Heat-induced and spontaneous expression of Hsp70.1Luciferase transgene copies localized on Xp22 in female bovine cells

Background Expression of several copies of the heat-inducible Hsp70.1Luciferase (LUC) transgene inserted at a single X chromosome locus of a bull (Bos taurus) was assessed in females after X-chromosome inactivation (XCI). Furthermore, impact of the chromosomal environment on the spontaneous expression of these transgene copies before XCI was studied during early development in embryos obtained after in vitro fertilization (IVF), when the locus was carried by the X chromosome inherited from the bull, and after somatic cell nuclear transfer (SCNT) cloning, when the locus could be carried by the inactive Xi or the active Xa chromosome in a female donor cell, or by the (active) X in a male donor cell. Findings Transgene copies were mapped to bovine Xp22. In XXLUC female fibroblasts, i.e. after random XCI, the proportions of late-replicating inactive and early-replicating active XLUC chromosomes were not biased and the proportion of cells displaying an increase in the level of immunostained luciferase protein after heat-shock induction was similar to that in male fibroblasts. Spontaneous transgene expression occurred at the 8-16-cell stage both in transgenic (female) embryos obtained after IVF and in male and female embryos obtained after SCNT. Conclusions The XLUC chromosome is normally inactivated but at least part of the inactivated X-linked Hsp70.1Luciferase transgene copies remains heat-inducible after random XCI in somatic cells. Before XCI, the profile of the transgenes' spontaneous expression is independent of the epigenetic origin of the XLUC chromosome since it is similar in IVF female, SCNT male and SCNT female embryos.


Background
Menck and colleagues have reported a luminescent screening system based on the integration of a transgene composed of scaffold attachment regions flanking the murine HSP70.1 gene promoter linked to firefly luciferase cDNA [1]. Among the transgenic fetuses obtained, one male carried a cluster of 20 to 30 copies of the transgene [1]. Later, somatic cell nuclear transfer (SCNT) cloning with cells from this fetus generated a healthy and fertile bull for which we have localized the transgenic cluster on the X chromosome (this report). Thus an interesting animal model was available to investigate the inactivation/activation status of transgenes in bovine female fetuses from this bull. Indeed, dosage compensation between male and females is achieved after X-chromosome inactivation (XCI) in mammalian female cells, i.e. one of the two X, the inactive X (Xi) chromosome, is in great part transcriptionally silent [2,3]. At least in domestic mouse, XCI occurs in two waves early during development (reviewed in [2,3]). First, both X chromosomes are transcriptionally active during a short developmental window of the cleavage phase. Then, most studies agree that the paternally inherited X P chromosome becomes inactivated by the blastocyst stage [2,3] and also in most placental cells. In the epiblast cells, both X chromosomes are again transiently active, before random XCI during gastrulation [4]. This results in a mosaic of two somatic cell types expressing X-linked genes inherited either from the mother or the father. Some X-linked genes maintain bi-allelic expression in female cells [2,3]. On the human submetacentric X chromosome, 5% [5] to 15% [6] of the genes escape inactivation; they are preferentially found in clusters and more frequently on the short arm than on the long arm [6,7]. On the mouse acrocentric X chromosome, homologs of the human genes escaping inactivation are mostly inactivated and only two non-clustered genes with no homolog on the Y have been shown to escape inactivation [8]. Thus, the phenomenon of inactivation escape may depend on genomic context, including either the absence of sequence elements necessary for silencing spreading [9,10] or the presence of insulators/ barriers that prevent XCI-coupled silencing [9,11].
Similarly, XCI-related silencing of X-linked transgenes may depend on the insertion site, the transgene's intrinsic properties or other unknown factors. Furthermore, it may vary between cell lineages or during development ( [11][12][13][14][15] and references therein), as reported for 10% of the human X-linked genes [6,16] and one mouse Xlinked gene [17].
Expression of an X-linked transgene has rarely been observed during early development and only in the domestic mouse [15,18] in which surprisingly, one Xlinked transgene has been shown to display delayed expression when paternally inherited [15].
Analysis of SCNT cloned embryos can provide further insight on how XCI influences gene expression. For an Xi-associated transgene, silencing reversion has been reported in SCNT cloned early mouse embryos [18] but for an autosomal insertion, transgene-related and/or position effect-related silencing was found unchanged in SCNT cloned cattle [19].
Indirect evidence suggests that bovine and mouse XCI profiles are quite similar. De La Fuente and colleagues [20] have shown that in some cells from bovine embryos the two X chromosomes replicate asynchronously, one early and one late in S phase, thus XCI is established at the blastocyst stage 7 days after in vitro fertilization (IVF) in cattle. Indeed, late replicating regions including the Xi are generally transcriptionally inactive while early-replicating regions including the Xa are generally transcriptionally active (except in the mouse immediately after imprinted XCI; [3]). Furthermore, two reports clearly indicate that the paternally-inherited X is preferentially inactivated in the placenta [21] or in the chorion only [22], suggesting that imprinted XCI takes place earlier in the associated cell lineage(s), i.e., at least in the trophoblast, at the blastocyst stage. In somatic bovine cells, both X are inactivated, [21,22], suggesting that XCI occurs randomly in the bovine epiblast during gastrulation as in the mouse. To date, it has not been established whether some genes escape inactivation on the bovine X.
Several studies have shown that the expression of the Hsp70.1Luciferase transgene mimics that of the murine Hsp70.1 gene, i.e. the level of luciferase activity increases after heat-shock (HS) induction in both mouse and bovine transgenic embryonic and somatic cells and also spontaneously during embryonic genome activation (EGA) in early mouse transgenic embryos ( [1] and references therein). To investigate whether some copies of the X-linked transgene remained inducible, we first analyzed the HS-induced luciferase activity and/or protein level in transgenic female somatic cells and blastocyst embryos. Second, we took advantage of the spontaneous activity of the transgene in early embryos before XCI and the relative success of SCNT cloning in cattle [23] to measure the influence on gene expression of the chromosomal environment inherited from spermatozoa or from male and female cells in early IVF embryos and SCNT embryos respectively.

