Trophectodermal cell-specific gene transfer methods

Abstract

The present inventors discovered that genes could be introduced specifically into trophectodermal cells with high efficiency, by infecting blastocysts with viral vectors carrying an arbitrary polynucleotide, or by using a nucleic acid transfection reagent in blastocysts, from which zona pellucida (extracellular matrix covering preimplantation early embryos to protect them from infection of viruses and the like) is removed. This method has no risk of infecting cells of the inner cell mass, which develops into a fetus in the future, with the introduced polynucleotide because the trophectoderm serves as a barrier. The present invention provides methods for introducing foreign genes into only placenta but not fetus, which enables rescue of genetically mutant animals from embryonic lethality due to placental abnormality and allows their birth. Furthermore, it is possible to analyze expression and effect of genes that regulate placental formation or placental function by using these methods.

1 .- 21 . (canceled) 
     
     
         22 . A method for introducing a polynucleotide specifically into a trophectodermal cell, which comprises the steps of:
 a) extracting a blastocyst from an animal;   b) removing zona pellucida from the blastocyst; and   c) introducing a polynucleotide into trophectodermal cells of the blastocyst by placing the blastocyst comprising trophectodermal cells obtained in step (b) in a solution comprising viral vectors;   wherein the viral vectors introduce the polynucleotide only into the trophectodermal cells of the blastocyst.   
     
     
         23 . The method of  claim 22 , wherein the viral vector is selected from the group consisting of a retroviral vector and adenovirus vector. 
     
     
         24 . The method of  claim 22 , wherein the polynucleotide is a polynucleotide encoding a protein selected from the group consisting of: Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esx1, Arnt, Tcfeb, and Gjb2. 
     
     
         25 . The method of  claim 22 , wherein the animal possesses abnormality in a protein essential for fetal development or in the regulation of expression of the protein. 
     
     
         26 . A method for introducing a polynucleotide specifically into a trophectodermal cell, which comprises the steps of:
 a) extracting a blastocyst from an animal;   b) removing zona pellucida from the blastocyst; and   c) introducing a polynucleotide into trophectodermal cells of the blastocyst with a nucleic acid transfection reagent;   wherein the polynucleotide are introduced only into the trophectodermal cells of the blastocyst.   
     
     
         27 . The method of  claim 26 , wherein the polynucleotide is a polynucleotide encoding a protein selected from the group consisting of: Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esx1, Arnt, Tcfeb, and Gjb2. 
     
     
         28 . The method of  claim 26 , wherein the animal possesses abnormality in a protein essential for fetal development or in the regulation of expression of the protein. 
     
     
         29 . A method for introducing a polynucleotide specifically into a trophectodermal cell, which comprises the steps of:
 a) extracting a blastocyst from an animal;   b) removing zona pellucida from the blastocyst; and   c) using a viral vector to introduce a polynucleotide only into trophectodermal cells of the blastocyte, wherein the polynucleotide encodes a protein selected from the group consisting of Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, Grb2 hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, ERK1, ERK2, ERK5, MEK1, MEKk3, p38α, p38β, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esx1, Arnt, Tcfeb, and Gjb2;   wherein the viral vectors introduce the polynucleotide only into the trophectodermal cells of the blastocyst.