METHOD FOR RECONSTRUCTING A NON-HUMAN ANIMAL EMBRYO

Abstract

The present invention refers to a method for reconstructing a non-human animal embryo, to a method for generating a nonhuman animal and to a non-human animal or non-human animal reconstructed embryo obtainable by the methods according to the invention. The method of somatic cell nuclear transfer (SCNT) has a step of transiently expressing or inducing the uptake of a protein involved in male sperm maturation, such as protamine or transition protein, in the somatic donor cell before nuclear transfer.

Claims

1. A method for reconstructing a non-human animal embryo, comprising: a) transiently expressing into a non-human donor cultivated cell a protein involved in male sperm maturation or a synthetic or recombinant portion thereof able to confer to the chromatin of said cell a spermatozoa like chromatin conformation; or a) inducing the uptake from the cultivating medium of a non-human donor cultivated cell of at least a protein involved in male sperm maturation or a synthetic or recombinant portion thereof able to confer to the chromatin of said cell a spermatozoa like chromatin conformation, b) identifying the nuclei wherein the chromatin has a spermatozoa like chromatin conformation; c) transplanting the identified nuclei into a non-human animal enucleated oocyte to get reconstructed embryo and optionally; and d) culturing said reconstructed embryo in vitro or in vivo.

2. The method according to claim 1, further comprising the steps of: e) transferring said reconstructed embryo into a suitable non-human foster mother/shell.

3. The method according to claim 1, wherein the protein involved in male sperm maturation belongs to the group consisting of: very basic DNA-interacting non-histones proteins/peptides, and transition proteins (TPS).

4. The method according to claim 3, wherein the very basic DNA-interacting non-histones protein is protamine 1 or protamine 2.

5. The method according to claim 1, wherein the non-human donor cultivated cell is a somatic cell and/or a fetal cell.

6. The method according to claim 5, wherein the somatic cell is a fibroblast.

7. The method according to claim 1, wherein said non-human donor cultivated cell has been previously genetically modified.

8. The method according to claim 7, wherein said genetic modification includes the insertion of at least one a heterologous DNA sequence and/or the deletion of a homologous gene and/or the modification of at least one a homologous gene.

9. The method according to claim 1, wherein the non-human donor cultivated cell and the non-human oocyte belong to the same species.

10. The method according to claim 9, wherein said species is selected from the group consisting of domestic animals laboratory, and companion animals, and non mammalian species.

11. A method for generating a non-human animal, comprising: a) transferring a reconstructed non-human animal embryo into a suitable non-human recipient animal or intermediate host or eggshell; b) causing said non-human animal embryo to develop to term; and c) further breeding the resulting non-human animal.

12. A non-human animal obtainable by the method according to claim 11.

13. Non-human animal reconstructed embryo obtainable by the method according to claim 1.

14. The method according to claim 10, wherein said domestic animal is a farm animal.

15. The method according to claim 10, wherein said non-mammalian animals are selected from the group consisting of avian, amphibian, fish and reptiles .

Description

[0066] The present invention will be illustrated by means of non-limiting examples referring to the following figures:

[0067] FIG. 1. Expression of protamine 1 gene in adult fibroblasts AProtamine mRNA expression in sheep adult fibroblasts (RT-PCR). Prm1-RFP: fibroblasts transfected with protamine tagged with RFP. CTR: fibroblasts not transfected. NTC: no template control; BProtamine protein expression in adult fibroblasts (Western blot, WB). Prm1-RFP: fibroblasts transfected with pPrm1-RFP. pTag-RFP: fibroblasts transfected with empty plasmid-RFP. CTR: fibroblasts not transfected. Immunoblotting with anti-tRFP, anti-actin were used as loading control; Tracking the RFP tag in cells transfected with pTag-RFP (C, D, E) and pPrm1-RFP (F, G, H); Nuclei (Hoechst stained), (C, F), RFP (D) or Prm1-RFP localizations (G), merge (E, H). Scale bar=10 m; Elongated nucleus in pPrm1-RFP transfected cell (I, J, K). Scale bar=10 m.

