1. Use microinjection technique in Reproductive, Cellular andUse microinjection technique in Reproductive, Cellular and
Molecular biologyMolecular biology
Some essays from the personal skills and experienceSome essays from the personal skills and experience
/A.Madich/A.Madich

2. Main directions to applyMain directions to apply
 Animal reproductionAnimal reproduction – Embryo bisection and identical twins production in bovine,– Embryo bisection and identical twins production in bovine,
Morulae aggregation and chimeras calves productionMorulae aggregation and chimeras calves production
 Transgenic technologyTransgenic technology –– DNA injection into pronuclear and 2-cell embryos inDNA injection into pronuclear and 2-cell embryos in
sheep and bovinesheep and bovine
 Biology of early developmentBiology of early development – Use trophoblastic vesicles cell culture prepared– Use trophoblastic vesicles cell culture prepared
micro surgically by biopsy from bovine blastocysts in the attempt to improve themicro surgically by biopsy from bovine blastocysts in the attempt to improve the
development of half-embryosdevelopment of half-embryos in vitroin vitro
 Reproductive BiotechnologyReproductive Biotechnology – Use bovine oviduct epithelial cells tissue culture– Use bovine oviduct epithelial cells tissue culture
(BOECs) and cell subculture to promote the viability and development of thawed(BOECs) and cell subculture to promote the viability and development of thawed
and dissected Bovine embryos as well as IV fertilized sheep oocytesand dissected Bovine embryos as well as IV fertilized sheep oocytes
 Transgenic technology /Reproductive BiotechnologyTransgenic technology /Reproductive Biotechnology -- Chimera production by morulaeChimera production by morulae
aggregation in minkaggregation in mink to get the offspring with GLT transmission resistance toto get the offspring with GLT transmission resistance to
Aleutian disease.Aleutian disease.
 ES cells BiologyES cells Biology - Combination of the Microinjection technique and Immune- Combination of the Microinjection technique and Immune
fluorescence /Confocal microscopy (dying) to confirm pluripotential features offluorescence /Confocal microscopy (dying) to confirm pluripotential features of
the hair follicles cells.the hair follicles cells.

3. Micromanipulation systems belonging to different generations:Micromanipulation systems belonging to different generations:
KM-3 And KM-5, Russian (Fonbrune’s prototype) 1985-1989, Narishige Manipulator 1992-KM-3 And KM-5, Russian (Fonbrune’s prototype) 1985-1989, Narishige Manipulator 1992-
1995 and1995 and three-dimensional manual manipulatorthree-dimensional manual manipulator Leica 1997-2007Leica 1997-2007

4. Dissecting the inner cell mass ofDissecting the inner cell mass of rabbitrabbit blastocysts with glassblastocysts with glass
capillary (1985)capillary (1985)
pronuclear removal from rabbit oocyte (1986)
 The pronuclear component was pulled out with aThe pronuclear component was pulled out with a
part of the cytoplasm from rabbit oocyte.part of the cytoplasm from rabbit oocyte.
 A mucine coat covering rabbit embryos at the allA mucine coat covering rabbit embryos at the all
stage of pre-implantation development makesstage of pre-implantation development makes
them difficult for micromanipulation techniquesthem difficult for micromanipulation techniques

5. Bisection ofBisection of cowcow early blastocysts. Identical twins produced microsurgically (1986-1989,early blastocysts. Identical twins produced microsurgically (1986-1989,
1990-1995) :1990-1995) : Holstein DairyHolstein Dairy and Red Dutch Milk Cattle (English selection) twinsand Red Dutch Milk Cattle (English selection) twins
Surviving embryos after dissection: 60-70%.Surviving embryos after dissection: 60-70%.
Implantation rate after nonsurgical E Transfer:Implantation rate after nonsurgical E Transfer:
–– two halves transferred unilaterally 45-56%two halves transferred unilaterally 45-56%
- two halves transferred bilaterally 42-45%- two halves transferred bilaterally 42-45%
- two halves transferred separately to two different- two halves transferred separately to two different
recipients 40-45 %recipients 40-45 %
- Black–white stains can have different configuration- Black–white stains can have different configuration
but similar spotted segments located on the skinbut similar spotted segments located on the skin

6. Triple-coloured Chimera calves derived from dissected three morulae andTriple-coloured Chimera calves derived from dissected three morulae and
aggregated then in a common zona pellucida and then transferred surgically toaggregated then in a common zona pellucida and then transferred surgically to
the recipient heifers:the recipient heifers:
-- cow 8 cell morulae belonging to Holstein Dairy, Simmental and Ayrshire milk cattle havecow 8 cell morulae belonging to Holstein Dairy, Simmental and Ayrshire milk cattle have
been usedbeen used
- Surgical transfer as a single embryo unilaterally- Surgical transfer as a single embryo unilaterally
- Inevitable increasing of inner cell mass as a result of the morulae aggregation leads to- Inevitable increasing of inner cell mass as a result of the morulae aggregation leads to
high embryo surviving and foetuses viability – 43-65% pregnancyhigh embryo surviving and foetuses viability – 43-65% pregnancy

