Generation of Transgenic Mice

Generation of Transgenic MiceMicroinjection Microinjection 显微注射显微注射 Viral transgenesis Viral transgenesis 病毒转染病毒转染ES aggregation ES aggregation 干细胞融合干细胞融合MicroinjectionMicroinjection( (显微镜下进行的显微镜下进行的微注射微注射）Pronuclear DNA microinjection Pronuclear DNA microinjection ( (原核注射）原核注射）Blastocyst ES microinjection Blastocyst ES microinjection （囊胚注射）（囊胚注射）The Microinjection ExperimentPronucleus microinjection Pronucleus microinjection of gene of gene constructsconstructs （DNA原核注射） e.g. Strain InformationTypeType： Transgenic; Description：Transgenic mice overexpress Green Fluorescent Protein under the control of the astrocyte-specific glial fibrillary acidic protein promoter. Bright fluorescence is observed in the cell bodies and processes of unfixed or fixed astrocyte preparations throughout the CNS of hemizygous mice. In addition retinal Mullers cells expressed the GFP transgene in response to degeneration of neighboring photoreceptors. These mice provide a method to follow changes in astrocyte morphology during development or disease processes. -from JAX LabStock No.003257Flow Chart for Pronucleus MicroinjectionDay 4/5: oviduct transfer to pseudo-pregnant foster mothersDay 1:PMSG injectionDay 3:HCG injection and matingDay 3/4:mating of foster mothers with vasectomized malesDay 4:isolation of oocytes and micro-injection of DNADay 46: tail tipping and analysis of offspringDay 23: birth of offspringRelated ConceptsPronucleus Pronucleus (原核）：The nucleus of an ovum or a spermatozoon after fertilization but before fusion of the chromosomes to form the nucleus of the zygote.在卵子受精后，二性染色体融合前，从精子或卵子发育而来的单倍体核样结构。 雄原核雄原核，雌原核雌原核Holding pipettePolar bodyInjection needleMale pronucleusFemale pronucleusNucleoliZona pellucidaDonorsDonors(供体鼠）: :female mice for mating to produce fertilized eggs for DNA injection为DNA注射提供受精卵的母鼠 Age:4-6-week-old Strains: F1 hybrids: e.g., B6C3F1inbred strains: e.g., C57BL/6,BALB/cJ,FVB/Noutbred strains: e.g., ICRStuds Studs (种公鼠）:fertile stud male mice to mate with donors用于和供体母鼠交配的雄鼠 Age:2-12-month-old Strains:same as the donors, generallyadministering gonadotropins to donors prior to mating, to increase the number of oocytes that are ovulated.为了增加实验时可操作的受精卵，通过人为注射激素，来诱导供体鼠多排卵。 PseudopregnancyPseudopregnancy(假孕）: : mating a female mouse with a infertile male, the stimulus of mating elicits the hormonal changes needed to make her uterus receptive. 发情期的母鼠在与无精子的公鼠交配后的一定时间内，黄体素会持续存在，而使它自己的行为和身体处于适合胚胎种植，及养育子鼠的状态。Receptions Receptions (受体鼠）:Female mice to serve as pseudopregnant recipients and foster mothers接受DNA注射过受精卵的假孕母鼠 Age:6-20-week-old Strains:random-bred mice or F1 hybrid micee.g., Swiss webster, B6CBA F1 or CD1Vasectomized male miceVasectomized male mice(结扎公鼠）: :Sterile stud male mice to mate with receptions for the production of pseudopregnant females输精管结扎过的公鼠。具有正常的交配能力，由于输精管中没有精子，与母鼠交配后，可使母鼠处于假孕状态。 Two Methods to Vasectomized Male MiceVaginal plug Vaginal plug ( (阴栓）阴栓）：The precence of the vagianl plug, a harden plug deposit by a male mouse into a female mouse genital tract, indicates the successful mating小鼠交配后，精液在凝固腺的作用下，会凝固形成栓塞，堵塞在阴道口与子宫口之间。它可以做为母鼠成功交配过的依据。 Transgenic miceTransgenic mice（转基因小鼠）： the mice which are determined to the presence of the injected gene 所有携带有外源基因的小鼠的总称 Founder mice:Founder mice: transgenic mice that develop from the injected eggsTransgenic lines:Transgenic lines: derived from these founders Basic rule:One founder, one line, no crossing with each otherSummary of Preimplantation DevelopmentSummary of Preimplantation Development壶腹部：卵子，精子受精输卵管：单细胞及分裂后 至桑葚期子宫： 囊胚期及之后Flow Chart for Pronucleus MicroinjectionDay 4/5: oviduct transfer to pseudo-pregnant foster mothersDay 1: PMSG injectionDay 3: HCG injection and matingDay 3/4:mating of foster mothers with vasectomized malesDay 4: isolation of oocytes and micro-injection of DNADay 46: tail tipping and analysis of offspringDay 23: birth of offspringOviduct Dissection and Zygotes IsolationMorphology of Preimplantation Mouse Development3,4 extrusion of first polar body (- 10 hr post HCG)5,6 extrusion of second polar body; formation of the male pronuclear ( - 17-33 hr post HCG)7 7 Formation of the female pronuclear (- 26 hr post HCG)8 8 migration of pronuclei to enter of zygote9,109,10 formation of visible nucleoli within both pronuclei. DNA replication (complete 28hr post-HCG mostly)Integration of Injected DNA ConstructsSYNGAMYSYNGAMYINTERGRATIONINTERGRATIONRANative DNAInjected ConstructTransgenic EmbryoMosaic EmbryoDelayed Integration 5%30%70%Flow Chart for Pronucleus MicroinjectionDay 4/5: oviduct transfer to pseudo-pregnant foster mothersDay 1:PMSG injectionDay 3:HCG injection and matingDay 3/4:mating