I.

Topic 1: Assisted Reproductive Technology (ART) - all techniques help for

infertile couples.
1. In Vitro Growth
- In Vitro growth is the method to bring the primordial or small follicle or small egg to
become a full growth follicle, or bring primordial spermatogonia to become mature
sperm.
- In Vitro growth is used when young ladies have cancer. So first, the doctor will take
out the ovaries and then freeze them. Then, the ladies will be treated with
chemotherapy or radiation to cure the cancer disease. When the ladies want to have
the babies, we can apply the in vitro growth to bring small follicles to full growth
follicles.
- During the growth. we have 2 method: in vitro growth, in vivo growth or
transplantation
+ In vitro growth: the important thing is we need to open the follicle. Moreover,
we need to use the poly vinyl pyrrolidone (PVP) or poly vinyl alcohol (PVA)
to make the extracellular matrix to increase the growth of the follicle.
+ In vivo growth or transplantation: is done in case of a young cancer lady.
When young ladies have cancer. So first, the doctor will take out the ovaries
and then freeze them. Then, the ladies will be treated with chemotherapy or
radiation to cure the cancer disease. When the ladies want to have the babies,
they transplant under the skin of the patients to get a germ cell line.
Xenotransplantation is a term used in the transplant of another species to the
Severe combined immunodeficient mice (SCID mice).
2. In Vitro Maturation:
- In Vitro maturation, we need to collect the oocyte cumulus-granulosa-complexes
(OCGCs) and then culture for specific times such as cows (24 hours), pigs (48 hours),
human (24 hours). During culture, we need the hormone: estrogen. If we don’t have
the estrogen, we use the theca cell, which is the cell that can produce estrogen.
- How to distinguish whether the oocyte that mature or not?
+ Base on the cumulus cell expand, after removing the cumulus expand by the
hydrolase enzyme, we can see the first polar body
- Application: Some ladies can not get the babies, and she has the high level of the
concentration of estrogen and a low level of luteinizing hormone. This may lead to the
girl's beauty and the overgrowth of the follicle. So, we need to break the follicle.
3. In Vitro Fertilization:
- In vitro fertilization is used when the couple is infertile, which means they cannot get
the babies for 1 years.
- Before doing the IVF, we must induce the sperm capacity and activity by pre-incubate
the sperm for 1 hours. It also helps the sperm penetrate in the zona pellucida easily.
- IVF need the artificial activation to activate the egg
4. Intracytoplasmic sperm injection (ICSI)
- ICSI is done when the male has few sperm.
- In ICSI, the sperm is injected directly to the egg.
- The methodology of ICSI:
 5. ROSI:
- ROSI is done when the male does not have mature sperm (often is the cause of
mumps disease). Normally, the mature sperm will be collected at the epididymis.
However, in ROSI, we will collect the round sperm at the testid by applying the
biopsy technique. We collect the sperm by on their size, mainly sperm is haploid and
already 2 meiosis
- The artificial activation must occur due to the round sperm unable to activate the egg.

6. Embryo Transfer:
- We need to follow the nature:
+ From 1 cell to morula: transfer in oviduct
+ Blastocyst: transfer in uterus
7. Preimplantation genetic diagnosis
- Pre-implantation genetic diagnosis is the genetic profiling of embryos prior to
implantation, and sometimes even of oocytes prior to fertilization. Scientists will use
the late 8 cell embryo to the blastocyst, which has the pluripotent characteristic to
avoid facing the ethical problem. Because, totipotent have the potential to develop
into new organisms (full-term development), meanwhile Pluripotent stem cells have
ability to differentiate and become almost any cell in the body.

TOPIC 2: Embryo

Fertilization

There are four stages to fertilization:

As the sperm approach the egg, they bind to the zona pellucida in a process known as sperm binding.

This triggers the acrosome reaction, in which the enzymes of the acrosome are freed. These enzymes then begin
to digest the zona pellucida and allow the sperm to tunnel toward the egg’s plasma membrane.

When the sperm cell finally reaches the egg cell, the plasma membranes of the two cells fuse together and the
sperm releases its genetic material into the egg..

The Stages of Early Cleavage

 - 1st Cleavage: Resulting embryo is 2 cells (i.e., 2 blastomeres or 2 cleavage cells)

- 2nd Cleavage: Embryo is 4 cells

- 3rd Cleavage: Embryo is 8-cells. Embryo will now undergo compaction just prior to the next division
leading to the 16-cell stage.

