BIO265 GMOs

Genetically modified organisms commonly used in research

• fruit fly

• mice (best mammal model)

• zebrafish (good model for development & biotoxicology)

• C. elegans/roundworm

Drosophila melanogaster

• 2nd multicellular organism to have genome sequenced (2000)

• recently published larval connectome (2023) and adult connectome (2025)

What is a connectome?

• map of neurons and connections

• lines represent neuronal projections

• can use connections to study behaviors

What are advantages associated with using Drosophila melanogaster?

• large community of researchers

• gold standard in genetic tools

• short generation time

• cheap/inexpensive

• couple hundred thousand neurons → much simpler system to work out

What are disadvantages associated with using Drosophila melanogaster?

• limited behavioral repertoire (some surprising behaviors - males drinking ethanol post repeated rejection)

• fewer shared genes with humans compared to rodents

Mice (Mus musculus)

• 2002 genome published

What are advantages associated with using mice?

• really only mammalian model with well developed genetic tools (directed tissue expression of target genes)

• wide behavioral repertoire (babies’s palates, fear behaviors)

• share many genes and pathways with humans: applicability to human disease

What are the disadvantages associated with using mice?

• significantly more difficult (and costly) to house than most the other transgenic models - hundreds of dollars/day

• also have to wait for development to study adults

What is a genetically modified/engineered mouse model?

a mouse that has had its genome altered through the use of genetic engineering techniques

What things are important to know about genetically modified mice?

• mice are the most common genetically modified animal used for research or as animal models of human diseases and are also used for research on genes and genetic pathways

• e.g., reporter assays to locate gene promoters spatially

How can we study the function of genes expressed throughout various tissues in an organism?

• removing genes (knockouts)

  • disrupt function, can be done simply with frameshifts → truncated non functional protein

• expressing transgenes

  • marking cells that express a gene or are active

  • reducing gene expression (e.g., RNA intereference with siRNAs)

  • activating or inhibiting cellular populations experimentally (optagenetics - light, thermogenetics)

  • controlling cell types under regulatory paradigm

Luciferase gene expressed under control of two different promoters in a transgenic mouse

• different activity in different tissues

• spatial intensity and regulation

What is a knockout?

removal/inactivation of specific gene

What can knockouts do?

• provide information about a gene’s function

• comparing knockout and wild-type phenotypes (looking for phenotypic differences) can provide information on the knockout gene’s function - most common way to study knockouts, typically done using organisms of the same strain

Keratin knockout

• comparison of wild type mouse skin cross section to keratin mutant cross section

• discovery: keratin helps maintain tissue integrity

Leptin knockout

• leptin is a satiety peptide produced by adipose tissue

• lack of satiety signal leads mouse to keep eating → increase in mass

• humans rarely have deficiencies in leptin signaling

What are homology arms?

• long segments of genomic DNA flanking (upstream and downstream) the sequence to be inserted that facilitate recombination

• tricks it into thinking its a sister chromatid

What are the steps to making genetically engineered mice through homologous recombination?

1. make a DNA construct where gene of interest is interrupted by a “marker” or selector - generating knockout (e.g., neo - Neomycin resistance gene (antibiotic resistance gene) is often used as marker/selector)

2. electroporate DNA construct into cultured embryonic stem cells (ES) - electric shock to drive DNA into cell

3. allow homologous recombination to take place (using homology arms, homologous recombination - LOW FREQUENCY EVENT)

4. select cells that incorporated marker (using selector gene)

5. inject stem cells into mouse blastocyst and implant into foster mother mouse

6. mice homozygous for mutated gene (knockout) can be obtained through mating - by mating two chimera (two heterozygous individuals)

Generating knock in mice

• uses the same techniques as generating knock outs

• gene to be “knocked in” flanked by homology arms

• homologous recombination will lead to the incorporation of gene to be “knocked in” at the location of homology arm sequences in genome (can select for cells that incorporated the gene only)

• humanized mice

What are humanized mice?

