Generation of Transgenic Mice

1
Generation of Transgenic Mice

• Microinjection ????
• Viral transgenesis ????
• ES aggregation ?????

2

• Microinjection(??????????)
• Pronuclear DNA microinjection (????)
• Blastocyst ES microinjection (????)

3
The Microinjection Experiment

• Pronucleus microinjection of gene constructs
  (DNA????)

--- to generate transgenic mice
4
e.g. Strain Information

• Type Transgenic

DescriptionTransgenic 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 Lab Stock No.003257
5
Flow Chart for Pronucleus Microinjection
Day 4/5 oviduct transfer to pseudo-pregnant
foster mothers
6
Related Concepts

• Pronucleus (??)
• The nucleus of an ovum or a spermatozoon after
  fertilization but before fusion of the
  chromosomes to form the nucleus of the zygote.
• ??????,????????,???????????????????

7

• ???,???

Polar body
Injection needle
Holding pipette
Male pronucleus
Female pronucleus
Nucleoli
Zona pellucida
8

• Donors(???)
• female mice for mating to produce fertilized
  eggs for DNA injection
• ?DNA??????????
• 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

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• Studs (???)
• fertile stud male mice to mate with donors
• ????????????
• Age 2-12-month-old
• Strains same as the donors, generally

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• Superovulation(???)

• administering gonadotropins to donors prior to
  mating, to increase the number of oocytes that
  are ovulated.
• ??????????????,????????,??????????

PMSG(????)to mimic the oocyte maturation effect
of FSH HCG( ?????????) to mimic the ovulation
induce effect of LH
11

• Pseudopregnancy(??)
• mating a female mouse with a infertile male, the
  stimulus of mating elicits the hormonal changes
  needed to make her uterus receptive.
• ???????????????????????,????????,????????????????
  ???,?????????

12

• Receptions (???)
• Female mice to serve as pseudopregnant
  recipients and foster mothers
• ??DNA???????????
• Age 6-20-week-old
• Strains random-bred mice or F1 hybrid mice
• e.g., Swiss webster, B6CBA F1 or CD1

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• Vasectomized male mice(????)
• Sterile stud male mice to mate with receptions
  for the production of pseudopregnant females
• ???????????????????,??????????,??????,???????????
  

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Two Methods to Vasectomized Male Mice
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• Vaginal plug (??)
• The precence of the vagianl plug, a harden plug
  deposit by a male mouse into a female mouse
  genital tract, indicates the successful mating
• ?????,??????????,???????,????????????????????????
  ?????

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• Transgenic mice(?????)
• the mice which are determined to the presence of
  the injected gene ???????????????
• 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
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• Summary of Preimplantation Development

• ?????,????
• ?????????? ????
• ?? ??????

18
Flow Chart for Pronucleus Microinjection
Day 4/5 oviduct transfer to pseudo-pregnant
foster mothers
19
Oviduct Dissection and Zygotes Isolation
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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 visible nucleoli within both
  pronuclei. DNA replication (complete 28hr
  post-HCG mostly)

21
Integration of Injected DNA Constructs

• INTERGRATION

• SYNGAMY

5
RA
Injected Construct
Native DNA
30
70
Transgenic Embryo
Mosaic Embryo
Delayed Integration
22
Flow Chart for Pronucleus Microinjection
Day 4/5 oviduct transfer to pseudo-pregnant
foster mothers
23
Oviduct Transfer(?????)
24
Flow Chart for Pronucleus Microinjection
Day 4/5 oviduct transfer to pseudo-pregnant
foster mothers
25

• Blastocyst microinjection of genetically altered
  embryonic stem (ES) cells
• (?????????)

--- to generate gene targeting mice
(KO/KI) ???????????
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• Knock out
• a mouse with a particular gene knocked out in
  only certain cells ???????????????????
• Knock in
• one of the mouse's own genes replaced with a new
  gene that the investigator wishes to study
• ?????????????????????????

27
e.g. Strain Information

• 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 Lab Stock No.002994
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Flow Chart for Blastocyst Microinjection
Day 1 PMSG injection
Day 3 HCG injection and mating
Day 5mating 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

• ????
• ??????????????????????
• ????????????????

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
Day 5mating 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
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Blastocyct Collecting
33
Development stage and quality of blastocysts for
injection

• A,B,C early stage blastocyst
• D almost 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 5mating 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 line transmission achieved by crossing
Chimera with wt mice
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• Chimera(?????)
• they have somatic cells composed of both
  transgenic ES cells and host cells????????,??????
  ??
• ???,??????ES??????

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.
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Flow Chart for Blastocyst Microinjection
Day 1 PMSG injection
Day 3 HCG injection and mating
Day 5mating 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
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• Germ line transmission(?????)
• 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
???????ES?????????????
?????
?????
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The microinjection set-up

• Arrangement of microscope, micromanipulators,
  transjector for injection method on an air table

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A Typical Laboratory for the Fabrication of
microtools
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• THE END

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DNA Concentration and Injection Buffer Used for
Microinjection
Plasmid DNA BAC DNA
Injecting Concentration Injecting Concentration 2 ng/ul 0.25ng/ul (0.005-1.5 ng/ul)
Injection Buffer Tris-HCL 10mM, pH 7.4 10mM, pH 7.4
Injection Buffer EDTA 0.1mM 0.1mM
Injection Buffer NaCl --- 100mM
Injection Buffer Ployamine --- 70 µM spermidine, 30 µM spermine