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Generation of Transgenic Mice

Generation of Transgenic Mice. Microinjection 显微注射 Viral transgenesis 病毒转染 ES aggregation 干细胞融合. Microinjection ( 显微镜下进行的 微注射 ） Pronuclear DNA microinjection ( 原核注射） Blastocyst ES microinjection （囊胚注射）. The Microinjection Experiment.

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Generation of Transgenic Mice

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  1. Generation of Transgenic Mice • Microinjection 显微注射 • Viral transgenesis 病毒转染 • ES aggregation 干细胞融合

  2. Microinjection(显微镜下进行的微注射） • Pronuclear DNA microinjection (原核注射） • Blastocyst ES microinjection （囊胚注射）

  3. The Microinjection Experiment • Pronucleus microinjection of gene constructs（DNA原核注射） --- to generate transgenic mice

  4. e.g. Strain Information Type：Transgenic; Description：Transgenicmice 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 Lab Stock No.003257

  5. Day 1:PMSG injection Day 3:HCG injection and mating Day 3/4:mating of foster mothers with vasectomized males Day 4:isolation of oocytes and micro-injection of DNA Day 46:tail tipping and analysis of offspring Day 23:birth of offspring Flow Chart for Pronucleus Microinjection Day 4/5:oviduct transfer to pseudo-pregnant foster mothers

  6. Related Concepts • 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。

  7. 雄原核，雌原核 Polar body Injection needle Holding pipette Male pronucleus Femalepronucleus Nucleoli Zona pellucida

  8. Donors(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., B6C3F1 inbred strains: e.g., C57BL/6,BALB/cJ,FVB/N outbred strains: e.g., ICR

  9. 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, generally

  10. Superovulation(超排卵）: administering 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。 PMSG(孕马血清）:to mimic the oocyte maturation effect of FSH HCG（ 人绒毛膜促性腺激素）: to mimic the ovulation induce effect of LH

  11. Pseudopregnancy(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。

  12. 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 mice e.g., Swiss webster, B6CBA F1or CD1

  13. Vasectomized 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。

  14. Two Methods to Vasectomized Male Mice

  15. Vaginal plug (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。

  16. Transgenic 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:transgenic mice that develop from the injected eggs •Transgenic lines:derived from these founders Basic rule: One founder, one line, no crossing with each other

  17. Summary 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

  18. Flow Chart for Pronucleus Microinjection Day 1: PMSG injection Day 3: HCG injection and mating Day 3/4:mating of foster mothers with vasectomized males Day 4: isolation of oocytes and micro-injection of DNA Day 46:tail tipping and analysis of offspring Day 23:birth of offspring Day 4/5:oviduct transfer to pseudo-pregnant foster mothers

  19. Oviduct Dissection and Zygotes Isolation

  20. Morphology of Preimplantation Mouse Development • 3,4 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 Formation of the female pronuclear ((- 26 hr post HCG) • 8 migration of pronuclei to enter of zygote • 9,10 formation of visiblenucleoli within both pronuclei. DNA replication (complete – 28hr post-HCG mostly)

  21. Integration of Injected DNA Constructs SYNGAMY INTERGRATION ~ 5% RA Injected Construct Native DNA ~30% ~70% Mosaic Embryo Delayed Integration Transgenic Embryo

  22. Flow Chart for Pronucleus Microinjection Day 1:PMSG injection Day 3:HCG injection and mating Day 3/4:mating of foster mothers with vasectomized males Day 4:isolation of oocytes and micro-injection of DNA Day 46:tail tipping and analysis of offspring Day 23:birth of offspring Day 4/5:oviduct transfer to pseudo-pregnant foster mothers

  23. Oviduct Transfer(输卵管移植）

  24. Flow Chart for Pronucleus Microinjection Day 1:PMSG injection Day 3:HCG injection and mating Day 3/4:mating of foster mothers with vasectomized males Day 4:isolation of oocytes and micro-injection of DNA Day 46:tail tipping and analysis of offspring Day 23:birth of offspring Day 4/5:oviduct transfer to pseudo-pregnant foster mothers

  25. Blastocyst microinjection of genetically altered embryonic stem (ES) cells (胚胎干细胞囊胚注射） --- to generate gene targeting mice (KO/KI) 基因敲除或基因敲入小鼠

  26. 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 in one of the mouse's 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

  27. e.g. Strain Information Type Targeted Mutation DescriptionMice homozygous for the Baxtm1Sjkmutation 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 Lab Stock No.002994

  28. Flow Chart for Blastocyst Microinjection Day 1: PMSG injection Day 3: HCG injection and mating Day 5:mating of foster mothers with vasectomized males Day 7.5: isolation of blastocyst and micro-injection of ES cells Day 7.5:uterus transfer to pseudo-pregnant foster mothers After Day 23:birth of offspring; chimera selection Germ line transmission achieved by crossing Chimera with wt mice

  29. Select appropriate donors to provide host blastocyst • 挑选原则： • 注射后发育得到的小鼠毛色要易于观察嵌合体小鼠 • 两个小鼠品系间要具有良好的相容性

  30. Superovulated donors Naturally mated donors --- higher quality blastocyst

  31. Flow Chart for Blastocyst Microinjection Day 1: PMSG injection Day 3: HCG injection and mating Day5:mating of foster mothers with vasectomized males Day 7.5: isolation of blastocyst and micro-injection of ES cells Day 7.5:uterus transfer to 2.5D pseudo-pregnant foster mothers After Day 23:birth of offspring; chimra selection Germ line transmission achieved by crossing Chimera with wt mice

  32. Blastocyct Collecting

  33. Development stage and quality of blastocysts for injection • A,B,C early stage blastocyst • Dalmost expanded blastocyst • E fully expanded blastocyst • F Over expanded blastocyst • G hatching blastocyst • H hatched blastocyst

  34. Uterus transfer Blastocyst ES microinjection

  35. Flow Chart for Blastocyst Microinjection Day 1: PMSG injection Day 3: HCG injection and mating Day 5:mating of foster mothers with vasectomized males Day 7.5: isolation of blastocyst and micro-injection of ES cells 7.5:uterus transfer to pseudo-pregnant foster mothers After Day 23:birth of offspring, chimera selection Germ linetransmission achieved by crossing Chimera with wt mice

  36. Chimera(嵌合体小鼠）: they have somatic cells composed of both transgenic ES 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

  37. host ES host host ES host C57BL/6J-Tyrc-2J/J C57BL/6(B6) chimera: B6 ES cells were injected into albino B6 blastocysts. 129 svj chimera: 129 ES cells were injected into B6 blastocysts.

  38. Flow Chart for Blastocyst Microinjection Day 1: PMSG injection Day 3: HCG injection and mating Day 5:mating of foster mothers with vasectomized males Day 7.5: isolation of blastocyst and micro-injection of ES cells Day 7.5: uterus transfer to pseudo-pregnant foster mothers Day 23:birth of offspring, chimera selection Germ line transmission achieved by crossing Chimera with wt mice

  39. Germ line transmission(生殖系传递）: should have germ cells composed of both transgenic ES cells and host cells host ES identified by crossing chimera with WT mice e.g. agouti offsprings indicates germline transmission (ES cells from some 129 substrains ) chimera 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

  40. The microinjection set-up Arrangement of microscope, micromanipulators, transjector for injection method on an air table

  41. A Typical Laboratory for the Fabrication of microtools

  42. THE END

  43. DNA Concentration and Injection Buffer Used for Microinjection

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