• mice where certain genes have been replaced with homologous human gene

• confers different properties where phenotypic differences in construct can be observed

Pronuclear injection - generating genetically engineered mice

• low throughput

• DNA construct injected as linear piece of DNA into male pronucleus (bigger and easier to access) of recently fertilized egg → injected into pseudopregnant female mouse

• DNA may be incorporated into genome and replicated

• Screen offspring to determine which express transgene

• eliminates some downstream mating & screening processes

What is CRISPR?

• Clustered Regularly Interspersed Short Palindromic Repeats

• used by some bacteria to defend against viruses

What does CRISPR allow for?

• specifically targeted alterations as small or as large as needed → inducing reparation of breaks we generate

• circumvents the “randomness” of mutations and homologous recombination → tricking cell into using what we generate

• makes anything a “model system” because only a small part of the genome needs to be known

What is Cas9?

• “CRISPR associated protein”, endonuclease that acts as the “scissors”

• targeted by sgRNA

What is single guide RNA (sgRNA)?

• targets specific gene sequence in genome

• nucleotides in guide RNA are complementary to nucleotides in DNA sequence (induces cut by Cas9)

Why is CRISPR-Cas9 is a big deal?

• simple & precise

• can be used to knock out (NHEJ) or knock in genes (homologous recombination with generated construct)

How CRISPR works?

1. The Cas9 protein forms a complex with guide RNA in a cell

2. This complex attaches a matching genomic DNA sequence adjacent to a spacer

3. The Cas9-RNA complex cuts the double strands of the DNA

4. Programmed DNA may be inserted at the cut

DNA editing using CRISPR-Cas9

• Two DNA sequences introduced to cell - one codes for Cas9 protein, other codes for sgRNA

• Cas9 can only digest DNA near PAM sites (always cuts 3 nt upstream of PAM site, can predict where break will occur)

• sgRNA contains 20bp sequence specific for sequence in genome immediately 5’ to endogenous PAM site

What is a PAM site?

• protospacer adjacent motif

• NGG

• multiple PAM sites in a single gene

CRISPR-Cas9 can be used to knock out and knock in genes

• increases efficiency

• double stranded breaks can be repaired through two mechanisms: NHEJ & HDR

• can be used for therapeutic or research purposes

CRISPR-Cas9 knock outs

• Non-homologous end joining (NHEJ) - broken DNA ends are rejoined, often includes insertion or deletion

• frameshift resulting from indel often leads to premature stop codon

• no introduction of donor construct

CRISPR-Cas9 knock ins

• Homology-directed repair (HDR) - DNA construct with flanking homology arms is used as a template for homologous recombination

RNAi-mediated mRNA degradation

• phenomenon first described after injecting single-stranded RNAs into petunias to alter flower color

• inexplicably led to loss of color…

• Craig Mello & Andrew
  Fire w/ C. elegans

• Antiviral response; new paradigm for gene regulation

Mello & Fire w/ C. elegans

• observed something similar to what occurred with the petunias

• they showed that a dsRNA (with sequence region of mRNA) potently induced the degradation of that mRNA (perfect complementarity)

RNA interference - “knockdown” of target genes

• RNA interference (RNAi) constructs allow researchers to lower or “knock down” target gene expression without removing the gene from the genome

• can mimic knock out phenotype BUT IS NOT A KNOCKOUT

• can be controlled temporally and spatially

• transcripts degraded → so low levels in the cell → minimal/negligible expression

RNA interference pathway

• dsRNA (natural of artificial) is cleaved by the endonuclease Dicer

• RISC complex is recruited to siRNA duplex - unwinds to form ssRNA

• RISC complex scans to find mRNA complementary to associated ssRNA

• once found, component of RISC cleaves mRNA, leading to its degradation

• not a knockout, altering amount of gene product, not the gene itself