METHODS AND COMPOSITIONS FOR MONITORING AND ENHANCING EARLY EMBRYO DEVELOPMENT
Method for determining developmental fate of early embryos comprises measuring the expression level of a gene selected from the group consisting of CDKN1C, IGF2R, MAGEL2, MKRN3, NAP1L5, NDN, PEG3, PHLDA2, TSSC4, and UBE3A genes. Also disclosed is a method for improving pregnancy rate, wherein early embryos whose expression level of the MKRN3, NDN, PEG3, PHLDA2, TSSC4, or UBE3A gene is not increased, or the expression level of the CDKN1C, IGF2R, MAGEL2, or NAP1L5 gene is not decreased, are selected for planting into a suitable uterus for further development. Also disclosed are methods for increasing the likelihood of an early embryo to develop successfully into full-term pregnancy, wherein a suitable amount of siRNA corresponding to the PHLDA2 gene is injected into a fertilized egg which is in turn cultured further and planted into a suitable uterus.
1 . A method of selecting an embryo for planting into a uterus for further development, the method comprising i) obtaining a supply of embryos, and growing the embryos to a stage ready for planting into a uterus; ii) obtaining a cell from a pre-planting embryo; iii) determining the expression level in the cell of at least a gene selected from the group consisting of CDKN1C, IGF2R, MAGEL2, MKRN3, NAP1L5, NDN, PEG3, PHLDA2, TSSC4, and UBE3A, and iv) planting the embryo into a suitable uterus if it does not show an increased expression level of the MKRN3, NDN, PEG3, PHLDA2, TSSC4, or UBE3A gene, or does not show a decreased expression level of the CDKN1C, IGF2R, MAGEL2, or NAP1L5 gene.
2 . The method according to claim 1 , wherein the embryo is a bovine embryo.
3 . The method according to claim 2 , wherein the embryo from which a cell is extracted is not more than 8 days post fertilization.
4 . The method according to claim 1 , wherein the embryo is in an embryonic developmental stage not later than a morula stage, or before the blastocyst stage, wherein there is no differentiation among the cells.
5 . The method according to claim 3 , wherein the embryo is in an embryonic developmental stage before hatching and is ready for planting.
6 . The method according to claim 3 , wherein the embryo has developed to comprise not more than about 32 cells.
7 . The method according to claim 6 , wherein the embryo has developed to comprise not more than about 150 cells.
8 . The method according to claim 1 , wherein the gene expression level is determined by real time qRT-PCR.
9 . The method according to claim 1 , wherein the gene expression level is determined by real time qRT-PCR on mRNA extracted from the single cell.
10 . An in vitro fertilization method for breeding animal, the method comprising obtaining embryos via an in vitro fertilization procedure, selecting a suitable embryo according to claim 1 , and planting into a suitable uterus to allow further development of embryos that do not show an increased expression level of the MKRN3, NDN, PEG3, PHLDA2, TSSC4, or UBE3A gene, or embryos that do not show a decreased expression level of the CDKN1C, IGF2R, MAGEL2, or NAP1L5 gene.
11 . A method for selectively animal breeding using a multiple ovulation and embryo transfer procedure (MOET), the method comprising superovulating a female animal, collecting eggs from said superovulated female, in vitro fertilizing said eggs and allow the fertilized eggs to develop into INF embryos, selecting embryos according to claim 1 , and planting into a uterus only embryos that do not show an increased expression level of the MKRN3, NDN, PEG3, PHLDA2, TSSC4, or UBE3A gene, or embryos that do not show a decreased expression level of the CDKN1C, IGF2R, MAGEL2, or NAP1L5 gene.
12 . A method of improving the likelihood of a bovine embryo to develop successfully, the method comprising i) obtaining a supply of fertilized eggs, ii) micro-injecting a suitable dosage of siRNA of corresponding to a PHLDA2 gene into the fertilized eggs, wherein the PHLDA2 gene expression level is inhibited; and iii) continuing to cultivate the fertilized eggs until they are ready for planting into a uterus.
13 . The method of claim 12 , wherein the siRNA comprises a duplex of PHLDA2 antisense [Phos]AGUAGCACCGGGCUAUAUCdTdT (SEQ ID No. 1), and PHLDA2 Sense GAUAUAGCCCGGUGCUACUdTdT (SEQ ID No. 2).
14 . The method of claim 13 , wherein about 100 μM of the siRNA is injected per fertilized egg.