Cleavage, Compaction, Blastocyst Formation and Hatching

- As cleavage continues (compaction is not shown), fluid appears in between the cells after the morula
stage resulting in the early blastocyst. This fluid accumulates in an increasingly larger cavity called the
blastocoel as differences in the cell populations become evident. The blastocyst hatches from the zona
pellucida preparing it for implantation.

Compaction

- At the 8 cell stage the human embryo undergoes a process called compaction.

- It results from cells adhering together more tightly

- The embryo becomes more compact, but the cells still remain separate from each other

- The cell adhesion protein, E-cadherin appears at the time of compaction and causes the cells to adhere
together more tightly than previously.

Formation of the Blastocyst

- Fluid begins to appear between blastomeres

- Process is called "Cavitation"

- Begins ~4 Days after fertilization

- Produces "Blastocoele": the fluid-filled cavity of blastocyst

Hatching of the Blastocyst

- Hatching occurs just prior to implantation

- Embryo breaks through ZP due to proteases secreted by blastocyst

- Inability to hatch is a reason for infertility; could be due to altered zona pellucida or to the absence of
essential protease for zona digestion

- Often assisted hatching is done in vitro during ART procedures

- In mutant mouse embryos lacking ZP1, the zona pellucida is weak and the embryos hatch prematurely.

Formation of the Inner Cell Mass

TE cells synthesize the T-box transcription factor eomesodermin and the homeodomain containing, caudal-like
transcription factor Cdx2. Cdx2 is responsible for downregulating Oct4 and Nanog, two transcription factors of
the ICM cells.

Oct4, Stat3 and Nanog are critical for maintaining the pluripotency of ICM cells.
1. Oct4 is expressed first, and blocks cells from taking on the trophoblastic fate

2. Nanog is expressed late and prevents ICM cells from becoming hypoblast cells.

3. Stat3 (phosphorylation) stimulates self-renewal of ICM cells.

The pluripotency of these stem cells is dependent on their retaining the expression of these three transcription
factors.

LIF is used in mouse embryonic stem cell culture. It is necessary to maintain the stem cells in an
undifferentiated state.

Removal of LIF pushes stem cells toward differentiation, but they retain their proliferative potential or
pluripotency.

- Kind of embryo:

+ Fertilized embryo ( IVF) : can produce ES cell

+ Parthenogenetic embryo: can produce ES cells. Trophectoderm cells develop poorly in

placenta + consist only of maternal gene, lose paternal gene in ICM => cannot become
human. In order to produce diploid embryo, NT embryo is treated with CB for 6 hr
immediately after activation

+ Androgenetic embryo: can produce ES cell. Consist only paternal gene, lose maternal
gene => cannot become human
+ Tetraploid embryo: CANNOT establish ES cell, it establishes placenta => cannot produce
baby

+ Cloning embryo: -SCNT: can produce ES cell

=> Fertilized embryo & cloning embryo can form baby

- Zygote teaching activation: ZGA

- To detect ZTA, we use total mRNA to apply the RT- PCR or we can use western blotting to
get the mRNA. However, we can find out the new mRNA synthesize, that mean the nascent
mRNA

- During embryo development, zygote teaching activation also important: 1st 2nd day use
mRNA of mother, explain for the bigger of egg

+In humans, cows: from 8 cells to 16 cells.

+ In mouse: 2-cell embryo

- Expression of nascent mRNA in 2-cell embryos (include Br-UTP, DAPI, Merge)

+Cloned embryos: 2-4% -> low level of nascent mRNA, low zygote teaching activation

+Fertilized embryos: 70-80%

- The low success rate (1-2%) in cloning animals is believed to be associated with: Abnormal
gene expression (oct3/4, nanos….) , Abnormal epigenetic modifications

- Abnormal gene expression were found in placentas and livers of clones by RT-PCR,
Northern blotting, and microarray

- However, these methods are useful for analysis of the total mRNAs present, different from
those of newly synthesized mRNA This lack of information about zygotic transcription => It’s
important to examine the nascent mRNA at each stage of embryo development

The transcriptional activity of nascent mRNA during preimplantation development of mouse

embryos

- Application for 2 cell-embryo (totipotency): can develop into a full term baby
(totipotency can develop into full term body), split embryo to produce more babies.

- Application: PGD (preimplantation genetic diagnosis): take 1 cell to analyze the

disease => bioethical issue: after 1st differentiation, they lose potent to become baby, they are
pluripotent (can differentiate into any part of body) -> waiting until late 8-cell embryo stage
and perform PGD

+ Use blastocyst to produce chimeric animal and ES cell

+ In a transgenic animal, foreign genes should be transferred into MALE pronucleus because:
male pronucleus has a high level of gene expression (chromosome decondense) , so we transfer
foreign DNA to male pronucleus. Appy to male and female pronucleus to produce the
transgenic animal, apply to totipotent to increase the number of blastocyst by splitting 2-cell
embryo to make 2 animals. The 4 cells make 4 animals.

