Establishment Of IGF-Ⅰ Transgenic Mice Model Expressed Specially In The Central Nerve System By Ecdysone-induction

Insulin-like growth factor-1 (IGF-1 )can act on many tissues and organs as one kind of pleiotropic trophic factor, which has been paid more attention since 1950's. Because of specificity and complexity of the central nerve system(CNS) structure, the research about the accurate biological role and mechanism of IGF- I in CNS are limited largely.Though many experiments on IGF-1 biological effect in CNS have been exploited, nerve cells in vitro separated from CNS are mostly used. These "sick" and unnormal cells can't represent real nerve cell in living organism. Transgenic animal models can simulate the real circumstance so furthest in which certain genes take effect that the biological role and mechanism of these genes can be investigated easily and truly, therefore these modles are welcomed largely.In order to study IGF-1 biological role and mechanism conveniently and truly during CNS's growth and development or under the conditions of irritabilities, stresses or diseases, one transgenic mouse model that can express hIGF- I instantly in the CNS under different temporal phases is to be established urgently.One promoter that can direct exogenous protein to express exclusively in CNS is needed firstly.Glial fibrillary acidic protein(GFAP) named as proprietary marker protein of glial cell is predominant skeleton protein and expressed exclusively in astrocyte in CNS.GFAP gene regulation region was unknown, then we must separate its 5' terminal regulation sequence.In this investigaton we have screened 129sv mouse genomic DNA library by PCR, the positive phages in which GFAP gene is carried have been separated from library after two rounds of PCR-Screening and one round of bacteriophage plaque in situ hybridization.At last we have gotten the-3-2353bp 5' terminal sequences of GFAP gene including the 1.8 Kb 5' terminal regulation region and partial coding region.The sequences have been embodied by GeneBank (GeneBank Accession Number: AY279974).Compared with other strain mice's GFAP gene, we find the different translation start site and amino acid sequences in N terminal of GFAP between 129sv mouse and other mice.We have utilized the 1.8 Kb 5' terminal regulation region of GFAP gene to construct transgenic vector pGFAP-Cre-hGH. 7 transgenic mice have been gotten by microinjection and named as GFAP-Cre, and the pGFAP-Cre-hGH vectors have been integrated into their genomic DNA by PCR and Southern's testing.GFAP-Cre mice are mated with ROSA26 mice to test the expression of Cre recombinase in GFAP-Cre mice, the double-positive mice carrying two gene are picked out by PCR. 13d embryos and two-week mice tissues of the double-positive mice have been chosen to make slices and staine the slices with LacZ-Staining.The results show that the active Cre recombinases are expressed widely and only in brain with good tissue-specification, especially in the choroid plexus region of interbrain.Transgenic vector pCE-IGF-I included with neomycin blocking sequence floxed by two LoxP sequences, ecdysone-inducible expression system and ablated hIGF-I expression frame has been constructed.In order to test the validity of the vector, the vectors were transformed to AMI bacteria integrated with Cre recombinase gene, Cre recombinase delete neomycin gene to make the pCMV free,Then the recombined plasmids were transfected to COS7 and the transfected COS7 cells were induced to express hIGF-1 with Muristerone AWestern blotting results show hIGF- I has been expressed successfully in COS7. pCE-IGF-1 was microinjected into fertilized eggs, 6 positive transgenic mice have been gotten by PCR and Southern testing and named as CE-IGF-1 .GFAP-Cre mice and CE-IGF-1 mice were crossbred, the double-positive mice carrying two gene are picked out by PCR.This kind of double-positive mice are our aim-mice models.The deletion of neomycin gene floxed by LoxP sites mediated by Cre recombinase has been identificated by PCR.The transcription-activated factors VgEcR and RXR of ecdysone-inducible expression system have been transcribed after deletion of blocking sequ...