Use microinjection technique in Reproductive, Use microinjection technique in Reproductive, Cellular and Molecular biology Cellular and Molecular biology

Some essays from the personal skills and experienceSome essays from the personal skills and experience

/A.Madich/A.Madich

Main directions to applyMain directions to apply

Animal reproduction Animal reproduction – Embryo bisection and identical twins production in – Embryo bisection and identical twins production in bovine, Morulae aggregation and chimeras calves productionbovine, Morulae aggregation and chimeras calves production

Transgenic technologyTransgenic technology – – DNA injection into pronuclear and 2-cell DNA injection into pronuclear and 2-cell embryos in sheep and bovine embryos in sheep and bovine

Biology of early development Biology of early development – Use trophoblastic vesicles cell culture – Use trophoblastic vesicles cell culture prepared micro surgically by biopsy from bovine blastocysts in the prepared micro surgically by biopsy from bovine blastocysts in the attempt to improve the development of half-embryos attempt to improve the development of half-embryos in vitroin vitro

Reproductive Biotechnology Reproductive Biotechnology – Use bovine oviduct epithelial cells tissue – Use bovine oviduct epithelial cells tissue culture (BOECs) and cell subculture to promote the viability and culture (BOECs) and cell subculture to promote the viability and development of thawed and dissected Bovine embryos as well as IV development of thawed and dissected Bovine embryos as well as IV fertilized sheep oocytesfertilized sheep oocytes

Transgenic technology /Reproductive Biotechnology Transgenic technology /Reproductive Biotechnology - - Chimera production Chimera production by morulae aggregation in mink by morulae aggregation in mink to get the offspring with GLT to get the offspring with GLT transmission resistance to Aleutian disease.transmission resistance to Aleutian disease.

ES cells Biology ES cells Biology - Combination of the Microinjection technique and - Combination of the Microinjection technique and Immune fluorescence /Confocal microscopy (dying) to confirm Immune fluorescence /Confocal microscopy (dying) to confirm pluripotential features of the hair follicles cells.pluripotential features of the hair follicles cells.

Micromanipulation systems belonging to different generations:Micromanipulation systems belonging to different generations:KM-3 And KM-5, Russian (Fonbrune’s prototype) 1985-1989, Narishige KM-3 And KM-5, Russian (Fonbrune’s prototype) 1985-1989, Narishige

Manipulator 1992-1995 and Manipulator 1992-1995 and three-dimensional manual manipulator three-dimensional manual manipulator Leica 1997-Leica 1997-20072007

Dissecting the inner cell mass of Dissecting the inner cell mass of rabbitrabbit blastocysts with glass blastocysts with glass capillary (1985)capillary (1985)

pronuclear removal from rabbit oocyte (1986)

The pronuclear component was pulled out The pronuclear component was pulled out with a part of the cytoplasm from rabbit with a part of the cytoplasm from rabbit oocyte. oocyte.

A mucine coat covering rabbit embryos at A mucine coat covering rabbit embryos at the all stage of pre-implantation the all stage of pre-implantation development makes them difficult for development makes them difficult for micromanipulation techniquesmicromanipulation techniques

Bisection of Bisection of cowcow early blastocysts. Identical twins produced microsurgically early blastocysts. Identical twins produced microsurgically (1986-1989, 1990-1995) : (1986-1989, 1990-1995) : Holstein Dairy Holstein Dairy and Red Dutch Milk Cattle (English and Red Dutch Milk Cattle (English

selection) twinsselection) 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 - two halves transferred separately to two different recipients 40-45 % different recipients 40-45 %

- Black–white stains can have different - Black–white stains can have different configuration but similar spotted segments configuration but similar spotted segments located on the skinlocated on the skin

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

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.

After Gene injection embryos were cultured in vitro and transferred surgically into After Gene injection embryos were cultured in vitro and transferred surgically into oviducts of the sheep foster mothers oviducts of the sheep foster mothers

. .

Gene Injection into cytoplasmae of each Gene Injection into cytoplasmae of each blastomere of 2-6-cell morulaeblastomere of 2-6-cell morulae

500-2500 gene copies per embryo, 500-2500 gene copies per embryo, injection volume 1-2 pl, DNA injection volume 1-2 pl, DNA concentration-2ng/mlconcentration-2ng/ml

Surgical unilateral transfers of 3-5 Surgical unilateral transfers of 3-5 embryos per recipient provided 75% embryos per recipient provided 75% implantationimplantation

In simple circumstances of feeding one In simple circumstances of feeding one male from 10 lambs had high growth male from 10 lambs had high growth rate and was considerably bigger than rate and was considerably bigger than his sisters and brothers. But on 6-his sisters and brothers. But on 6-months age his intensive growth months age his intensive growth stopped.stopped.

Analyses of the blood, skin and wool Analyses of the blood, skin and wool samples were a part of other samples were a part of other investigationinvestigation

Attempt to replicate the embryo microenvironment in the culture dishAttempt to replicate the embryo microenvironment in the culture dish

Embryo microenvironment of Embryo microenvironment of so-called niches so-called niches is important condition for embryo viability is important condition for embryo viability and development. Building up an Embryo niche in vitro suggests at least two and 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 cultured Second – it make available the opportunity to study the interaction of cultured embryos with their niche components and model states that result when these embryos 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 an One simple and well-used approach toward building up an in vitro in vitro niche involves co-niche involves co-culture embryos with oviduct epithelial cells situated around cleaving embryos culture embryos with oviduct epithelial cells situated around cleaving embryos in in vivo.vivo.

We had used bovine oviduct epithelial cells (BOECs) as organ specific feeding cells to We had used bovine oviduct epithelial cells (BOECs) as organ specific feeding cells to support a culture of thawed and dissected bovine embryos. That co-system support a culture of thawed and dissected bovine embryos. That co-system mimicked specific environment for embryo trophy function re-initiation in which the mimicked 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 attached There 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 systemically mechanical 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 .

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

for Embryonic potentialfor Embryonic potential

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