| 摘要 |
<p>This invention provides a method for the efficient introduction of cloned, very high molecular weight DNA into the germline of mice, whereby large genes can be expressed appropriately in transgenic mice. The β-amyloid precursor protein (APP) is known to be a complex gene consisting of 18 exons with total size estimates greater than 170 kb encoding three major RNA splicing forms. According to this invention, a neomycin resistance cassette is introduced into one of the arms of a 650 kb yeast artificial chromosome (YAC) which contains the entire unrearranged APP gene within 400 kb. Following gel purification, the YAC is introduced into embryonic stem (ES) cells by lipid mediated transfection. Neomycin resistant ES lines are isolated with the human APP gene stably integrated in an unrearranged state and expressing properly initiated and spliced full length human APP mRNA and APP human protein. Mouse chimeras generated from these ES lines transmit the YAC to their offspring, generating novel APP YAC transgenic mice. These transgenic mice express human APP gene products at significant levels in brain and peripheral tissues that mirror the expression of endogenous mouse APP gene products. This procedure will have great utility for transgenic studies of gene expression involving large genes and gene complexes.</p> |