7. PN injection into Mice zygotes and 2-4 cell Sheep embryos
Target: to study the expression of two highly homologous genes.
-PN injection into pronuclear or cytoplasm of each cell of 2-4-cell embryos. The
attempt to overcome two-cell embryo block division.
-human Growth Hormone (hGH-N) and bovine Growth Hormone-variant (bGH-V).
-DNA Construction Design: each gene was inserted into a bovine papillomavirus
shuttle vector Bovine Growth hormone gene as lineal form of plasmid pUC-19
consisted MT-1 promoter, structure part of bovine or human HGG, 3’-part of
viruses SV-40.
-under transcriptional control of the mouse metallothionein (MT-1) gene promoter.

8. After Gene injection embryos were cultured in vitro and transferred surgically into oviducts of theAfter Gene injection embryos were cultured in vitro and transferred surgically into oviducts of the
sheep foster motherssheep foster mothers
..
 Gene Injection into cytoplasmae of eachGene Injection into cytoplasmae of each
blastomere of 2-6-cell morulaeblastomere of 2-6-cell morulae
 500-2500 gene copies per embryo, injection500-2500 gene copies per embryo, injection
volume 1-2 pl, DNA concentration-2ng/mlvolume 1-2 pl, DNA concentration-2ng/ml
 Surgical unilateral transfers of 3-5 embryosSurgical unilateral transfers of 3-5 embryos
per recipient provided 75% implantationper recipient provided 75% implantation
 In simple circumstances of feeding one maleIn simple circumstances of feeding one male
from 10 lambs had high growth rate and wasfrom 10 lambs had high growth rate and was
considerably bigger than his sisters andconsiderably bigger than his sisters and
brothers. But on 6-months age his intensivebrothers. But on 6-months age his intensive
growth stopped.growth stopped.
 Analyses of the blood, skin and wool samplesAnalyses of the blood, skin and wool samples
were a part of other investigationwere a part of other investigation

9. Attempt to replicate the embryo microenvironment in the culture dishAttempt to replicate the embryo microenvironment in the culture dish
Embryo microenvironment ofEmbryo microenvironment of so-called nichesso-called niches is important condition for embryo viabilityis important condition for embryo viability
and development. Building up an Embryo niche in vitro suggests at least twoand development. Building up an Embryo niche in vitro suggests at least two
purposes:purposes:
 First – it may to provide a better conditions for culture and supply a quick re-First – it may to provide a better conditions for culture and supply a quick re-
expansion of bisected and/or injected Blastocysts.expansion of bisected and/or injected Blastocysts.
 Second – it make available the opportunity to study the interaction of culturedSecond – it make available the opportunity to study the interaction of cultured
embryos with their niche components and model states that result when theseembryos with their niche components and model states that result when these
interactions are aberrant.interactions are aberrant.
One simple and well-used approach toward building up anOne simple and well-used approach toward building up an in vitroin vitro niche involves co-niche involves co-
culture embryos with oviduct epithelial cells situated around cleaving embryosculture embryos with oviduct epithelial cells situated around cleaving embryos inin
vivo.vivo.
We had used bovine oviduct epithelial cells (BOECs) as organ specific feeding cells toWe had used bovine oviduct epithelial cells (BOECs) as organ specific feeding cells to
support a culture of thawed and dissected bovine embryos. That co-systemsupport a culture of thawed and dissected bovine embryos. That co-system
mimicked specific environment for embryo trophy function re-initiation in which themimicked specific environment for embryo trophy function re-initiation in which the
embryo-maternal signal could be generated.embryo-maternal signal could be generated.
There is suggesting that many components of a niche include soluble and attachedThere is suggesting that many components of a niche include soluble and attached
signaling molecules, cell-cell interactions, cell-extracellular matrix interactions,signaling molecules, cell-cell interactions, cell-extracellular matrix interactions,
mechanical forces that can be renewed in three dimension system and systemicallymechanical forces that can be renewed in three dimension system and systemically
regulated by small molecules such as metabolites and oxygen .regulated by small molecules such as metabolites and oxygen .

10. Use bovine oviductal epithelial cell monolayer (Use bovine oviductal epithelial cell monolayer (BOECs) as in vitro nichesBOECs) as in vitro niches to promote theto promote the
viability, growth and development of cow demy-embryos. The right environment is a keyviability, growth and development of cow demy-embryos. The right environment is a key
for Embryonic potentialfor Embryonic potential

11. Investigations of early embryo development in mink.Investigations of early embryo development in mink.
Morulae Aggregation and chimera production.Morulae Aggregation and chimera production.
- Different stage morulae from Black and Grey genotypes have been aggregated in the same zona
pellucida. Or the halves of morulae were microinjected into Blastocysts
- Surgical transfer of 10-12 aggregates unilaterally per recipient gave chimeras puppies