TOPIC 3: human recombinant protein (pharmacy)

-Pharmacy is the science and technique of preparing, researching, and distributing medicinal
drugs. Pharmacy as a career instructs one about how to prepare medicines, and recommend
dosages that patients should receive so that they can recover from their illness, or remain
healthy.

-In pharmin, we use animal body or bacteria to produce human recombinant protein

-Therapeutic proteins are used to treat patients suffering from cancers, heart attacks, strokes,
cystic fibrosis, diabetes, anemia and hemophilia. These proteins are produced by using
microbial fermentation on cell cultures, in transgenic animals and in transgenic plants.

-Method 1: Classic method to produce human therapeutic proteins from bacteria:

+Synthesis of the DNA containing the nucleotide sequences of the A and B polypeptide
chains of insulin.

+Plasmid + restriction enzyme

+Insertion of the insulin gene into plasmid (circular DNA)

+ Restriction enzymes cut plasmid DNA

+ DNA ligase agglutinates the insulin gene and the plasmid DNA Plasmid + insulin gene

+ Introduction of recombinant plasmids into bacteria: E. coli

+ Bacterium reproduces => the insulin gene replicates along with plasmid E. Coli

+Formed protein partly of a by product the A or B chain of insulin

+Extraction and purification of A and B chain

+Connections of A- and B-chain by reaction forming disulfide cross bridges results in Pure
synthetic human insulin
-Method 2: Novel method to produce human therapeutic proteins by produce transgenic
animal:

+We can inject human recombination of protein gene into the male pronucleus

+ Or we can combine the human recombinant protein gene with sperm have break
down the membrane by ICSI. We can inject sperm and foreign gene into mature oocyte
⇒ improve the success of transgenic animal

-Method 3: The other method to produce transgenic animal to produce recombinant protein
by SCNT :

+We start with human recombinant protein genes and inject into the somatic cell.

+ From the somatic cell, we use Somatic cell nuclear transfer embryo to produce full term
development:

+Firstly, breakdown somatic cell membrane and injects its nucleus inside enucleated oocyte

+Afterwards, we need to Activation to induce MPF down

==>Application of SCNT:

- SCNT is used to produce transgenic animals. We start with human recombinant

protein genes and inject into the somatic cell.
- Preservation of the genome, especially endangered animals or extinct animals.

+ For example: We can apply SCNT to bring back the mammus. First, we can take
the somatic cell from the mammus (freeze in Russia). Then, we transfer the somatic
cell (already breaking down the cell membrane) to the matured oocyte from another
elephant to get the cloning animal. Moreover, we can apply the same technique to
rescue the Bos Gaurus from the oocyte from the Bos Taurus. So, SCNT can occur
between different species with different chromosomes.

- human recombinant protein:

+ started with the forein gene into the somatic cell using SCNT to produce the
transgenic cloning-> produce the human-recombinant protein
- Process of human-bio organ
+ firstly, knock out the organ-genetic related gene
+ secondly, know-out cell for cloning -> produce organ involved in gene
knocked out cloning blastocyst
+ induce chimeric by injection Human IVF cell in that blastocyst
- SCNT can be used to improve the quality of meat and milk. By selecting the good
trait from the animal, the scientist will culture this fibroblast cell. Then, the scientist
will use this fibroblast cell to inject the matured oocyte from another cow. Finally, the
scientist can get the cow that have a good trait

EX: there are many successes in therapeutic proteins:

+Human protein C
+Glutamic acid decarboxylase

+Anpha-lactalbumin

+CFTR (Cystic fibrosis transmembrane conductance regulator

- Vaccination is a simple and effective way of protecting people against harmful diseases,
before they come into contact with them. It uses your body’s natural defenses to build
resistance to specific infections and makes your immune system stronger. Vaccines train your
immune system to create antibodies.

- Most vaccines are given by an injection, but some are given orally (by mouth) or sprayed
into the nose.

**How do vaccines work?

- Vaccines work by training and preparing the body’s natural defences – the immune system
– to recognize and fight off viruses and bacteria. When a person gets vaccinated against a
disease, their risk of infection is also reduced.

+Vaccines contain either killed or weakened versions of the virus that causes the disease or
a small part of it, such as a protein or nucleic acid. When you get a vaccine, your immune
system recognises these as foreign. It responds by creating memory cells and antibodies
that protect you against future infection.

