Genetic engineering in mice

• Genetic engineering involves altering an organism's DNA or adding a foreign gene.

Genetically Modified Mice

• Knockout mice lack a gene for a specific protein and are used to study gene functions by comparing behavior and drug response to normal mice.
• Knockin mice have a modified inserted gene, producing a slightly different protein (often by one amino acid), used to study protein structure-function relationships.
• In neuropharmacology, knockin mice are used to study receptor subtypes or enzymes.
• Transgenic mice have one gene substituted for another and can model human diseases like Huntington's and Alzheimer's.
• Genome editing techniques have replaced older transgenic procedures.

Potential Problems with Transgenic Mice

• Behaviors are controlled by multiple genes.
• Compensation by other genes may mask the effect of a mutation.
• Altered gene function in all tissues at all developmental stages can cause behavioral changes.
• Environmental factors can affect gene expression in developing mice.

Cre-LoxP System

• The Cre-LoxP system controls gene expression.
• It relies on Cre recombinase and 34bp lox sequences in DNA.
• It allows deletion (excision), inversion, and translocation.
• Cre causes recombination and cyclization recombinase.
• The LoxP site, is the locus of X(cross)-over in P1.
• It consists of 13 bp palindromic recognition sites separated by an 8 bp spacer that provides directionality.
• Deletion (excision) occurs when loxP sites are in the same orientation.
• Inversion occurs when loxP sites are in opposite orientations.
• Translocation occurs when loxP sites are on separate DNA molecules.
• Removing a gene (KO) can be problematic if the gene is essential in some or all tissues, or for development.
• This system allows for targeting specific tissues or cell populations and temporal control of recombination (inducible cre).
• Examples of tissue-specific targeting include Albumin-cre for the liver and Sox9-cre for the pancreas.
• Cell-type-specific targeting examples are PV-cre for parvalbumin interneurons and Slc17a6-cre for excitatory neurons.
• Gene deletion happens in the tissues or cell types that express that particular promotor.
• Other delivery methods include stereotaxic injection of AAV/Cre and electroporation of Cre-expressing plasmids.
• Temporal control can be achieved using Tamoxifen or Tetracycline/Doxycycline.

Other Cre-LoxP System Applications

• Cre-ON systems block gene expression until cre recombination removes a stop cassette.
  • Can be combined with inducible cre.
• FLEX (flip excision) switches are based on modified lox sequences.
• FLEX switches: DIO (Double-Floxed Inverted Open reading frame).

The Flp-Frt System

• It has the same principle as the Cre-LoxP system.
• Flippase is obtained from Saccharomyces cerevisiae.
• Recognizes Frt sequences.
• Can be combined with the Cre - LoxP system.

Viral Vectors

• Viral vectors are used in neurobiology for brain targeting.
• Types include HSV-1, HIV-1 (LV), Ad, AAV, and a-virus (SFV - sindbis - EEV).
• Lentiviral vectors and adeno-associated vectors are popular.
• Lentiviral vectors pseudotyped with VSVg mediate local transduction.
• Lentiviral vectors pseudotyped with Rbg transduce both the soma of local neurons and afferents.
• AAV mediates larger physical spread.
• A potential use of Cre-lox and cell-specific promoters target gene expression both spatially and temporally.
• This happens through lentiviral vector injected in the hippocampal dentate gyrus of a nestin-cre mouse.
• Cre expressed in nestin-positive neural progenitor cells reverses mCherry and expresses new neurons as they differentiate in granule neurons.

Other Info

• Optogenetics integrates optics and genetics to activate or inhibit specific neurons.
• This includes methods for delivering optical tools into specific cells, light delivery technologies, and integrating optical control with readouts.
• Opsins are light-modulated ion channels, including Bacteriorhodopsin, Halorhodopsin, and Channelrhodopsin.
• Designer Receptors Exclusively Activated by Designer Drugs (DREADD) are used in chemo-genetics.