[0068] FIG. 2. Timing of incorporation of protamine in somatic nuclei.Schematic representation of post transfection incorporation of protamine in somatic nuclei. pPrm1-RFPprotamine plasmide tagged with RFP; ANucleus of fibroblasts before the transcription of protamine; BIncorporation of protamine in nucleus 16-20 h post transfection visible as spots; CComplete incorporation of protamine in nucleus 40-48 h post transfection, complete overlapping nucleus/protamine; E, F, GTransmission electron microscope (TEM) analysis of adult sheep fibroblasts transfected with pPrm1-RFP; FNucleus of fibroblasts before the transcription of protamine., GNucleus of fibroblasts after 16-20 h post transfection. Visible partial compaction of chromatin; HNucleus of fibroblasts after 40-48 h post transfection. Evident the complete chromatin compaction. Bars=8 m (A), 2 m (F); D, HNucleus of spermatozoa stained with Hoechst (E) and analyzed by TEM (I). Scale bar: 500 nm

[0069] FIG. 3. Nuclear remodelling of protaminized somatic fibroblasts. A-Displacement protamine during pronucleus formation after Nuclear Transfer (NT); scale bar=20 m. Nucleus/Prm1 (overlapping), 3 h 30 m (displacement Prm1 from nucleus), 6 h (protamine disappears and formation Pronucleus, PN); B- Incorporation TH2B in Pronucleus (PN) after NT of protaminized nuclei. Pronucleus (PN), TH2B , Merge (TH2B/PN). Scale bar=20 m; CSomatic cell nuclear transfer (SCNT) of fibroblast transfected with pPrm1-RFP; aPicture represents protamine (arrows) positive fibroblasts used as donors for SCNT; a1protaminized nucleus in injected capillar before nuclear transfer into enucleated MII sheep oocytes, a2Percentage of blastocysts produced by SCNT using control (CTR) and protaminized (Prm) sheep fibroblast as donor.

EXAMPLE

Methods

Plasmids Construction

[0070] Sperm protamin 1 (Prm1) cDNA (accession N: NM_002761.2 [SEQ ID NO:1]) was amplified from a human testis cDNA library with appropriate primes and cloned into a pTagRFP vector (Evrogen, Milan, Italy). The identity of the cloned cDNA and its in frame cloning C-terminal to RFP was verified by sequencing.

Source of Cells and Transient Transfection.

[0071] Sheep Adult Fibroblasts (SAF) were derived from ear biopsy of three female Sarda breed sheep (2 years old). Primary cultures were establishment from the biopsy and fibroblasts were used for transfection experiments between second and eighth passage. SAF were maintained during the culture in Dulbecco's modified eagle medium (DMEM) (Gibco, Life Technology, Milan, Italy) containing 10% FBS (Fetal Bovine Serum), 2 mM Glutamine, 3.7 g/l NaHCO3 and 0.5% Gentamicin. SAF at 80% confluence were transfected with 3 g of pPrm1-RFP and pRFP (transfection CTR) (pTagRFP vector, FP141, Evrogen, Milan, Italy) by Lipofectamine 2000 (Invitrogen, Oslo, Norway), according to the manufacturer's instructions. The post-transfection medium was changed for DMEM containing 5 nM Trichostatin A (TSA) 4 h post transfection, and SAF were cultured for additional 16, 20, 40, 48 h.

[0072] Cells were cultured for 48 hours and checked for phenotypic changes following protamine expression.

[0073] The expression of protamine was additionally demonstrated by RT-PCR and western blotting analysis of transfected and control fibroblasts, as described below.

RNA Isolation and Reverse Transcription (RT-PCR)

[0074] Poly (A) +RNA was isolated from cells using Dynabeads mRNA DIRECT kit (Invitrogen Dynal AS, Oslo, Norway) according to the manufacturer's instructions. RT was performed using 80% of the eluted Poly (A) +RNA in a total volume of 20 l using the QuantiTect Reverse Transcription Kit (Qiagen, Milan, Italy). cDNAs were diluted 1:3 in H.sub.2O. The PCR reactions were performed using the PCR Master Mix (Promega, Milano, Italy). PCR conditions: 95 5 (95 C., 30, 58 30 C., 72 C. 30)35 cycles, 72 C. 10. Primers were: FW; atggccagataccgatgct (SEQ ID NO: 9), RV; cagcatcttcgcctcctc (SEQ ID NO: 10); amplicon: 160 bp.