Materials and methods
All samples were generated according to the International Guiding Principles for Biomedical Research involving animals of experimental farms. The research work on cloned animals was approved by COMEPRA (Ethical and Precaution Committee for Agronomic Research Application) in December 1999.
The remaining of this section is found in [additional file 1: Material and methods].

Results and discussion
Transgenes are located on the X chromosome of the transgenic bull After hybridization with a probe specific for the whole transgene, a unique strong signal was observed on the short arm of the bovine submetacentric X in chromosome preparations from the transgenic bull, precisely in the early-replicating R band Xp22 ( Figure 1A). The bovine Xp region is conserved with part of the human Xq [24,25] while the human Xp22 region, in which about 30% of the genes may escape inactivation [6], is conserved with part of the bovine Xq [24].

In XX LUC somatic female cells inactivation frequencies of both X are similar
Since XCI occurs randomly in the somatic bovine lineages [21,22], about half of the transgenic female somatic cells are expected to have an active X LUC chromosome inherited from the bull. Presence of a strong bias would indicate preferential inactivation of one of the X or preferential survival of the cells that inactivate one X. Although normal random XCI is reported for mouse and bovine clones [18,21,22,26], it was important to check whether this was the case in IVF females carrying the X of the transgenic bull since the bull was obtained by SCNT cloning, a technique which can result in developmental anomalies generally associated with abnormal epigenetic processes [23].
Analysis of metaphase chromosome spreads prepared from synchronized female XX LUC fibroblast cells cultured in the presence of BrdU during late S phase showed no bias. An equivalent number of BrdU-labeled X chromosomes or inactive Xi (N = 22) and partly BrdU-labeled X chromosomes or active Xa (N = 21) carried copies of the transgene. Furthermore, the transgenes' presence had no visible influence on Xp22 inactivation/activation since the X LUC p22 region replicates late on the Xi ( Figure 1B) and on the normal X [27]. Thus, inactivation of the X carried by the bull's sperm is normal, which indicates that the presence of multiple copies of the transgene on either Xi or Xa is not toxic (counter-selected) to cell physiology and does not interfere with random XCI.
Proportions of cells expressing the luciferase protein after heat-shock induction are similar in X LUC Y male and XX LUC female population of cultured fibroblasts We compared the level of luciferase activity and protein after heat-shock (HS) induction in somatic female cells derived from two fetuses generated by IVF with the sperm of the transgenic original bull and referred to as F616 and BSF731 cells, and in male cells from the original transgenic bull (referred to as OV7060 cells). HS-induced luciferase activity ranged from 0.99 × 10 6 RLU.μg protein -1 .min -1 in female BSF731 fibroblast cells to about 2.60 × 10 6 RLU.μg protein -1 . min -1 in both male OV7060 and female F616 fibroblast cells (Table 1), corresponding to a many-fold increase in the three cell populations. Since in the BSF731 cells the numbers of Xi LUC and Xa LUC are similar after random XCI (see above), we compared the proportions of luciferase-positive cells after HS induction and immunostaining in this female cell population and the male OV7060 cell population (Table 1; Figure 2) They were very similar (Figure 2 and [additional File 2 Additional data]). It indicated that the X-linked luciferase transgene was expressed in most cells, strongly suggesting that at least some transgene copies remain active on the Xi in BSF731 cells. In tissue biopsies of three fetal organs tested from the F616 female fetus, a strong increase in the level of luciferase activity after HS-induction was observed (Table  2). Overall, these data strongly suggest that the presence of 20 to 30 transgene copies [1] does not prevent the HS-induced expression of at least some of them in the   During normal development, between the one-cell and the 4-8-cell stages, the level of luciferase activity was null in at least 80% of the IVF and SCNT transgenic bovine embryos and very low in the remaining 20% ( Figure 3; Table 3). Since the sperm used here was obtained from a bull hemizygous at the X-linked transgenes, statistically half of the IVF embryos, all female, should be transgenic. As expected, about 50% of the "8-16-cell" IVF embryos displayed a high level of luciferase activity at days 3 and 4, suggesting that the transgenes are expressed in most female IVF embryos. At the morula stage, 32% of the IVF embryos remained luciferasepositive but the level of luciferase activity per embryo had already decreased significantly (P < 0.05). At the blastocyst stage, less than 10% of the IVF embryos displayed luciferase activity, and all at a low level. Moreover, the percentages of luciferase-positive embryos differed significantly between the 8-16-cell stage and the 4-8-cell or blastocyst stages in the three embryo types (Table 3). Before nuclear transfer, the BSF731 female donor cells carried the transgenes either on the Xa or the Xi with a similar probability (see above). However, in female BSF731-derived SCNT embryos, we found no evidence for two sub-populations displaying two different levels and patterns of luciferase activity and the standard error to the mean was similar to that observed in the two Table 2 Rate of increase in luciferase activity in fetal and placental female tissues after heat shock a