of foster mothers with vasectomized malesDay 4:isolation of oocytes and micro-injection of DNADay 46: tail tipping and analysis of offspringDay 23: birth of offspringOviduct Transfer(输卵管移植）Flow Chart for Pronucleus MicroinjectionDay 4/5: oviduct transfer to pseudo-pregnant foster mothersDay 1:PMSG injectionDay 3:HCG injection and matingDay 3/4:mating of foster mothers with vasectomized malesDay 4:isolation of oocytes and micro-injection of DNADay 46: tail tipping and analysis of offspringDay 23: birth of offspringBlastocyst microinjection Blastocyst microinjection of genetically of genetically altered embryonic stem (ES) cells altered embryonic stem (ES) cells (胚胎干细胞囊胚注射） Knock out a mouse with a particular gene knocked out in only certain cells 是使小鼠体内的某个基因在特定细胞中缺失 Knock inone of the mouses own genes replaced with a new gene that the investigator wishes to study是用我们想研究的某个特定基因代替小鼠原有的某个基因e.g. Strain InformationType Type Targeted Mutation DescriptionMice homozygous for the Baxtm1Sjk mutation are viable but display lineage-specific aberrations in cell death. Thymocytes and B cells from homozygous mutant mice display hyperplasia. Ovaries contain unusual atretic follicles with excess granulosa cells while Bax-deficient males are infertile. There is an accumulation of atypical premeiotic germ cells and no mature haploid sperm found in seminiferous tubules. Multinucleated giant cells and dysplastic cells accompany massive cell death. -from JAX LabStock No.002994Flow Chart for Blastocyst MicroinjectionDay 7.5: uterus transfer to pseudo-pregnant foster mothersDay 1: PMSG injectionDay 3: HCG injection and matingDay 5:mating of foster mothers with vasectomized malesDay 7.5: isolation of blastocyst and micro-injection of ES cellsGerm line transmission achieved by crossing Chimera with wt miceAfter Day 23: birth of offspring; chimera selectionSelect appropriate donors to provide host blastocyst挑选原则：1.注射后发育得到的小鼠毛色要易于观察嵌合体小鼠2.两个小鼠品系间要具有良好的相容性Superovulated donorsSuperovulated donorsNaturally mated donors - higher quality blastocystFlow Chart for Blastocyst MicroinjectionDay 7.5: uterus transfer to 2.5D pseudo-pregnant foster mothersDay 1: PMSG injectionDay 3: HCG injection and matingDay 5:mating of foster mothers with vasectomized malesDay 7.5: isolation of blastocyst and micro-injection of ES cellsGerm line transmission achieved by crossing Chimera with wt miceAfter Day 23: birth of offspring; chimra selectionBlastocyct CollectingDevelopment stage and quality of blastocysts for injectionA,B,C early stage blastocystD D almost expanded blastocystE E fully expanded blastocystF Over expanded blastocystG hatching blastocystH hatched blastocystUterus transferBlastocyst ES Blastocyst ES microinjectionmicroinjectionFlow Chart for Blastocyst Microinjection 7.5: uterus transfer to pseudo-pregnant foster mothersDay 1: PMSG injectionDay 3: HCG injection and matingDay 5:mating of foster mothers with vasectomized malesDay 7.5: isolation of blastocyst and micro-injection of ES cellsGerm line transmission achieved by crossing Chimera with wt miceAfter Day 23: birth of offspring, chimera selectionChimeraChimera(嵌合体小鼠）: : they have somatic cells composed of both transgenic transgenic ES cellsES cells and host cells嵌合体小鼠的毛色，是在供体来源小鼠 毛色中，带有工程改造ES来源小鼠毛色hostC57BL/6(B6) chimera: B6 ES cells were injected into albino B6 blastocysts.129 svj chimera: 129 ES cells were injected into B6 blastocysts.C57BL/6J-Tyrc-2J/J ESEShosthosthostFlow Chart for Blastocyst MicroinjectionDay 7.5: uterus transfer to pseudo-pregnant foster mothersDay 1: PMSG injectionDay 3: HCG injection and matingDay 5:mating of foster mothers with vasectomized malesDay 7.5: isolation of blastocyst and micro-injection of ES cellsGerm line transmission achieved by crossing Chimera with wt miceDay 23: birth of offspring, chimera selectionGerm line transmissionGerm line transmission(生殖系传递）: should have germ cellsgerm cells composed of both transgenic ES cellstransgenic ES cells and host cellsidentified by crossing chimera with WT micee.g. agouti offsprings indicates germline transmission (ES cells from some 129 substrains )在后代中得到与ES来源小鼠品系相同毛色的小鼠杂合子小鼠纯合子小鼠EShostchimeraThe microinjection set-up Arrangement of Arrangement of microscope, microscope, micromanipulators, micromanipulators, transjector for transjector for injection method injection method on an air tableon an air table A Typical Laboratory for the Fabrication of microtools THE THE ENDENDDNA Concentration and Injection Buffer Used for MicroinjectionPlasmid DNABAC DNAInjecting Concentration 2 ng/ul 0.25ng/ul(0.005-1.5 ng/ul)Injection BufferTris-HCL10mM, pH 7.410mM, pH 7.4EDTA0.1mM0.1mMNaCl-100mMPloyamine-70 M spermidine,30 M spermine