Western Blotting

[0075] Cell protein extracts were denatured by heating at 95 C. for 5 min in 1% (v:v) sodium dodecyl sulphate (SDS), 1% (v:v) -mercaptoethanol, 20% (v:v) glycerol in 50 mM TrisHCl at pH 6.8. Samples were subjected to electrophoresis in 10% SDS polyacrylamide gels. After electrophoresis proteins were transferred to nitrocellulose membranes. Membranes were blocked in TBS-T (0.2% (v:v) Tween-20 in 20 mM Tris, 137 mM NaCl at pH 7.6) with 5% (w:v) skimmed milk, and then washed three times in TBS-T at room temperature. Membranes were incubated overnight (o/n) at 4 C. with the primary antibody anti-tRFP (1:300, Evrogen, Milan, Italy) and anti--Actin as the loading control (1:1000; sc-1615, Santa Cruz Biotechnology, Santa Cruz, USA) diluted in blocking solution. After three washes with TBS-T, membranes were incubated for 1 h at room temperature with the secondary antibody (anti-goat or anti-rabbit IgG HRP-labelled, Santa Cruz Biotechnology, Santa Cruz, USA) diluted 1:1000 in blocking solution. After three washes in TBS-T, the final detection was performed by enhanced chemiluminescence using the ECL Plus Western Blotting Detection System (Amersham-Pharmacia, Piscataway, USA). Image acquisition was carried out using the ChemiDoc System (Bio-Rad, Milan, Italy). Non transfected cells and transfected with only RFP tag (20 g/lane) were used as positive controls.

TEM Analysis

[0076] Transfected cells were fixed and processed for TEM (Transmission Electron Microscopy) as described in the following. Cells were washed twice with PBS and fixed in glutaraldehyde (2.5% in 0.1 M cacodylate buffer, pH 7.4) for 24 h. After washing in ddH2O, cells were post-fixed in 2% OsO4 in ddH2O for 4 h and washed three times in ddH2O. Next, cells were dehydrated through a graded series of ethanol solutions (30%-10 min, 50%-15 min, 70%-24 h, 80%-10 min, 96%-10 min, 100%-10 min, acetonetwice for 15 min) and were infiltrated with graded concentrations of EPON resin in 100% acetone (1:3-20 min, 1:1-24 h, 3:1-2 h), infused twice for 1 h in pure EPON resin and polymerized at 65 C. for 24 h. Next, 60 nm sections were prepared and examined using a LEO 912AB electron microscope. Images were captured using a Slow Scan CCD camera (Proscane) and EsiVision Pro 3.2 software (Soft Imaging Systems GmbH).

Oocyte Maturation

[0077] Sheep oocytes recovered from local ovine slaughterhouse were matured in vitro in bicarbonate-buffered TCM-199 medium (Gibco) (275mOsm) containing 2 mM glutamine, 100 mM cysteamine, 0.3 mM sodium pyruvate, 10% foetal bovine serum (FBS) (Gibco), 5 mg/ml FSH (Ovagen), 5 mg/ml LH, and 1 mg/ml estradiol in a humidified atmosphere of 5% CO2/air at 39 C. for 24 h (Ptak G. et al, 2002).

Nuclear Transfer

[0078] Methods of in vitro embryo production were adapted from those previously described (Ptak et al., 2002). Oocytes were matured in vitro in a humidified atmosphere of 5% CO2/air at 39 C. for 24 h. Oocytes were incubated in Hepes-buffered TCM-199 medium containing 4 mg/ml BSA, 7.5 mg/ml Cytochalasin B and 5 mg/ml Hoechst 33342 in an incubator for 15 minutes. Oocytes manipulation was carried out with a piezo-driven enucleation/injection pipette (PiezoXpert, Eppendorf). Enucleation was carried out in Hepes-buffered TCM-199 medium with 0.4% (w/v) BSA and Cytochalasin B with a Narishighe micromanipulator fitted to a Nikon Eclipse inverted microscope. No DNA vital dyes/UV irradiation was used to locate the chromosomes in the oocytes, but a blind aspiration of the cytoplasm surrounding the first polar body was conducted, and enucleation was confirmed later by Hoechst staining and UV irradiation of the aspired cytoplasmic fragments (Iuso et al., 2014). Enucleate oocytes were allowed to recover from the Cytochalasin B treatment and then directly injected with a nucleus, either from CTR or a Prm1-RFP fibroblasts suspended in PBS with 6% Polyvinylpyrrolidone (Sigma). Reconstructed oocytes were activated in Hepes-buffered TCM-199 medium containing 5 mg/ml Ionomycin for 5 minutes and then incubated in SOF medium plus antibiotics and 0.8% BSA containing 10 mM Dimethylaminopurine and 7.5 mg/ml Cytochalasin B for 3-5 hours and cultured for 10-12 hours in SOF enriched with 1% (v:v) minimum essential medium (MEM) nonessential amino acids (Gibco, Milan, Italy), 2% (v:v) basal medium Eagle (BME) essential amino acids, 1 mM glutamine, and 8 mg/ml BSA covered with mineral oil pre-washed in SOF. Cultures were checked for embryonic development every 24 hours till day 7 post activation.

