Part I

Basic Experiment in
Embryology Laboratory
Bui Hong Thuy, Ph.D.
Associated Professor
School of Biotechnology,
International University
Email: bhthuy@hcmiu.edu.vn

1
How to kill a mouse for using in experiment?
Quick and humane sacrifice of a mouse
A. The cervical dislocation method
1. Place the mouse on the top of cage, break its neck
humanely by firm pressure at the base of the skull,
while at the same time pulling backward on the tail.
3
1
2

B. The CO2 inhalation method

2
 Dissection of reproductive organs of
a female mouse

A B

3 2

3
Dissection of reproductive organs of
a female mouse

4
Superovulation & Collection
of mature mouse oocytes

5
 Induce superovulation by hormone PMSG and hCG

• PMSG (Pregnant Mare Serum Gonadotropin): Increase the number of full-grow follicles.
• hCG (Human Chorionic Gonadotropin): Induce oocytes ovulation.

1. Schedule of hormone injection for collection of mature oocytes.

Day 1 Day 2 Day 3 Day 4
5h PM 5h PM 7-9h AM

PMSG hCG Oocytes

collection

2. Schedule of hormone injection for collection of fertilized embryos.

Day 1 Day 2 Day 3 Day 4 Day 4 Day 5
5h PM 5h PM 8-9h AM 7-8h PM 9-10h AM

PMSG hCG & Check Collect Collect

mate with plus 1-cell 2-cell
male embryo embryo
6
 Collection of mouse oocytes
Reposition forceps
Ovary
Cut
Pull back

Oviduct
Right
Uterus Left Infundibulum
Uterus Fat may be attached

Cut end of uterus

Eggs with
cumulus cells

Dissection of
reproductive organs
in a female mouse
Coils of oviduct
Swollen ampulla
With ridges in epithelium
12 hrs post-ovulation 7
 Collection of mouse oocytes
HESPES
medium 1 1
HEPES +
hyaluronidaze 2 3
Hyaluronidase là HESPES
enzyme có tác dụng
cắt polymer của
3 medium
hyaluronic acid
trong chất gắn kết Swollen ampulla
giữa các tế bào

2 2
2-3 min

8
 Collection of mouse oocytes
HESPES
medium 1

This drop is
HEPES + 2 3
hyaluronidaze
Remove HESPES
cumulus cells 3 medium
by pipeting

In vivo mature mouse oocytes

9
Collection of in vivo fertilized mouse embryos
Transfer oviducts into
drop of HEPES medium
Infundibulum

Embryos

Swollen ampulla
With ridges epithelium

Prepare HEPES
drops in culture dish
and cover with
mineral oil

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 Collection of mouse
spermatozoa for in vitro
fertilization (IVF)
and ICSI

11
Collection of mouse spermatozoa

12
 Collection of mouse spermatozoa

4 5

2
1

6 7 Incubate at
1 ml tube 37.5o for 1 hr
13
Culture fibroblast cells
from mouse tail

14
Collection and culture of mouse tail cells
Clean mouse tail by alcohol 70% and throw off its skin & fur 1

Wash three times with PBS and keep in the dish for 10 min 3

Culture dish is covered with 0.1% gelatin (at least 1 h before use) 4

Put bone of tail in the dish and cover with slide glass. 5
Keep in clean bench for 10 min.

Add culture medium (DMEM + 10% FCS) and culture 6

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 Culture fibroblast cells from tail of mouse
Tail skin

Cover glass

Tail bone
Culture for 1 week at 370C, 5% CO2. Replace old medium by fresh medium if
the culture medium change from pink to yellow. Culture medium: DMEM + 1
16
0% FCS. Note: Tail bone is good develop more than Tail skin.
Mouse fibroblast cells from tail

8-10 days we can get fibroblast cells

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Micromanipulation
• Intracytoplasmic Sperm
Injection (ICSI)
• Somatic cell nuclear
transfer (SCNT): Cloned
animals
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Equipments for ICSI and Nuclear Transfer

Microscope and micromanipulators system

Stereomicroscope Making micropipette Micropipetting microscope Co2 incubate

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 How to make micropipette

1
Making micropipette

3
Micropipetting microscope

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How to prepare injection dish

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How to inject sperm into oocyte cytoplasm
A mature oocyte (mice)
First polar body Metaphase II

900 7-8
100-200
micrometer
micrometer

Holding pipette

Inject sperm into oocyte cytoplasm near the holding

pipette and make an angle 900 with the metaphase II.