**For example: A vaccine prevent covid-19:

+The coronavirus that causes COVID-19 has spikes of protein on each viral particle. These
spike proteins allow the virus to attach to cells and cause disease.

+The vaccines in development help the body to “recognise” these spike proteins as foreign
and fight the coronavirus that has them. The vaccine will protect a person who receives it by
lowering their chances of getting COVID-19 if they encounter the coronavirus.

⇒ If you have already had COVID-19 and recovered, you may have some natural immunity to
contracting the disease again
TOPIC 4: HUMAN-BIO ORGAN

- Use Xenotransplantation – use organs from different species to the human and
explain the method to knock out 1,3 alpha – galactosyltransferase of the cell line. Then
scientists use cell lines already knocked out by 1,3 alpha galactosyltransferase to make
the cloning and nuclear transfer into the pig and nuclear transfer writing in detail. For
example, after enucleation, the pig, injection with alpha galactosyltransferase knock out
the cell line and then the activation treatment with the cytochalasin B and then they go
to the zygote and then development and then we can get the pig with knock out alpha
galactosyltransferase

Transplants from pigs to primates are subject to vigorous immunologic rejection involving
both innate and adaptive immune responses. The strong effects are from products of α (1,3)-
galactosyltransferase in pigs. To obtain transplantable organs from the animal donor, the Gal
α (1,3) Gal antigen must be removed from xenograft cell surfaces. The best method to rid off
Gal α (1,3) Gal epitopes is the inactivation of the gene encoding GGTA1, which acts as a
catalyst for the Galα(1,3)Gal epitope forming reaction. Genetic recombination can be used to
replace the wild-type GGTA1 gene with a mutant variant, preventing the production of the
enzyme. =>.knocked out the GalT gene by homologous recombination in cultured fetal
fibroblasts, followed by nuclear transfer into enucleated ova and implantation of the resulting
modified embryos into receptive sows. The process was repeated, to knock out the second
allele for this enzyme, and the resulting offspring no longer expressed Gal. With this method,
we use Lentiviral vectors with siRNA, which are capable of infecting a wide variety of
dividing and non-dividing cells, integrate stably into the host genome, and result in long term
expression of the transgene. Pig organs were no longer affected by high titers of anti-Gal in
primate recipients, thus largely eliminating the problem of hyperacute rejection (HAR) and
markedly extending xenograft survival.

Further advances in techniques for genome editing, including the use of Zinc Finger
nucleases, Talens (transcription activator-like effector nucleases) and most recently,
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and their
associated RNA-guided DNA endonuclease #9), have made it increasingly feasible to change
the pig genome in ways intended to improve the success of xenotransplantation. All of these
techniques have been used to modify the genome through nuclear transfer of nuclei from
gene-edited somatic cells. (cái này ghi cũng được, không ghi cũng được =)))

- How to improve the success of cloning in this technique

That mean improve the histone acetylation by treatment with histone deacetylation inhibitor
for 10h of somatic cell nuclear transfer (SCNT) and we can increase the zygote teaching
activation, increase the success of embryo development to blastocyst -> increase the
frequency of cloning embryo to the full term.

SCNT: 1 technique of ET
- Need: recipient egg, donor cell
- Take out DNA of the recipient egg called Enucleation (take a cell out of the
membrane but not break it.
+ In mouse, we can see correctly location of Metaphase II because of low lipid in
cytoplasm => cytoplasm has white color
+ In pig, cow, cytoplasm is dark color, because high lipid in cytoplasm =>cannot see
the spindle, need to base on the location of 1st polar body
+ Normally, the chromosome located near the 1st polar body
- After enucleation, from somatic donor cells , based on size should select cell 2n:
Selective cell: select small cell at G0-G1 phase and transfer into enucleated oocyte.
And need to break down the cell membrane by pipetting and injecting the somatic cell
into the nucleus oocyte and incubating for 1h.

after incubate 1hr: we need to do the activation method: this method is different for
each type of species

+ mouse using the chemical activation as STRONTIUM ( The ability of strontium to

provoke calcium oscillations appears to be more physiologically sound than alternative
methods of oocyte activation that produce a monotonic rise in calcium. this treated
calcium oscillations lead to improved blastocyst composition

+ pig: using the electron activation

 + cow: using the 6-DAMP

- During activation, treating the mature oocyte with CB for 6h is enough to prevent
polarization. CB: cytochalasin B: inhibit the polymerization, CB will inhibit function
of micro actin =>, inhibit cell division => produce diploid embryo

