Cell Cycle Role in Genetics & Development

1. Mitosis & meiosisa. Reproductive cloning Somatic cell nuclear transfer (SCNT)b. Therapeutic cloning (stem cells)

2. Meiosisa. Independent assortment Recombinant chromosomesb. Crossing over Recombinant chromosomes (Linked genes)c. Non-disjunction Aneuploidy (chromosome number)

Reproductive Cloning: Somatic Cell Nuclear Transfer (SCNT)

I. What is cloning?

Clones: a group of genetically identical individuals

a. Descended from the same parent by asexual reproduction (occur naturally)b. Produced by mitotic division from an original cell (occur naturally)c. Produced by 'embryo splitting'. (can occur naturally at the two cell stage to give identical twins; in cattle)

Reproductive cloningd. The creation of one or more genetically identical animals

By transferring the nucleus of a body cell into an egg from which the nucleus has been removed. Somatic Cell Nuclear Transfer (SCNT)

Advantageous of producing genetically identical clones

a. For research purposes such as biological controlsb. Commercial applications (uniformity of meat products & animal management)

II. Somatic Cell Nuclear Transfer

SCNT is the principle method in embryo cloning Involves the interaction of donor nucleus and recipient cytoplasm (enucleated unfertilized one-cell egg) to reconstruct an embryo (proposed by Spearman,

1938)o Certain nuclei could direct development to a sexually mature adult

Donor cell (Karyoplast) –

Cellular nucleus with small amount of cytoplasm

Recipient cell (Cytoplast) – Metaphase II oocyte / Zygotes - Enucleated (no nucleus)

Two predominant methods of nuclear transfer ; based on different cytoplast used (Collas & Barnes, 1994 ; Ritchie & Campbell, 1995) a. Use metaphase II oocytes as cytoplast / recipient cellb. Use zygotes as cytoplast

Blastomeres as source of donor cells. The donor cells forced into the Gap Zero, or G0 cell stage, a dormant phase, which causes the cell to shut down but not die.

A. Metaphase II (M2) Oocytes Cytoplast B. Two pronuclear (2PN) Zygotic Cytoplasti. Incubate M2 oocyte in medium containing microfilament inhibitor cytochalasin D & DNA-specific fluorochrome Hoechst 3332 microfilament disrupted & cell membrane become elastic

ii. Enucleation: portion of cytoplasm containing 1 st polar body & metaphase II plate is aspirated out (using Enucleation pipette)

aspirated cytoplasm checked under UV to confirm

i. Incubate zygote medium containing microfilament inhibitor cytochalasin D & microtubule inhibitor colchicine

in bovine/porcine : zygotes have to be centrifuged (no damage)

the pronuclei in zygotes can be confirmed under direct interference contrast (DIC microscopy)

ii. Enucleation: the pronuclei are aspirated (using enucleation pipette)

iii. using enucleation pipette, the karyoplast is inserted into the hole at zona pellucida

iv. karyoplast is expelled into the perivitelline space contact with the cell membrane of enucleated oocyte/zygote (cytoplast)

v. Cell fusion : induced by direct current electric pulse at 90o to the plane of contact activate the oocyte/zygote

vi. The activated egg cell/zygote is then quickly to begin forming an embryo.

Purpose I. To harvest stem cells Embryonic stem cells (ESCs) The egg containing the transferred nucleus is encouraged to divide until it reached the blastocyst stage, The cells of the inner cell mass are removed and cultured. These are known as embryonic stem cells, or ESC's.).Use for therapeutic cloning

Purpose 2: To produce identical animal cloneThe embryo is transplanted into a surrogate motherA perfect replica of the donor animal will be born

Cell Cycle: Oocytes Fertilized egg two pronuclei zygote Blastomeres (2-cell)

- Oocytes are developmentally arrested at the germinal vesicle in Prophase I (meiosis I)- By appropriate stimulation, resume meiosis, produce first polar body and arrest at Metaphase II (mature oocyte – unfertilized egg)- After fertilization, complete meiosis II and produce second polar body- After fertilization, male & female chromatin decondense produce two pronuclei zygote (male nucleus & female nucleus)- DNA replication mitosis (equal segregation of genetic material)- Zygote cleaves into two daughter blastomeres

Issue 1: Importance of cell cycle co-ordination in embryo reconstruction by nuclear transfer

C ytoplasmic states/environment of different cytoplast recipients Influenced by the level of maturation promoting factor (MPF) in cytoplast

a. In M2 oocytes, high MPF activity as oocytes are arrested at metaphase IIIn pronuclear zygote, low MPF activity as the cell cycle phases are completed

b. When nuclei is transferred into cytoplast with high MPF activity (M2 as cytoplast) Nuclear envelope breakdown (NEBD) Premature chromosome condensation (PCC)

DNA replication in reconstructed embryos (only at S-phase: 2C 4C)a. Chromosomal DNA only replicate once & only onceb. When nuclei transfer into high MPF activity cytoplast

Regardless of any phase, will perform DNA synthesis (DNA replication) Re-replication of replicated chromosome (G2) Produce incorrect DNA content in daughter cells Adverse effect to mitosis Chromosome damage, unequal segregation & chromosomal abnormalities (aneuploidy, polyploidy)

Only nuclei that in G1 phase can be transferred into M2 oocytes (cytoplast)

When nuclei is transferred into cytoplast with low MPF activity (eg Pronuclear zygotes)a. No NEBD and PCCb. All nuclei phases (G1, S and G2) perform replication depends on the cell cycle

Stage of the transferred nucleusc. The nuclei in daughter cells are in phase (4C 2C)d. The activated cytoplast is known as Universal Recipient

Sufficient time is needed to allow complete DNA replication in the donor nucleusto be initiated and completed before mitosis.