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ICSI Video

23
How to enucleate an oocyte

Diameter of e
nucleation pip
ette: 7-8 µm

40 µm

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How to remove donor cell membrane

25
How to inject somatic nucleus into oocyte

26
Full-video of SCNT in the mouse

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 Procedure of Nuclear Transfer in Mice
Oocyte
donor

Nuclear Activation with

transfer CB for 6 hrs
Oocyte
collection
Enucleation

Subsequently Embryo
culture for 1-2 hr culture

Donor cells Embryo

collection Surrogate transfer
mother
Embryo
culture
Caesarian section at
Somatic cell Remove donor 19.5dpc
nucleus donor cell cytoplasm

Blastocyst
Cloned mice ntES cell
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In vitro development of mouse embryo

2-cell embryo at 24 after ICSI 4- to 8-cell embryo at 36 after ICSI

Morula at 60 after ICSI Blastocyst at 72-80 after ICSI

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In vivo preimplantation development of
embryo

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 Embryo transfer
Transfer 2-4 cell embryo (into oviduct)

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Embryo transfer into oviduct

32
 Embryo transfer
Transfer blastocyst (into uterus)

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Embryo transfer into uterus

34
Transgenic Animals

Transgenic Animals by
gene targeted ES cells &
blastocyst injection

35
 Embryo transfer into oviduct

1. Diploid fertilized
blastocyst injection.
Injection of gene targeted ES cells in to normal
fertilized embryo at the expanded blastocyst stage

36
 Transgenic animals by blastocyst injection
What do we need?
ES cells DNA
Transfection
Positive, negative selection
Diploid blastocyst
(2n)

Negative
(GFP-) Positive
(GFP+) Diploid expanded
blastocyst injection

Proliferation of ES cells
carrying targeted gene

25-30 ES cells for 1 blastocyst

37
Video of blastocyst injection by piezo-action

Note: Only small ES cells are selected for injection

Optimal number of ES cells is 25-30/blastocyst 38
Video of 8-cell injection by XY laser
 How to produce chimeric mice
- Injection of ES cells into diploid blastocysts

B6D2F1 Blastocyst injection

ICR
Chimeric mice received from
B6D2F1 ES cells injected into
ICR diploid blastocysts 40
From ES cells, can we produce full-term
development?
- Injection of ES cells into tetraploid blastocysts

GFP mouse Blastocyst injection

ICR
Full-term development
derived from ES cells
Yes, we can produce the full-term development
from ES cells injected into tetraploid blasotycts 41
 Blastocyst
Blastocyst injection vs
injection VS 8-cell
8-cell injection?
injection

Blastocyte injection 8-cells injection

Using piezo action Using XYclone laser

Contribution of ES cells into chimera?

42
8-cell embryo injection in the mouse

8-cell embryos injected with GFP ES-cells developed to the blast cyst stage
Demonstration of ES Cell line
Blastocyst injection VS 8-cell injection?
Blastocyte injection 8-cells injection

45
 Eight-cell Embryo biopsy for
Preimplantation Genetic Diagnosis (PGD)

46
 Embryo aggregation
1. Remove zona pellucida by tyrode acid (pH: 2.5)
Before (intact After (without
embryos) zona pellucida)
CZB or Tyrode
KSOM acid

Few
second

Few
second
Tyrode CZB or 4-cell Embryo
acid KSOM

Note: The timing for Tyrode acid

treatment is very important, just few
seconds enough for zona pellucida
disappear (observe under stereo
microscope)
8-cell Embryo 47
Embryo aggregation in the aggregation plate
How to prepare aggregation plate

Use 35 mm culture dish

300–500 micrometer

Success rate more than 30-40% 48

Embryo aggregation by micromanipulator
Keep few seconds

Keep few seconds

Success rate more than 95% 49

Embryo aggregation

Chimeric mouse

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Transplantation of testicular
pieces under the skin

51
Xenotransplantation of testes

Honaramooz et al., Nature 2002; 418: 778-781

Mouse oocyte
52
Transplantation of germ cells
into the seminiferous tubules

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Epididymis Efferent duct

Rete testis

Seminiferous
tubules

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Pipette for transplantation

Injection into seminiferous

tubule

55
56
Open the injection site Insert micropipette
←Epididymis

←Testis

Start injection After injection

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Germ cells transplanted into
seminiferous tubules

58
4 months after TP Under excitation light

Donor: GFP transgenic mouse

Recipient: wild mouse treated with busulfan
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Transplantation of a whole
gonad in the recipient testis

60
Implantation of a whole gonad in the
recipient testis
Donor

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1. Transplantation of 12.5 dpc male
gonad into recipient testis

Progeny

10 weeks after TP ICSI

Testicular section
62
 Transplantation of PGCs with
gonadal somatic cells under the
kidney capsule

63
Video of Transplantation of PGCs with gonadal
somatic cells under the kidney capsule

64
A) Schematic shows the transplantation procedure was used.
Two pairs of sex-matched gonads at 12.5 dpc were
dissociated, reaggregated, and transplanted under the
kidney capsules of ovariectomized female recipient mice.