-After transfer and incubate, MPF down regulation, which is dephosphorylation. So,
chromosome condense, nuclear membrane break down, spindle establish,
chromosome go to metaphase like-structure (not metaphase because we choose cell at
G0-G1 phase so centromere still not duplicated yet so it mono polar)

- If not break down donor cell membrane: protein cannot move inside => cannot
reprogram
SCNT has become a unique and powerful tool for epigenetic reprogramming research
and gene manipulation in animals since "Dolly," the first animal cloned from an adult
cell was reported in 1997.
-Somatic cell reprogram to become totipotent cell by egg: SCNT cloned animal

Remove cytoplasm of donor cell because the injection of somatic cytoplasm into
oocytes before ICSI causes a decrease in preimplantation development.
Improve histone acetylation: 2 steps
- Step 1(10h for enhance chromosome relax): After SCNT, during activation,↑

histone acetylation (=deacetylation inhibitor) to make chromosome more relax

and can receive a lot of reprogram factors of egg, treat with CB for 6h is enough

to prevent polar body. CB: cytochalasin B: inhibit the polymerization, CB will

inhibit function of micro actin => produce diploid embryo

+ We treat with CB at G0 – the DNA is 2n. Waiting until DNA synthesis => G1 =>
G2. After that we treat for 6h => chromosome separate (anaphase, metaphase)
- Step 2: Wait until zygote teaching activation, treat with histone deacetylation of 8-
cell in cow, pig
- Inhance of reprogram cell: induce the chromosome relax, increase histone
acetylation by inhibit the histone deacetylation, induce histone methylation

-Method use pig body to produce human recombinant protein and method base on the
success of mouse with the rat pancreas:

We use the pig cell, after that we culture the pig fibroblast cell line and knock out the
organ involving tissue. And then we use this cell line for cloning and when go to the
blastocyst, we can collect the human cell and after that, we use the iPS technique to
reprogram the human differentiated cell become pluripotent cell (iPS cell), and then
we injection to the cloned pig have already knock out organ related gene and then if
success the cloning, and get the the human recombinant protein.

- Application of iPS technique.

For the patients who have complex blindness. First, we take the somatic cell from this
patient, then we reprogram it to become iPS cells, which have pluripotency
characteristics. After that we can differentiate into the retinal layer. The retinal cell
membrane is the cell type in the eyes that helps for vision. If successful for the retinal
layer membrane, and then transplant to tissue again to the blindness patient.
The process of iPS cell reprogramming

In mammals, germ cells undergo a different epigenetic modification process from

somatic cells throughout the developmental stage. This process continues until after
fertilization, at which point the epigenetic modification is completed. This final
epigenetic change allows the zygote to enter a state of totipotency such that a new
cycle of normal development to form an entire organism can be initiated. In the case
of nuclear transfer, the egg nucleus carrying the haploid number of chromosomes is
replaced by the diploid somatic nucleus. Example: The creation of Dolly the Sheep by
somatic cell nuclear transfer

A somatic cell from the patient is used to derive induced pluripotent stem cells
(iPSCs). The iPSC colonies are characterised to ensure pluripotency markers are
present, they form teratoma or embryoid body and they have stable chromosomes. It
may take up to three months to derive and validate iPSC lines. The validated iPSC
colonies are differentiated to form optic vesicle structures, which contain retinal
pigment epithelium and neural retinal cells. Mature retinal cells can be used for
confirming the pathogenicity of newly-discovered genetic variants, modelling of
developmental or degenerative retinal disease, testing of pharmacologic agents or
gene therapy and autologous cellular therapy
1. Creating iPSC from Patients:

Using Pluripotent Stem Cells: An alternative method to obtain patient-specific retinal cells is to
use patient-derived adult stem cells for differentiation into retinal lineages.

Induced Pluripotent Stem Cells :Since the original description of the iPSC protocol by
Yamanaka, there has been significant development in the reprogramming approach, and many types
of somatic cells have been successfully induced into a pluripotent state

Validation of Human iPSC Line: The key defining features of iPSC are the self-renewal capacity
and the ability to produce all three germ layers.

2. Creating Retinal Tissue from iPSC

Derivation of Retina Lineages: there are two broad approaches: one by default differentiation of

iPSC into neuroectodermal lineages and through directed differentiation .

iPSC to Photoreceptor Cells: deriving photoreceptor cells using human iPSC from dermal
fibroblast to generate retinal.

- Ethical: how to overcome the ethical problem:

1/ knock out the spermatogenesis so that human iPS cells cannot contribute to the germ cell.

