Cloning and Making Mice

tightly linked

positional cloning/chromosome walk key word

make genomic library

First step of positional cloning/ chromosome walk

Perform colony life with known gene as probe

second step of positional cloning/ chromosome walk

make new probe from end of first clone and rescreen until desired gene

third step of positional cloning/ chromosome walk

must have a way to recognize gene

fourth step of of positional cloning/ chromosome walk

temp sensitive, fungi

Key word(s) for functional complementation

prepare cDNA library from wild type, insert DNA into plasmid

first step of functional complementation

introduce plasmids to mutant cells (each mutant cell gets one different normal gene)

second step of functional complementation

look for rescued cell/fungi after grow in conditions where mutant fails

third step of functional complementation

identify the gene that “fixes” the mutation

fourth step of functional complementation

synthesized antibody

Key word for Antibody probe

convert mRNA to cDNA

first step of antibody probe cloning

put cDNA into expression vector, created cDNA library

second step of antibody probe

colony lift (grow bacteria, transfer, break cells open)

third step of antibody probe

use antibody as probe

fourth step of antibody probe

DNA sequence unknown

Nucleic acid probe key word

prepare cDNA or genomic

first step of nucleic acid probe

colony lift

second step of nucleic acid probe

add nucleic acid probe

third step of nucleic acid probe

screen with gene as proble using a label (fluorescent)

fourth step of nucleic acid probe

Protein/amino acid sequence is known

Degenerate Primers Key Word

Screen DNA by colony lift using degenerate primers synthesized based on the amino acid sequence of the protein as your probe

Degenerate Primers Steps

making orgamism with mutation

transgenic organism key word

engineer mutant form of target gene and put into plasmid

first step of transgenic organism

place mutant downstream from promoter

second step of transgenic organism

allow replication to occur

third step of transgenic organism

where gene is expressed

reporter transgene key word

fuse regulatory sequence to a reports (gpf)

reporter transgene first step

use lacZ, gpf, or luciferase and introduce into mice

second step of reporter transgene

get human eggs and remove nuclear

Clone Human first step

fuse enucleated eggs with somatic cells

Clone human second step

allow cells to develop

clone human third step

different environment, age difference, random developmental events

Reason human clone is not exact copy

phenotype of offspring depends on mom’s nuclear genotype

Maternal Effect

phenotype depends on organeller DNA from mom

Extranuclear Inheritance

infect ES cells from black mouse with vector

First step of making mouse (knock out model)

transgene with mutation

what is vector of mouse knock out model

select for recombination in Neo and gan

second step of making mouse (knock out model)

verify deletetion by PCR

third step of making mouse (knock out model)

fourth step of making mouse (knock out model)

identify chimeric mice

fifth step of making mouse (knock out model)

cross chimeric and white mice, identify black furred offspring

sixth step of making mouse (knock out model)

determine which black offspring are hets

seventh step of making mouse (knock out model)

cross hets to get homozygotes

8th step of making mouse (knock out model)