Mouse Genetics part 1 - 4

goals: Mendelian inheritance, generation of inbred strains in mice, identify the different methologies to create transgenic animals, tools to regulate transgene expression

Genotype

an individuals genetic constitution

Phenotype

what is observed physically or clinically

Gene

a section of DNA that encodes for a certain trait

Autosomal gene

a gene located on one of the non-sex chromosomes

Allele

a varient form of a gene

• each autosomal gene has two alleles

Homozygous

the two alleles for a gene are the same

Heterozygous

the two alleles for a gene are different

Hemizygous

an allele for a gene is present in only a single copy in an otherwise diploid organism

Dominant allele

an allele that always appears phenotypically

Recessive allele

must carry two recessive alleles in order for phenotype to show

Mendelian Genetics

in 1866, Gregor Mendel deduced several important genetic principles from his well-designed experiments with garden peas, that would lay the foundation for the discipline of genetics

Punnett square

parental genotype on the outside of the box, potential genotypes of offspring inside the box

Autosomal Dominant Inheritance

a way a trait or genetic condition is passed down from one generation to the next, where only one copy of a mutated gene from either parent is enough to cause the condition in the offspring

• if one parent carries a dominant gene mutation, each child has a 50% chance of inheriting the condition

Autosomal Recessive Inheritance

the risk for two carrier parents to produce offspring with two aa alleles is 25%

Sex linked inheritance

• X-linked inheritance: for alleles located on the X chromosome, males with mutations on the X never transmit to their son, but always to their daughter

• Y-linked inheritance: for alleles located only on the Y chromosome. only segregates in males

Abbie Lathrop

created many of the common inbred lines important to mouse geneticists today - including C57BL/6

William Castle

brought the fancy mouse into his laboratory in 1902, and began the development of innbred genetically homogeneous lines of mice

Why are mice ideal for genetic analysis

• small, short generation time, breed prolifically, docile, males do not harm their young, vaginal plug, pseudopregnancy

Inbred mating

defined as colonies produced by a minimum of 20 generations of brother-sister mating, traceable to a single founding pair

• results in animals that are genetically identical (homozygous across their entire genome) within each inbred strain (free of genetic variants that could increase variation in experimental results)

• easily available

• results are very consistent across time and the world

Specific inbred mice strains

• C57BL/6J has the Nnt mutation, which modulates metabolism and immune response

• C57BL/6J and C57BL/6N vary in their fear responses and how anesthetics affect their cardiac functions

• C57BL/6N mice and embryonic stem cells derived from them harbor the Rd8 mutation of the Crb1 gene, which causes developmental defects in photoreceptor segments

Incrosses

a cross between two animals of the same inbred strain

• serves primarily for maintaining strains of animals that are inbred or carry particular alleles of interest

Outcrosses

a cross between two animals from different inbred strains

• resulting offspring are called F1

• many experiments start with an outcross, in which at least one animal is carrying a mutation of interest

Intercross

a cross between two animals that have the same heterozygous genotype at a designated loci

• a cross between sibling F1 hybrids (Aa) that were derived from an outcross

Backcross

a cross of a hybrid animal with one of the two parental strains

• number of backcrosses are denoted as N

Wild-Type mice

“Normal” mice of a given strain. often referred as allele “+”

Transgenic mice

genetically modified mice that have a transgene introduced to their genome

Knockout mice

a type of transgenic mice in which a gene is functionally inactivated by deleting or silencing it

• help to understand the function of specific genes in an organism by blocking the expression of a gene

• issues: large delay due to backcrossing to a pure strain

Knockin mice

a type of transgenic mice in which a gene is inserted into a specific locus in the genome

• allows the creation of mutants that mimic exactly human mutation (ex. single nucleotide change) to substitute mouse gene by a human one

• Rosa26: locus found on chromosome 6 of the mouse genome that encodes nonessential RNA that replicates in every cell/tissue in the body that expresses it

  • provides a useful place for making gene insertions and studying how proteins ectopic or overexpression impact cells

ES cells

embryonic stem cells are derived from the inner cell mass of the blastocyst

Locus/Loci

specific physical location of a gene

How transgenic mice are made

CRISPR/Cas9

“Clustered Regularly Interspaced Short Palindromic Repeats”

• one repair mechanism can lead to knock outs, whereas the second can lead to knock ins

CRISPR-Cas9 vs Embryonic stem (ES) cell

CRISPR-Cas9 is faster (skip stage of breeding chimeric offspring with wild type mice) and has a higher success rate

CRE/Lox system *

used to regulate spatially and temporally the expression or the knockout of a gene (global knockout could be lethal)

• system allows generate tissue-specific and inducible knockouts to have control over the location and timing of gene expression

CreER/Tamoxifen system

Tamoxifen is an estrogen analog that binds to estrogen receptors

• in order to control WHEN the Cre enzyme will function, the Cre protein can be used with a modified estrogen receptor, that gets activated when bound with tamoxifen, and in turn activates the Cre protein (by moving it to the cell nucleus)