Download 11转基因鼠的产生

Generation of Transgenic Mice
 Microinjection
 Viral
显微注射
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

Pronucleus microinjection of gene
constructs （DNA原核注射）
--- to generate
transgenic mice
e.g. Strain Information
Type： 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 Lab
Stock No.003257
Flow Chart
for Pronucleus Microinjection
Day 1:PMSG
injection
Day 3:HCG
Day 3/4:mating of foster
injection and mating
mothers with vasectomized males
Day 4:isolation of
Day 4/5: oviduct
oocytes and microinjection of DNA
transfer to pseudopregnant foster mothers
Day 46: tail
Day 23:
tipping and
analysis of
offspring
birth of
offspring
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。
雄原核，雌原核
Polar body
Injection needle
Holding
pipette
Male pronucleus
Female pronucleus
Nucleoli
Zona pellucida
 Donors(供体鼠）:
female mice for mating to produce fertilized
DNA injection
eggs for
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
 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
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
 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。
 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 F1 or CD1
 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。
Two Methods to Vasectomized Male
Mice
 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。
 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

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
Flow Chart
for Pronucleus Microinjection
Day 1: PMSG
injection
Day 3: HCG
Day 3/4:mating of foster
injection and mating
mothers with vasectomized males
Day 4: isolation of
Day 4/5: oviduct
oocytes and microinjection of DNA
transfer to pseudopregnant foster mothers
Day 46: tail
Day 23:
tipping and
analysis of
offspring
birth of
offspring
Oviduct Dissection and Zygotes
Isolation
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)
Integration of Injected DNA
Constructs
INTERGRATION
SYNGAMY
RA
~ 5%
Injected
Native
DNA
Mosaic Embryo
Construct
~30%
Delayed Integration
~70%
Transgenic Embryo
Flow Chart
for Pronucleus Microinjection
Day 1:PMSG
injection
Day 3:HCG
Day 3/4:mating of foster
injection and mating
mothers with vasectomized males
Day 4:isolation of
Day 4/5: oviduct
oocytes and microinjection of DNA
transfer to pseudopregnant foster mothers
Day 46: tail
Day 23:
tipping and
analysis of
offspring
birth of
offspring
Oviduct
Transfer
(输卵管移植）
Flow Chart
for Pronucleus Microinjection
Day 1:PMSG
injection
Day 3:HCG
Day 3/4:mating of foster
injection and mating
mothers with vasectomized males
Day 4:isolation of
Day 4/5: oviduct
oocytes and microinjection of DNA
transfer to pseudopregnant foster mothers
Day 46: tail
Day 23:
tipping and
analysis of
offspring
birth of
offspring

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

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
e.g. Strain Information
Type Targeted Mutation
Description
Mice 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
Flow Chart
for Blastocyst Microinjection
Day 1: PMSG
injection
Day 7.5: isolation
of blastocyst and
micro-injection of
ES cells
Germ line
transmission
achieved by
crossing Chimera
with wt mice
Day 3: HCG
Day 5:mating of foster mothers
injection and mating
with vasectomized males
Day 7.5: uterus
transfer to pseudopregnant foster mothers
After Day
23: birth of
offspring;
chimera
selection
Select 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 donors
Naturally mated donors
--- higher quality blastocyst
Flow Chart
for Blastocyst Microinjection
Day 1: PMSG
injection
Day 7.5: isolation
of blastocyst and
micro-injection of
ES cells
Germ line
transmission
achieved by
crossing Chimera
with wt mice
Day 3: HCG
Day 5:mating of foster mothers
injection and mating
with vasectomized males
Day 7.5: uterus
transfer to 2.5D pseudopregnant foster mothers
After Day
23: birth of
offspring;
chimra
selection
Blastocyct
Collecting
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
Blastocyst ES
microinjection
Uterus transfer
Flow Chart
for Blastocyst Microinjection
Day 1: PMSG
injection
Day 3: HCG
Day 5:mating of foster mothers
injection and mating
with vasectomized males
Day 7.5: isolation
7.5: uterus transfer to
of blastocyst and
micro-injection of
ES cells
pseudo-pregnant foster
mothers
Germ line
transmission
achieved by
crossing Chimera
with wt mice
After Day
23: birth of
offspring,
chimera
selection
 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
host
ES
ES
host
host
C57BL/6J-Tyrc-2J/J
C57BL/6(B6) chimera:
129 svj chimera:
B6 ES cells were injected into
albino B6 blastocysts.
129 ES cells were injected
into B6 blastocysts.
Flow Chart
for Blastocyst Microinjection
Day 1: PMSG
injection
Day 7.5: isolation
of blastocyst and
micro-injection of
ES cells
Germ line
transmission
achieved by
crossing Chimera
with wt mice
Day 3: HCG
Day 5:mating of foster mothers
injection and mating
with vasectomized males
Day 7.5: uterus
transfer to pseudopregnant foster mothers
Day 23:
birth of
offspring,
chimera
selection
 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 )
在后代中得到与ES来源小鼠品系相同毛色的小鼠
chimera
杂合子小鼠
纯合子小鼠
The microinjection set-up
Arrangement of
microscope,
micromanipulator
s, transjector for
injection method
on an air table
A Typical Laboratory for the Fabrication
of microtools
THE
END
DNA Concentration and Injection Buffer
Used for Microinjection
Plasmid DNA
BAC DNA
~ 2 ng/ul
~ 0.25ng/ul
(0.005-1.5 ng/ul)
Tris-HCL
10mM,
pH 7.4
10mM,
pH 7.4
EDTA
0.1mM
0.1mM
NaCl
---
100mM
---
70 µM spermidine,
30 µM spermine
Injecting Concentration
Injection
Buffer
Ployamine