised to enable efficient, reproducible, and stable integration of even larger DNA constructs that will be of increasing importance in the manipulation and study of large genes and gene clusters in biomedicine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Alfonso Gutiérrez-Adán. FAX: +34913474014; agutierr{at}inia.es
3 Correspondence: Lluís Montoliu. FAX: +34915854506; montoliu{at}cnb.uam.es
4 Current address: Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro, 3. 28029 Madrid, Spain
Received: 8 June 2004.
First decision: 1 July 2004.
Accepted: 23 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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