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How-to 1How-to 1 FOR

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How-to 2How-to 2

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Future ofFuture of

BIOCHEM GENETICS CELL BIO.MOL. BIO

STEM CELLS,

CLONING

REC. DNA

CELL TYPE

3DSTRUCTURE

FERTILI-ZATION

SYSTEMS

VIRUSES

IMMUNE

LIFELIFE

NERVOUSCANCER

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Student question of the day

Q: In the last lecture, what allele was convertedfrom mRNA into cDNA?

A: Typically all of the alleles of all expressedgenes are converted into cDNA in the sametube. We can clone the resulting cDNAs tosort through them – the result is a cDNAlibrary.

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3http://de.wikipedia.org/wiki/5-Brom-4-chlor-3-indoxyl-%CE%B2-D-galactopyranosid

A white colony reports the presence of an insert in thelacZ gene causing X-GAL to not be cleaved

X-GAL + lacZ -> blue colony

Plate on growth media + ampicillin + X-GAL

4Fireflies making luciferase, from Life by Purves et al, 7th edition

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5http://www.pgec.usda.gov/Ow/A10-1986OwetalScience.pdf

Luciferase (in cells) + Luciferin (in media) -> Luminescence

6http://en.wikipedia.org/wiki/Green_fluorescent_protein

Hit the Beach! In 8 colors…

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ddA ddG ddC ddT

5’ GAGTAACCGGTATGCA3’

Separate DNA fragments of different length

3’ACGTATGGCCAATGAG5’

1.denature 2. gelelectro-phoresis

-

+

longest, slowest migrating

shortest, fastest migrating

sequence

8A control and a transgenic mouse expressing rat growth hormone, from Introduction to Genetic Analysis by Griffiths et al, 8th edition

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9http://clinicaldepartments.musc.edu/transgenicmouse

X-gal staining in the head region of a transgenicmouse embryo (day 16 of gestation) carrying a

Hoxc13-lacZ reporter gene.

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A chimeric mouse

http://intramural.nimh.nih.gov/tgc/photogallery.html

12GFP mice and blastocysts, from Molecular Cell Biology by Lodish et al, 5th edition

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13Nuturing defect in FosB mutant mice, from http://www.cell.com/content/issue?volume=86&issue=2Fos family proteins trigger neuronal responses in the brain and are expressed in response to environmentalstimuli

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Terms• Host• Virus• Reporter• Tissue specific promoter• DNA sequencing• Sanger method• Resequencing• dideoxyribonucleoside

triphosphate (ddNTP)• SNP• Haplotype• Oligonucleotide• Gene therapy• Host immune response

• Oncogene• Transgenic• Transgene

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DNA sequencing

1. What is this? Determine the base sequence of DNA fragment

2. Why?Coding capacity of a geneGene identificationDisease gene/ allele identificationEvolutionary relationshipsGenome analysis- promoters, centromeres..

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Dideoxy nucleotides terminate DNA polymerization

+dNTPsDNA polymerase

polymerizationterminateswhere a ddNTP is incorporated

polymerization ofwhole fragment

3’ 5’

5’ 3’

templateprimer

+dNTPs+ low level ddNTPs DNA polymerase

3’ 5’

5’ 3’

templateprimer

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Dideoxy DNA sequencing/ ddATP as example

3’ACTGGTACTCATTGGCCATACGTG5’5’TGACCAT3’

3’ACTGGTACTCATTGGCCATACGTG5’5’TGACCATGAGTAA

3’ACTGGTACTCATTGGCCATACGTG5’5’TGACCATGAGTAACCGGTA

3’ACTGGTACTCATTGGCCATACGTG5’5’TGACCATGAGTAACCGGTATGCA

A = ddATP (low)+dNTP (high)DNA polymeraseradioactive orfluorescent label

polymerizedfragmentsterminate whereddA incorporates

Length of terminated fragment indicates position of ANeed to do separate reactions containing

+ddATP, +ddCTP, +ddGTP, +ddTTP

18Making a knockout mouse, from Life by Purves et al, 7th edition

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Making knock-out mice(see Fig. 16.9)

3.5 dayembryo

ES cells(Embryonic Stem)

ES cells in culture

gene targeting selection

“Targeted” Aa ES cells

3.5 dayembryo/

blastocyst

AAcells

implant

chimericmouse

Aacells

embryos developprogeny born

AAcells

Aa

breed to wild typemouse

AaX aa ?

A Wild type allelea Knock out