Extrusion of polar body (?)

Two distinct protocols for embryo construction by nuclear transfer when using M2 oocytes as cytoplast.1. Nuclei in G1 phase activated enucleated M2 oocytes2. Nuclei in G1/S/G2 phase activated enucleated M2 oocytes after the disappearance of MPF activity (low MPF activity)

Issue 2: not significant overall freq. of development of reconstructed embryo to blastocyst stages between unsynchronized blastomeres with universal donor & M2 cytoplastsi. % of development = % of G1 phase in Blastomeres (synchronization of blastomere will be the limiting step)

Use of embryonic nucleus as donorii. Aged cytoplast, maybe the cytoplasts were not at MII phase during embryo reconstruction

III. There are two techniques which are the variation of SCNT

A. Roslin Technique cloning of Dolly (lamb)

Developed by Ian Wilmut and Keith Campbell from Roslin institute, Uni Edinburgh in 1996Rosalin method , they used donor nucleus from adult cells (mammary gland cells) and cultured the cells (Issue 2)

Important principle: Synchronize the cell cycles of the donor cell and recipient cell (Issue 1) to ensure the nucleus is ‘in phase’ for development of embryo with the right DNA chromosomal content

Methods:1. 2N Donor cells from udder cells of Finn Dorset sheep (white face) and culture the donor cells in vitro2. The cells were taken and starved in deprived nutrient media, only enough for cells to live force cells to

be dormant and paused at G0 phase3. The M2 oocyte (low MPF level) as universal recipient from black face ewe. The nucleus (M2

chromosome & polar body) were removed (enucleation)4. The donor cells (karyoplast) were in contact, near the enucleated oocyte5. Use electrical pulse to fuse the donor cell & enucleated oocyte (mimicking fertilization by sperm – not so

correct as not all activated oocyte embryo)6. Current use for fuse = current use to activate oocyte (oocyte is activated immediately after fusion)7. Activate the oocyte for zygote development reconstructed embryo Culture until reach morula /

blastocyst implant in the uterus of foster mother (black face ewe)8. Embryo developed Dolly (Finn dorset sheep white face)

Success rate: 1 in 277 trials

Significance:1. Dolly, the first animal cloned from a cell taken from cultured adult cells

2. enhance the production of GM animalsIncrease livestock, as animal breeding strategies

3. open the field of reprogramming of nucleus rapid expansion of stem cell biology & stem cell therapies

Limiitation: cloning extinct species?- require functional chromosomes (donor)- require appropriate supply of oocytes &

MII chromosome

suitable surrogate mother

B. Honolulu Technique cloning three generations of mice Developed by Wakayama and Yanagimachi from Hawaii University in July 1998Donor cells: used 3 types of adult somatic cells Sertoli & neuronal cell (always G0 phase) and cumulus cell (G0/G1 phase). Focus on cumulus cell (2N nuclei) due to high successful rate.

Methods:1. Isolate cumulus cell from donor

cumulus cells, ovarian cells that surround the egg and are shed with it on ovulationCumulus cell donor: female B6C3F1 mice were superovulated induced by hormone (equineCG & hCG) cumulus-oocyte complexesThe cells are kept in HEPES-CZB at RT for 3 hours before injectionThe cells used immediately without in vitro culturing

2. Isolate Oocyte from female mice (black/brown B6D2F1) Oocytes transfer into HEPES-CZB + Cytochalasin B (making CM oocte elastic to form hole)The M2 chromosome spindle complex was aspirated using enucleation pipette (application of piezo-pulses)Enucleated oocyte transfer into CZB – Cytochalasin B for 2 hours before injection

3. Microinjection : Fusion of cumulus nuclei with enucleated oocyte using piezo-impact pipette driving unitNucleus removed from karyoplast (minimized amount of cytoplasm) injected directly into the cytoplasmand fused with enucleated oocyteDelay: Incubate for 1-6 hours in CZB medium allow chromosome condensationFactors in oocyte cytoplasm facilitate nuclear changes essential for development

4. Jumpstart / activation of oocyte The oocytes were activated in culture medium containing strontium (Sr2+) & cytochalasin BSr2+ activate oocytesCytochalasin B prevent subsequent polar-body formation & chromosome expulsion maintain chromosome number (2N) of the zygotes (prevent 2N become N)Cumulus-derived chromosome segregate 2N pseudo-pronuclei (pronuclei formed after normal fertilization)Activated oocyte embryo; then transfer to CZB medium to continue incubation morula/blastocyst

5. Embryo transfer into surrogate mother (CD-1 albino female mice) The mice mated with vasectomized CD-1 males (enhance successful rate mother physiologically ready to receive embryo)Embryo transfer into the oviduct/uteriCaesarian section to the mother produce young (clones of B6DF1)

High success rate!By repeating the procedure, the team created second and third generations of genetically identical mice.