GEM KEY POINTS

What are Genetically Engineered Mice?

They are mice that have had their genomes altered by genetic engineering techniques.

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This process typically involves inserting, deleting, or modifying specific genes to study their functions during development and effects on physiological functions, or to be used as disease models for testing various treatments.

What is Superovulation?

Superovulation in mice refers to a process that female mice are induced to produce and ovulate many oocytes in a single ovarian cycle, typically through hormonal stimulation.

Hormones commonly used to induce superovulation in mice?

PMSG (pregnant mare serum gonadotropin) and HCG (human chorionic gonadotropin)

What is Pseudo pregnancy?

Pseudo-pregnant female mice are those that have been hormonally or behaviorally induced to exhibit the physiological signs of pregnancy without actually being pregnant.

• This state mimics pregnancy and is achieved through mating with vasectomized male mice or by hormonal treatment.

Day 0.5 embryos are called ____

One-cell stage embryo, zygote

Day 3.5 embryos are called ___

Blastocyst stage embryo

What are the 3 methods to introduce gene editing reagents into mouse embryos?

1. Microinjection of zygotes with DNA, RNA, Protein, Virus or Sperm into cytoplasm or pronucleus

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2. Microinjection of blastocyst stage embryos with ES cells

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3. Electroporation of CRISPR Cas9/sgRNAs/Donor DNA or rAAV into the pronucleus or cytoplasm of zygotes

What are the 3 approaches (systems) introduced in class to make Genetically Engineered Mice

1. Traditional Transgenic approach

2. Genome Edited ES cells

3. CRISPR/Cas9 Technology 

Using the Traditional Transgenic Approach for GEM

Random integration of the microinjected transgene DNA into the genome of early embryonic cells to generate transgenic mice

Using Genome-edited Embryonic Stem (ES) Cells to make GEM

Edit one or more genes in mouse ES cells in culture by gene targeting (homologous recombination), and then inject the ES cells into mouse blastocysts to generate chimeric mice with germ line transmission capability.

Using CRISPR to make GEM

Allows precise modification of DNA sequences in the mouse zygotes or ES cells. The Cas9 endonuclease creates double strand DNA breaks at the specific site determined by sgRNA.

What is Conditional knockout in mice?

Cre-LoxP and Flp/frt systems: binary systems of unique strains of mice (LoxP and Cre mouse lines) selectively disable a gene in specific cell-types/tissues or at certain developmental stages

What are the Selectable Markers and what are they for during ES Cell Genome Targeting?

Positive selection cassette (select for homologous recombination)

Negative selection cassette (select against random integration)

Upon CRISPR Cas9/gRNA-mediated double-strand DNA breaks, what DNA repair mechanisms are employed to introduce knockout and knock-in mutations?

1. NHEJ (Non-Homologous End Joining) for knock out: an error-prone repair system causing double-stranded DNA breaks that rejoins the broken ends, often leading to small insertions or deletions (INDEL) that disrupt the gene function.

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2. HDR (Homology-Directed Repair) is another cell’s repair system causing doublestranded DNA breaks that use a homologous DNA template to precisely repair double-stranded breaks, enabling the introduction (knock-in) of specific DNA fragment (genetic changes) into the genome.

What are the 3 common donor DNA template types for HDR (knockin) allele?

1. Single-stranded oligodeoxynucleotide (ssODN)

2. Long single stranded DNA (lssDNA)

3. Double stranded plasmid DNA