2/ Alternative proposals involving embryonic destruction. Two successive proposals were suggested
to forestall this possible rejection, both using SCNT:

(a) –‘therapeutic cloning’ or ‘research cloning’.

(b) – creation of human cytoplasmic hybrid embryos (cybrids).

2/ Alternative proposals to obtain hES‐like cells from embryos with no development potential

•   use of poor‐quality embryos rejected by IVF centres;

•   use parthenogenetically created embryos;

•   use created embryos that are defective and unable to implant: altered nuclear transfer, OAR.

3/ Alternative proposals that do not involve the destruction of embryos: obtain hES cells without
having to destroy human embryos. These are:

•  using germ stem cells;

•  using blastomeres harvested by embryo biopsy; and

•  reprogramming somatic cells to embryonic‐like cells

Topic 5: Cell therapy

-What is the stem cell & how many kinds of stem cell:

Stem cells are found in all of us, from the early stages of human development to the end of
life. All stem cells may prove useful for medical research, but each of the different types has
both promise and limitations.

Stem cells can be classified by the extent to which they can differentiate into different cell
types. These five main classifications are totipotent, pluripotent, multipotent, Oligopotent or
unipotent.

Type of stem Developmental potency Examples

cells

Totipotent Ability to differentiate into all cell zygote formed at egg fertilization
types and a functional organism and the first few cells that result
from the division of the zygote

eg: zygote, 2 cell embryos,4 cell

embryos, early 8 cell embryos,

Pluripotent Ability to differentiate into almost embryonic stem cells and cells that
all cell types but cannot form a are derived from the mesoderm,
functional organism endoderm, and ectoderm germ
layers that are formed in the
beginning stages of embryonic
stem cell differentiation, iPS cell

Multipotent The ability to differentiate into a hematopoietic (adult) stem cells

closely related family of cells. that can become red and white
blood cells or platelets, bone
marrow stem cell, tế bào gốc mỡ
adipose stem cell
 Oligopotent The ability to differentiate into a lymphoid (adult) that can
few cells. differentiate into various blood cell
such as B cell and T cell

Unipotent The ability to only produce cells Muscle (adult) stem cell,
of their own type, but have the epidermal stem cell
property of self -renewal required
to be labeled a stem cell.

Classification of stem cells on the basis of their sources: The easiest way to categorize stem
cells is by dividing them into two types: Early or embryonic and mature or adult.

- How to produce embryonic stem cell?

Produce ES cell:

• Removing the zona pellucida by acid tyrode for 10-20 second => zona pellucida free
blastocyst

• Preparing the 96 well dish:

- Put about 50 μl (1 drop) of 0.1% gelatin/per well and incubate for 30 min. Help the cell
attach to dish

- Take out of 0.1% gelatin by pipetting.

• Preparing fetus cell:

- take out fetus cell (kept in –80 - 180℃), mix with DPBS for thawing and then centrifuges
for 1000 rpm* 10 min. (put 9 ml DPBS with 1 ml fetus cell, and then centrifuge).

- Take out of PBS and then mix of fetus cell by 12 ml DMEM

• Preparing culture medium:

- DMEM. Put about 150 – 200 μl of fetus cells in DMEM medium per well and then incubate
for 1 day (So, the treatment of culture well dish with gelatin and fetus cells must do 24 h
before culture of morula or blastocyst.

• Change of medium: The color of the culture medium will change from pink to yellow (3-4
day after culture). Replace the old medium with a fresh medium.

- Take out of medium and then wash by PBS.

- Treat with 50 μl Tripsin per well for 3-5 min, then mix with 150 μl DMEM, after that mix
and transfer to a 24 well dish.

Culture the multi dish for 10-14 days in the incubator at 370C, 5% CO2. During this period,
trophectoderm (TE) cells will attach to the surface of gelatin and ICM can be seen to grow.

Therefore, LIF is used in mouse embryonic stem cell culture. It is necessary to maintain the
stem cells in an undifferentiated state.

Removal of LIF pushes stem cells toward differentiation, but they retain their proliferative
potential or pluripotency.

- iPS cell: established to pluripotent gene from somatic cell and can be reprogrammed to
become the pluripotent cell. iPS cells have pluripotency, iPS cells (contribute fetus) injected
into tetraploid embryos ( contribute placenta) will produce babies. iPS cell can differentiate
into the germ line cell (pluripotency) -> make embryo

- Expression of ES cell markers, chromatin methylation patterns, embryoid body formation,

teratoma formation, viable chimera formation, pluripotency and the ability to contribute to
many different tissues in vitro.

