Genetic manipulation method

Abstract

A method of inducing transposition in a transgenic embryo, sperm and egg is described, comprising the steps of (a) generating a first adult transgenic organism comprising within its genome one or more copies of a transposon; (b) generating a second adult transgenic organism comprising within its genome one or more copies of a gene encoding a transposase cognate for the transposon and/or a sequence capable of regulating expression of the gene encoding the transposase; (c) crossing the first adult transgenic organism with the second transgenic adult organism to provide a progeny which comprises, in the genome of one or more of its cells, both (i) one or more copies of the transposon and (ii) a gene encoding a transposase cognate for the transposon, wherein the gene encoding the transposase is under the control of one or more inducible regulatory sequences which permit expression of the transposase, and (d) expressing the gene encoding the transposase in the embryo, sperm or egg to cause mobilisation of the transposon within a portion of the tissues or cells of the progeny. Using the method, mobilisation of a transposon can advantageously be induced at predetermined stages of development of an embryo, sperm or egg and the mutated gene of a single cell may be replicated in subsequent cell divisions, resulting in groups of cells which are essentially homogenous for the transposed gene.

1 . A method of generating a non-human mammalian transgenic embryo or offspring by germ-line transmission wherein one or more insertional events are present in all cells and tissues of said embryos or off-spring comprising:
 a) Generating either (i) a first non-human transgenic adult male mammal wherein transposon transposition occurs in developing spermatocytes by the regulated expression of a cognate transposase such that the resultant sperm carry one or more insertional events, or (ii) a first non-human transgenic female mammal wherein transposon transposition occurs in developing oocytes such that the resultant oocytes carry one or more insertional events:   b) Crossing either (i) the first non-human transgenic adult male with a second non-human adult female mammal lacking a transposon and cognate transposase activity, or (ii) the first non-human transgenic adult female with a second non-human adult male lacking a transposon and cognate transposase activity to provide:   Non-human mammalian embryos or offspring comprising one or more insertional events in all cells and tissues contributed either by sperm or oocyte derived from either (i) the non-human transgenic adult male mammal, or (ii) the non-human transgenic adult female mammal of step (a).   
     
     
         2 . The method of  claim 1  wherein transposase activity in developing sperm is regulated by H1t regulatory sequences. 
     
     
         3 . The method of  claim 1  wherein transposase activity in the developing oocyte is regulated by ZP3 regulatory sequences 
     
     
         4 . The method of  claim 1 ,  2 , or  3 , wherein one or both of the first or second non-human adult transgenic mammals is prone to a disease state 
     
     
         5 . The method of  claim 1  wherein the second non-human adult mammal is generated by germ-line transmission 
     
     
         6 . The method of  claim 1  wherein as a result of a transposition event there is a measurable change in a phenotypic characteristic in any non-human embryo or progeny relative to the first or second adult non-human mammals 
     
     
         7 . The method of  claim 6  wherein said change in phenotype is correlated with a transposition event by a method comprising:
 (a) identifying in the transgenic embryo or offspring a variant phenotype;   (b) detecting the position of one or more transposon transposition events in the genome of one or more of said cells; and   (c) correlating the position of the transposition events with the observed variant phenotype, the position of the transposition events being indicative of the location of one or more genetic loci associated with the observed variant phenotype.   
     
     
         8 . A method according to  claim 6  wherein a transposition associated with a disease related phenotype is identified by a method comprising:
 (b) identifying in the transgenic embryo or offspring a disease related variant phenotype;   (c) detecting the position of one or more transposon transposition events in the genome of one or more of said cells; and   (d) correlating the position of the transposition events with the observed variant phenotype, the position of the transposition events being indicative of the location of one or more genetic loci associated with the observed variant phenotype.   
     
     
         9 . The method of  claim 7  or  8 , further comprising the step of sequencing genomic DNA adjacent to said transpositions, for the identification of one or more disease associated genes or regulatory sequences. 
     
     
         10 . A method for producing a library of transgenic embryos or offspring comprising generating a plurality of transgenic embryos and inducing transposition therein by the method according to  claim 6 . 
     
     
         11 . A library of transgenic non-human transgenic mammals produced according to the method of  claim 10 . 
     
     
         12 . A method according to  claim 9  where the step of sequencing genomic DNA adjacent to said transpositions occurs prior to phenotypic analysis of embryos or progeny 
     
     
         13 . A library of transgenic non-human transgenic mammals generated according to  claim 12  comprising off-spring each comprising a gene disruption in a different gene. 
     
     
         14 . The method according to  claim 1 , wherein the transposon comprises a nucleic acid sequence encoding a selectable marker. 
     
     
         15 . The method according to  claim 14 , wherein the selectable marker is a fluorescent or luminescent polypeptide. 
     
     
         16 . A method of  claim 14  or  15  wherein the selectable marker is incorporated in an exon trap 
     
     
         17 . A Method of  claim 14  or  15  wherein the selectable marker is incorporated in an enhancer trap 
     
     
         18 . A method of  claim 6  wherein the change in phenotype is defined by the enhanced expression of a selectable marker resulting from one or more insertional events