Origin of biopsy b
Rate of increase after HS c

Placenta 38
Heart 141 Muscle 282 Lung 2930 a the level of specific luciferase activity (RLU.μg protein -1 .min -1 ) was measured in explants of the transgenic female F616 fetus after HS induction and compared with the level measured in non-induced explants. b all biopsies were recovered from the transgenic female F616 fetus. c average value based on the measurement of luciferase activity on two independent extracts for each tissue and each condition. other embryo types (Table 3). This indicates that after cloning, the number of active genes was similar in these female SCNT embryos as expected if their XCI-dependent silencing in the donor cell was not achieved (as suggested from Table 1; Figure 2) or was reverted after cloning [18]. In turn, the variation in HS response of BSF731 fibroblast cells may result either from a variable number of inactivated transgenes on the Xi or from a variable HS response in the BSF731 cell population.
Since the Hsp70.1Luciferase transgene can be similarly active at the 8-16-cell stage in both IVF and cloned embryos, this further suggests that neither the paternal origin of the transgenic X LUC in the case of IVF embryos, nor the origin, male or female, of the somatic cells, in the case of SCNT embryos, prevented spontaneous, oocytedriven, expression of the X-linked transgenes.
We detected an increased level of luciferase activity after heat-shock induction in IVF female blastocysts [additional File 3 Additional Table S1] and placental tissue ( Table 2) in which imprinted XCI, i.e. inactivation of the paternallyinherited X LUC , is expected. However, we cannot yet conclude whether the transgenes escaped inactivation after imprinted XCI since the presence of cells in which XCI had not occurred or had occurred randomly is likely.
In conclusion, using a species other than mouse and different approaches we have investigated the expression of an X-linked transgene to determine its innocuousness as well as that of the transgene insertion site, and to test its sensitivity to XCI-dependent or -independent silencing. The results indicate that the transgenic X inherited from the cloned bull is normally inactivated/activated in somatic female cells and that at least some of the transgene copies at this locus escape XCI-coupled silencing in these cells. Whether this is due to HS-dependent [28,29] or HS-independent [30] properties of the transgene, to the insertion site and/or to the creation of a new genomic environment remains to be determined. Authors' contributions DLB and XV generated the bovine bull; XV and JLS generated cell cultures, JML, AB, MC, HH and JLS generated IVF embryos, performed FISH and immuno-detection, and measured luciferase activity and protein concentrations; DLB generated SCNT embryos, JML designed the experiments, performed statistical tests and wrote the draft. HH and XV edited the draft and all authors agreed on the final version of this report.

Competing interests
The authors declare that they have no competing interests.