Animal gene engineering Practical course Skoltech RAS Gene

Animal gene engineering Practical course Skoltech– RAS Gene Biology Alexei Deikin, Evgeniia Zotova Spring 2017

Why do we need to modify animal genomes? • Fundamental research • Biotechnology • Food industry

Which animals are modified and for what purpose?

Which modifications can be introduced into a genome? • Insertion of gene (knock-in) • Deletion of gene (knock-out) • ? ? ?

How modifications can be introduced? Gene engineering Permanent modifications Germline or embryos (GMO animals!) Temporary modifications Somatic cells

We create GMO animals: Microinjections What and where?

We create GMO animals: Embryonic stem cells Why so difficult?

Why clone sheep?

Difficulties with mammals • Harvest embryos • Put them back in a pseudopregnant female

Brief workflow of microinjection

Practical course • Animal handling • Determine stage of mouse estrous cycle • Harvesting of embryos • Microinjections • Cultivation of embryos • Embryo transfer

Practice: Animal handling • Housing • Methods of drug administration • Surgery with anesthesia

How to determine mice sex?

Intraperitoneal injection

Syringe scales are variable • Volume – 1 ml (is written down on syringe) • Scale can be 30, 40, 100 units • Scale interval = 1000 ul / n units

Practice: Determine stage of mouse estrous cycle • Vaginal smears • Mating with vasectomized male mice and observation of plugs • Vasectomy

Oogenesis, fertilization and pregnancy

Estrous cycle

Endometrium and uterus state

Vaginal smear composition

Vaginal smear composition (more realistic)

Copulation plug after mating with vasectomized male

Male urogenital system

Vasectomy • Performed under anesthesia • Skin and muscle cut (fur can be pulled) • Ductus deference cut with hot pincers • Muscle and skin sewed

Practice: Harvesting of embryos • Induction of hyperovulation • Mating • Surgical harvest of embryos • Cleaning and debris removal

Female urogenital system

Oviduct (fallopian tube) dissection

Oviduct (fallopian tube) dissection

Media for working with embryos outside of CO 2 incubator М 2, HEPES-KSOM • • HEPES Albumin Piruvic and acids, glucose Bicarbonate Various salts Phenol red Penicillin, streptomycin

Embryo collection from oviduct

Embryo collection from oviduct

A bit more realistic view of dissected oviduct

Corona radiata – remaining follicular cells

Cleaning of follicular cells with hyaluronidase

Washing procedure

Capillary preparation • Capillary (outer diameter 1 mm) • Pulling • Microforge (allows to chose diameter and mill the tip) • Best inner diameter > 100 um

Embryos after washing

Practice: Microinjections • Injection solution preparation • Microinstruments: transfer capillary, microneedle and sucker capillary • Handling pullers and microforge • Assembly of microinjection camera • Microinjections

Zygotes Zona pellucida (shell) Nucleoli Pronucleus Polar body

Microinjection camera • 2 halves of coverslip • Drop of M 2 medium • Paraffin oil

Microscope for microinjections • Inverted • Contrasting optic • 5 x + 40 x objectives • Microneedle and holder pipette manipulators • Pressure regulation device • CO 2 gas cylinder

Comparison of different contrasting

DIC – differential interference contrast

Microinjection procedure

Holding pipette • Outer diameter ~ 80 um • Cut with microforge • Melt the tip with microforge • Inner diameter ~ 10 um

Microneedle • Capillary with filament (outer diameter 1 mm) • Create needle using puller • Fill from blunt end • Filter or heat solutions to avoid clogging

Practice: Cultivation and analysis • Culture conditions • Visual determination of embryo viability • Analysis of modification after cultivation

Culture in CO 2 Incubator M 16, KSOM-AA

What happens during cultivation?

Distinguish good cells from bad ones

Distinguish good cells from bad ones

Distinguish good cells from dead ones

1 day of cultivation

2 days of cultivation: compaction

2 days of cultivation: anomalies

3 days of cultivation: cavitation

4 days of cultivation: hatching

Analysis • Washing of blastocysts before hatching • PCR with a whole blastocyst (2 -cell stage can be OK) • PCR with pre-extracted DNA

Blastocyst DNA extraction protocol • Add to the each tube 10 ul of BLB • Incubate 10 min at 56 o. C and 10 min at 95 o. C • Freeze and store at -20 before use • Use 5 ul of solution in PCR BLB • 100 ul of 1 M Tris-HCl p. H 8, 3 • 100 ul of 1 M KCl • 100 ul of 0, 2% gelatin • 4, 5 ul of Tween-20 • 6, 25 ul of Proteinase K 20 mg/ml solution • bring final volume to 1 m. L

Practice: Embryo transfer • Pseudopregnancy • Embryo transfer in the fallopian tubes • Embryo transfer into uterine horns • Pregnancy

Right stage of estrous cycle

Embryo transfer into oviducts • Single cell stage, 2 cell stage • Recipient in estrus

Surgery scheme

Tear in ovary capsule under binocular

Insertion of transfer capillary with embryos

One more way to do it

Embryo transfer into uterine horn • Blastocysts (with zona pellucida) • Diestrus (recipient was in estrus 2 days prior embryo transfer) 2 days before

Surgery scheme Incision spot

Insert transfer capillary into the uterine horn trough incision in oviduct

Pregnancy and embryonic development

Uterus, 8 th day

Pregnancy and embryonic development

Extraembryonic membranes

C-section

Newborn mouse pups

Feeding (~21 day)