B) Appearance of male and female gonads (g) with adjacent

mesonephros (m) of 12.5-dpc embryos.
The mesonephros acts as a structure similar to the kigney that, in
humans, functions between the sixth and tenth weeks of embryological
life.

C) Appearance of gonadal dissociates of 12.5-dpc female gonads.

Arrows and inset indicate PGCs.

D-F) Appearance of grafts at 4 wk after transplantation under

the kidney (kid) capsule is shown. Male and female grafts
formed testis-like (white arrowheads) and ovary-like (black
arrowheads) tissues, respectively, in both B6-GFP (D and
E) and BDF1 (F) strains.

G) A testis-like tissue generated by mixing gonadal cells from

different strains (GFP and non-GFP mice). The chimeric
composition of the entire tissue was confirmed by GFP
fluorescence in the gross appearance (upper right panel)
and in a histological section (lower panel).
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Collection and in vitro culture for mature of
pig oocytes

Ovaries were collected Follicles 4-6 mm in

from abattoir Dissection diameter were
dissected from
ovaries

Collection of oocytes : *Aspiration Oocyte-cumulus- Oocyte-cumulus-

*Dissection
granulosa complexes complexes (OCGCs)
(OCGCs)
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In vitro culture for maturation of pig oocytes

38-42 h
OCGCs Culture medium: Mature oocyte
(M II)
TCM 199 + 10% FCS + 0.1 mg/ml
Na pyruvate + 0.08 mg/ml kanamycin
+ 0.1 IU/ml hMG
First polar body First polar body

Chromosome
67
Collection of pig small oocytes

68
Nuclear morphology of pig oocytes isolated from follicles of
various sizes

0.2- <1

1 - <2

2 - <3
Denuded
Pig ovary oocytes
3 - <4
Cumulus-oocyte- Fixed
granulosa cell and
complexes stained
4 - 6 mm (OCGs)
with 1%
orcein
Antral follicles

69
Samples were mounted on slides

10 or 15 l medium for
Mouse or Pig oocytes ,
respectively

Glass slide

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 Aceto-orcein staining
1) Fixative (acetic acid:ethanol 1:3 v/v) was passed twice via
introduction from one side of the cover slip and removal
from the other by absorbing it with a piece of filter paper.

2) Staining solution (1% aceto-orcein in acetic acid:ethanol

1:3) was then passed once, albeit in a similar manner.

3) After 5-7 min of orcein staining, destaining solution (acetic

acid:distilled water:glycerol: 1:3:1) was passed thoroughly to
remove excess stain, making sure that the oocytes were not
washed away.

4) The cover slip was sealed and stained oocytes were

examined under a light microscope (differential interference
microscope) to determine the stages of chromosome
morphology. 71
Chromatin configuration of growing oocytes in pig

Filamentous chromatin Stringy chromatin Germinal vesicle

FC SC GVI

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 Chromatin morphology of pig oocytes after isolation
from ovary
A B C

D E F

Oocytes collected from various sized follicles were fixed in acetic alcohol (1/3),
stained with 1% orcein, and examined under a differential interference
microscope. Chromatin configurations of oocytes at the germinal vesicle stage
were classified into 6 categories as follow: A. Filamentous chromatin stage, B.
Stringy chromatin stage, C. Germinal vesicle I stage, D. Germinal vesicle II
stage, E. Germinal vesicle III stage, and F. Germinal vesicle IV stage.
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 Chromosome morphology of pig oocytes after culture
in maturation medium (from GVI to Metaphase I)
A B C

D E F

Fully grown oocyte (GV I) were collected and cultured for maturation.
After 42h, oocytes were fixed in acetic alcohol (1/3), stained with 1% orcein,
and examined under a differential interference microscope. Chromatin
Morphology of oocytes as follow: A. Germinal vesicle I stage, B. Germinal
vesicle II stage, C. Germinal vesicle III stage, and D. Germinal vesicle IV
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stage. E. Diakinesis, F. Metaphase I.
Chromosome morphology of pig oocytes after culture in
maturation medium (from Metaphase I to Metaphase II)
A B

C D

Fully grown oocyte (GV I) were collected and cultured for maturation.
After 42h, oocytes were fixed in acetic alcohol (1/3), stained with 1% orcein,
and examined under a differential interference microscope. Chromatin
configurations of oocytes after GVBD as follow:
A) Metaphase I, B) Anaphase I, C) Telophase I, and D) Metaphase II.
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