- iPS cells will inject into tetraploid embryos => produce baby

- 3 basic methods to reprogram somatic cells into totipotent stem cells or pluripotent
stem cells:

+ SCNT by injection of a somatic cell into an enucleated oocyte in order to produce

totipotent cell (cloned animals)

+ Fusion of somatic cell with embryonic stem cells

+ Introducing 2 to 4 pluripotent genes, Oct4, Sox2, Klf4 and c-MyC into somatic cells .

- How to distinguish the stem cell line?

First, based on the pluripotent protein expressed that means we use the staining technique
looking for the Oct4 expressed and Nanog expressed that means use the pluripotent marker.
Second, in vivo, another technique like take out the LIF

- In vivo system, we use the teratoma technique: transplant the stem cell line under skin and
after that you can make the differentiation become the 3 germ line: ectoderm, mesoderm, and
endoderm usually collected to distinguish that is the pluripotency characteristic cell line

- In vivo system yield:

Blastocyst injection to produce the chimeric. If they are the pluripotency, they can contribute
to the chimeric animal. Example: we have the es cell line is the black mouse inject in white
mouse blastocyst and we can get the chimeric that means your es cell line can contribute to
the different species and then your es cell line can contribute the organ into the other

- Oct-3/4 is a key transcriptional factor whose expression level governs inner cell mass and
embryonic stem (ES) cells.

- Inject ES cell to diploid => full- term development of ES cell

- Inject ES cell (contribute to fetus) to tetraploid(contribute to placenta)=> full- term

development

- Injection of ES cell into blastocyst (The method to produce chimera)

+The injection of embryonic stem cells into mouse blastocysts results in chimeric mice, in
which some cells in each tissue are derived from the ES cell lineage. If the ES cells have been
genetically modified, e.g., via gene targeting, those modifications can be passed onto
offspring from the chimeras

- Diploid blastocyst injection to produce chimera mouse

+ ES cells from B6D2F2 injected into blastocyst received from ICR mouse

+ ES cells received from B6D2F2-GFP mouse injected into tetraploid blastocyst received
from normal B6D2F1 mouse.

*GFP: Green Fluorescent Protein is collected the green protein from the jellyfish, glowing
under the uv light

=> This is strongest evidence that ES cell could differentiate to all functionally normal tissues

And the last technique is tetraploid injection of ES cell line, if you get the full term
development, that result more strongly demonstrates that the ES cell line is the perfect es cell
line and can contribute to all the organs in the body.

Topic 6: Application of SCNT

summarize the procedure of scnt :

SCNT: 1 technique of ET
- Need: recipient egg, donor cell
- Take out DNA of the recipient egg called Enucleation (take a chromosome out of the
membrane but not break it)
+ In mouse, we can see correctly location of Metaphase II because of low lipid in
cytoplasm => cytoplasm has white color
+ In pig, cow, cytoplasm is dark color, because high lipid in cytoplasm =>cannot see
the spindle, need to base on the location of 1st polar body
+ Normally, the chromosome located near the 1st polar body
- After enucleation, from somatic donor cells , based on size should select cell 2n:
Selective cell: select small cell at G0-G1 phase and transfer into enucleated oocyte.
And need to break down the cell membrane by pipetting and injecting the somatic cell
into the nucleus oocyte and incubating for 1h.

after incubate 1hr: we need to do the activation method: this method is different for
each type of species

+ mouse using the chemical activation as STRONTIUM ( The ability of strontium to

provoke calcium oscillations appears to be more physiologically sound than alternative
methods of oocyte activation that produce a monotonic rise in calcium. this treated
calcium oscillations lead to improved blastocyst composition)

+ pig: using the electron activation

+ cow: using the 6-DAMP or calcium ionophore

- During activation, treating the mature oocyte with CB for 6h is enough to prevent
polarization. CB: cytochalasin B: inhibit the polymerization, CB will inhibit function
of micro actin => inhibit cell division => produce diploid embryo

-After transfer and incubate, MPF down regulation, which is dephosphorylation. So,
chromosome condense, nuclear membrane break down, spindle establish,
chromosome go to metaphase like-structure (not metaphase because we choose cell at
G0-G1 phase so centromere still not duplicated yet so it mono polar)
- If not break down donor cell membrane: protein cannot move inside => cannot
reprogram
SCNT has become a unique and powerful tool for epigenetic reprogramming research
and gene manipulation in animals since "Dolly," the first animal cloned from an adult
cell was reported in 1997.

