creloxp转基因小鼠制作

1、1. Gene knock-out mice2. Conditional gene knock-out3. Transgenic mice44 ApplicationAdvantages of gene knock-out andtransgenic mice After large scale of DNA sequencing, ihc next frontier is the study of gene function 11 is one of the most powerful tool to study gene functiori Study gene fiinction in

2、an organism.Gene knock-out mouseChimeric m记e -Helerozygous miceHomozygous for the mutated genePhenotype analysis« i- »Homologous recombination The mechanism is not well understood A rare event Higher frequency if construct has free endsGenomic DNA Mouse genomic library screening GenBank; N

3、CBI (long PCR) Preferably > 10 kbRandom integrationBlcmih * Hj*Positive selection neo encoding neomycin pho§phot8nHfbii&c G41S (neomycin) selection Kill cells without constructNegative selection 11SV-TK: herpes simplex virus thymidine kinase Mechanism: TK activates acyclovir or gancyclov

4、ir (r ANC'X resulting in their incorpomtion into growing DNA, causing cell deathCreate a constructRandom integration G418 resistant GANC sensitiveHomologous recombination G418 resistant GANC resistantCreate a construct, Homologous sequence on both ends: > 5kb A positive selectable m2kein the

5、middle: eg neo gene A negative selectable maker outside the homologous region: eg HSV-TK A hybridization probe to identify homologous recombinantsCreate a constructansfection of ES cells In a typical experiment, about 107 EScells are transfected with the 8nstuct. About 500 colonies are expected afte

6、rboth positive and negative selection.Pi# bE&sChimeric mice 仃流的即Heterozygous miceHomozygous far the mutated genePhenotype analysis2. Conditional gene knock-outCre-MxP system is derived from the bacteriophage Pl Cre recombinase recognizes loxP sequence If a gene is flanked by two ioxP sequence, C

7、rc can delete the gene and leave one loxP in the original DN A.口弟日独1VConditional gene knock-out* Spatial control of knock-out* Temporal control of knock-outSpatial control of knock-out* Use cell-type specific promoters to express Cre* Add loxP sequence on both side of the targeted exon(s)XGOTemporal

8、 control of knock-out* If a gene is important in development,it is possible that no animal can be obtained* Using conditional expression system: e,g. interferon-inducible promoter; tet-an/off system etc.loxPX3. Transgenic miceMM,Transgenic mice* Stable insertion of foreign DNA into the mouse genome*

9、 Il can be created by microinjection or viral infectionTransgenic mice* 11 is random integration* Founder mouse can be identified by PCR screening or Southern blot* J he founder mouse is heterozygous fbr the transgene insertion locusCell specific transgenic mice* Use cell or tissue specific promoter

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