TH2B Immunostaining on Pronuclear Stage Embryos

[0079] The zona pellucida of embryos at pronuclear stage (10-12 h after oocyte activation) was removed by incubation in 0.5% (w/v) pronase and acid Tyrode's solution for 30s. Embryos were then fixed in 4% PFA for 15 min and subjected to immunofluorescence analysis as described in Torres-Padilla et al. (2006) with the rabbit anti-TH2B antibody (1:700, ab23913 Abcam, UK). Finally, zygotes were mounted with VectaShield mounting medium with 5 g/mL of DAPI.

Statistical Analysis

[0080] Fisher's exact test were used for comparing frequencies of transfection of somatic cells and development to blastocysts to enucleated oocytes injected with protaminized somatic cells.

[0081] Probability values lower than 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism 5.0 software.

Results

[0082] The transfected fibroblasts regularly expressed protamine (50%) in 34 replicates (FIG. 1, A,B). Expression of mRNA and protein Prm1 was confirmed by RT-PCR, western blotting and by tracking the RFP-tag (FIG. 1A, B, G). Prm1-RFP co-localized with nuclei stained by Hoechst (FIG. 1F, G, H) whereas in control, pRFP transfected fibroblasts, the red signal was diffused in cytoplasm and nucleus (FIG. 1C, D, E vs F, G, H). Prm1 translocate into the nuclei immediately after its translation and the end product of Prm1 assembly on somatic DNA is the acquisition of a nuclear morphology overlapping that found in elongating spermatids (FIG. 1I, J, K).

[0083] Protaminization started as focal points within the nucleus starting 10 hours post transfection, and induced radical nuclear transformation within 48 hours (FIG. 2), when a phenotype overlapping spermatocytes was acquired (FIG. 2C, D). TEM analysis confirmed the extreme compaction of the nuclear compartment (FIG. 2G).

[0084] It is concluded that the transient expression of protamine induces a radical chromatin reorganization of fibroblast nuclei (FIG. 2C, G), similar to that occurring in spermatozoa (FIG. 2D, H).

[0085] Hence, the method of the present invention confers the chromatin of a somatic cell the structure of a spermatozoa DNA, thus perfectly compatible with the reprogramming machinery of the oocytes.

[0086] The remodeling of protaminized cells following nuclear transfer into enucleated oocytes was then analyzed. The protamine red tag was lost following oocyte activation, and the nuclei expand into a normal pronucleus (size: 16.22.3 m) in 89% of the injected oocytes, FIG. 3A. Meanwhile, protamine progressively disappeared in 77% of oocyte injected (28/36, FIG. 3A) and oocyte specific histones variant TH2B began to be assembled in the pronuclei (FIG. 3B). TH2B has recently described as the unique histone variant that plays a key role for nuclear reprogramming (Shinagawa et al., 2014), as it guides the protamine-histone chromatin transition post fertilization (Montellier et al., 2013).

[0087] The enucleated oocytes reconstructed with protaminized somatic nuclei were further cultured in vitro in 7 separated replicates, indicating their full competence to direct early embryonic cleavages. Moreover, the developmental frequencies to the blastocyst stage of Prm1 nuclei/derived embryos were significantly higher than control ones (NT blastocysts/cleavage: Prm1, 30/200; 15%; CTR, 14/176; 7.9%) (P=0.0372; Fisher's exact test, FIG. 3, a2).

[0088] The protamine-induced reprogramming method object of the present invention is a major breakthrough for nuclear reprogramming. Moreover, the simplicity and the robustness of the protocol developed by the present inventions render the method easily repeatable in all non-human animals reproducible through Somatic Cell Nuclear Transfer.

REFERENCES

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