How to get a good successful embryo transfer?

we need to choose the right position which is the destiny of embryo will be placed

+from 1-cell embryo(zygote) to morula: we put it in the oviduct

+ from blastocyst: it is placed in the uterus

How to prepare a host mother for embryo transfer?

Application of SCNT?

- SCNT is used to produce transgenic animals. We start with human recombinant

protein genes and inject into the somatic cell.
+ From the somatic cell, we use the Somatic cell nuclear transfer embryo to
produce full term development: Firstly, breakdown somatic cell membrane
and inject its nucleus inside an enucleated oocyte. Afterwards, we need to
Activation to induce MPF down
- Preservation of the genome, especially endangered animals or extinct animals. For
example: We can apply SCNT to bring back the mammus. First, we can take the
somatic cell from the mammus (freeze in Russia). Then, we transfer the somatic cell
(already breaking down the cell membrane) to the matured oocyte from another
elephant to get the cloning animal. Moreover, we can apply the same technique to
rescue the Bos Gaurus from the oocyte from the Bos Taurus. So, SCNT can occur
between different species with different chromosomes.
- human recombinant protein:
+ started with the forein gene into the somatic cell using SCNT to produce the
transgenic cloning-> produce the human-recombinant protein
- Process of human-bio organ
+ firstly, knock out the organ-genetic related gene
+ secondly, know-out cell for cloning -> produce organ involved in gene
knocked out cloning blastocyst
+ induce chimeric by injection Human IVF cell in that blastocyst
- SCNT can be used to improve the quality of meat and milk. By selecting the good
trait from the animal, the scientist will culture this fibroblast cell. Then, the scientist
will use this fibroblast cell to inject the matured oocyte from another cow. Finally, the
scientist can get the cow that have a good trait
Topic 7: IVG

- In Vitro growth is the method to bring the primordial or small follicle or small egg to
become a full growth follicle, or bring primordial spermatogonia to become mature
sperm.
- In Vitro growth is used when young ladies have cancer. So first, the doctor will take
out the ovaries and then freeze them. Then, the ladies will be treated with
chemotherapy or radiation to cure the cancer disease. When the ladies want to have
the babies, we can apply the in vitro growth to bring small follicles to full growth
follicles.

- Male and female:

+ Male: in vitro growth and transplantation of primordial oocytes: under skin, under surface
of kidney. In vitro culture system and in vitro transplantation

+ In IVG of male germ cell: collect biopsy of tissue from testis, transplant piece of testis
under skin, under kidney, inside testis tube

- Method applied for IVG: in vitro culture

- Important point of IVG:we need to open the follicle and add PVA/PVP to mimic the
extracellular matrix to increase the growth of the follicle.

- In vivo: Xenotransplantation, use SCID mouse (severe combined immunodeficient mice)

Xenotransplantation is any procedure that involves the transplantation, implantation or

infusion into a human recipient of either (a) live cells, tissues, or organs from a nonhuman
animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact
with live nonhuman animal cells, tissues or organs. The development of xenotransplantation
is, in part, driven by the fact that the demand for human organs for clinical transplantation far
exceeds the supply.

- Location of male germ cell for transplantation: 3 locations: inside testis, kidney, under skin
of SCID mouse

- 2 method for transplantation: under skin, under kidney

- Compare the success of transplant under skin, under kidney, in surface of the kidney and
inside testis

- Transplant under skin is the most effective way

- Application:

- If some animals die very early during fetus development, the germ line develops very early.
Mouse pregnant are 19.5 days but in cloning animals, mostly they die at 9.5 days. But in 7.5
days, the germ cell is already established. We can collect the primordial germ cell from the
dead fetus and transplant it under the skin, inside the testis, in surface of the kidney. Then we
can get matured sperm but sometimes you can get only round sperm => ROSI techniques

- We apply ROSI when we don’t have matured sperm: no mature spermatozoa are obtained
during a testicular sperm extraction, mumps disease.

- Method to collect round sperm: use biopsy to collect small tissue in testis. Normally, the
mature sperm will be collected at the epididymis. However, in ROSI, we will collect the
round sperm at the testis by applying the biopsy technique. We collect the sperm by their
size, mainly sperm is haploid and already 2nd meiosis.

=> After injection, nothing will occur so it needs artificial activation (kích hoạt nhân tạo).
Round sperm cannot elongate to enter the egg so we need artificial activation. In mouse: use
strontium, in pig use calcium freeze medium, in cow use 6-DAMP

-ROSI has been proposed as a treatment for men in whom mature sperm forms (elongating
spermatids or spermatozoa) cannot be identified for intracytoplasmic sperm injection (ICSI)