Genomes, genomic libraries and mutant mice

Question 1

What is a genome ?

Answer 1

A genome is an organisms complete set of DNA (or RNA, in case of RNA viruses).

Question 2

How big is the genome of phage lambda ?
E.coli  (K12) ?               
Saccharomyces cerevisiae ?
Drosophila melanogaster ?
Mus musculus ?
Homo sapiens ?
Protopterus aethiopicus (marbled lungfish) ?
Human mitochondria ?

Answer 2

phage lambda : 48.5 kb
E.coli (K12) : 4.6  Mb
Saccharomyces cerevisiae : 12.2 Mb
Drosophila melanogaster : 130 Mb
Mus musculus : 2.7 Gb
Homo sapiens : 3.2 Gb
Protopterus aethiopicus (marbled lungfish) 130 Gb
Organelles human mitochondria : 16.5 kb

Question 3

Why do we swish to make genomic libraries with a sufficient number of independent clones ?

Answer 3

In order to make sure that we cover the genome with high probability.

Question 4

What are the maximum insert sizes that can be inserted into :

• a lambda phage ?
• a cosmid ?
• a BAC ?
• a YAC ?

Answer 4

Lambda : < 23 kb
Cosmid : < 30-42 kb
BAC : < 120-350 kb
YAC : 100-1000 kb

Question 5

What equation can we use to estimate the size N of our library ?
Why must we be cautious when using this formula ?

Answer 5

N = [ln(1-P)] / [ln(1-f)]
With :
N = size of library
P = probability that sequence is present (at least once)
f = fractional size (i.e. clone insert size / genome insert size)
Caveat : the above formula assumes that each fragment clones equally

Question 6

How can we produce fragments of a desired size range ?

Answer 6

• e.g. partial digestion with restriction enzyme

- average restriction enzyme site distance &laquo_space;than desired DNA fragment length (followed by size selection of fragments)

Question 7

What is a cosmid ?

What are they usually used for ?

Answer 7

A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. Cosmids (cos sites + plasmid = cosmids) DNA sequences are originally from the lambda phage.
They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They can contain 37-52 (normally 45) kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitable origin of replication: for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication or F1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance.

Question 8

How can cosmids be introduced into E Coli ?

Answer 8

By transduction.

Question 9

How long is SuperCos1 ?

What is it made of ?

Answer 9

• 7.9 kb
• pUC19 ORI + P SV40
• ampicilin + neomycin resistance
• BamHI + XbaI restriction site

Question 10

What is a BAC ?

Answer 10

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli.
F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome’s usual insert size is 150–350 kb. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

Question 11

What is pPeloBAC11 ?

Answer 11

pBeloBAC11 is an E. coli plasmid cloning vector designed for the construction of Bacterial Artificial Chromosomes (BACs). It is maintained in single copy, which allows the cloning and stable maintenance of very large DNA fragments (up to 300 kb).

Question 12

What is a YAC ?

Answer 12

Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking.

Question 13

What are YACs made of ?

Answer 13

The primary components of a YAC are the ARS (autonomously Replicating Sequence), centromere, and telomeres from S. cerevisiae. Additionally, selectable marker genes, such as antibiotic resistance and a visible marker, are utilized to select transformed yeast cells. Without these sequences, the chromosome will not be stable during extracellular replication, and would not be distinguishable from colonies without the vector.

Question 14

How can YACs be introduced into yeast ?

Answer 14

Electroporation.

Question 15

IS the BAC or YAC system more stable ?

Which is more labor-intensive ?

Answer 15

YAC stability often less than BACs
YAC system is more labor-intensive (and slower) than BAC system
But YACs are more easily manipulatable in their host cell by homologuous recombination.

Question 16

What is the advantage of manipulating the murine (animal in general) genome ?

Answer 16

Stable manipulation (i.e passed on to “progeny”) (compare to transient expression systems) :

• in cell lines
• in animals

Question 17

When manipulating the genome of animals, what cells must the manipulation go through ?

Answer 17

The germ cells or germ line.

Question 18

What are the different ways of manipulating the animal genome ?

Answer 18

• addition by random integration (in mice ~1982)
• manipulation by homologous recombination (~1990)
• manipulation by site-specific recombination (~ 1996)
• manipulation using guide RNA (~ 2013)

Question 19

What is addition by random integration ?
What is it used for ?
How efficient is it ?

Answer 19

• DNA introduced into the cell via various mechanisms, a small percentage of cell integrate added DNA into genome
• added DNA has a selection marker (e.g neo gene, early 1980ies)
• useful for establishing clonal cell lines
• efficiency (number of cells) 1-2 orders of magnitude lower than with transient expression
  In mice => new transgenic mouse line, [Palmiter et al (1982) Cell & Nature] injection of DNA into fertilize oocytes + re-implanted into foster –> pseudopregnant female mice
• often these mice then have new DNA in germline

Question 20

What are the limits of addition by random integration ?

Answer 20

• integration –> often as multiple copies
• integration locus not equally distributed throughout genome
• transgenic mice: often disappointing expression in CNS
• longer DNA fragments often express better (e.g. BAC DNA) –> poor expression for short fragment

Question 21

What is homologous recombination ?

What do cells use homologous recombination for ?

Answer 21

Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA.
It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks. Homologous recombination also produces new combinations of DNA sequences during meiosis.
Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.

Question 22

How can scientists use homologous recombination ?

Answer 22

• to generate engineered mice (Nobel prize 2007) manipulate embryonic stem cells (ES cells), generate a stable cell line may use double selection
• to generate a stable cell line (targeting may create an insertion, deletion or replacement)
• to bread chimeric mice : inject a few cells into mouse blastocysts implant injected blastocysts in pseudopregnant foster mother ⇒ chimeric mice, with certain cells derived from the manipulated ES cells
  Breed chimeric mice. If manipulated ES cells contribute to germline => new engineered mouse line

Question 23

What is site specific recombination ?

Answer 23

Site-specific recombination is a type of genetic recombination in which DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology.

Question 24

What are the main known site specific recombination systems ?
What is the main one used for ES cells ?

Answer 24

Many site-specific recombination systems are known (e.g. lambda integration into host)
For ES cells Cre/lox system from P1 phage is often used
A recombinase (Cre) is acting an a short sequence specific element (loxPsite).

Question 25

What is the Cre-Lox system ?
Why is it used ?
How does the Cre-lox system work ?

Answer 25

Cre-Lox is a system that can be used to introduce gene deletions, inversions, and translocations on specific target sites. It was first discovered in the P1 bacteriophage.
For instance you want to do a gene knockout study on mice. In many cases, systematically deleting the gene would produce an embryonic lethal phenotype, rendering your experiment useless because you can’t observe the effect of the deletion. You would want to use a Cre-Lox system where you have temporal and tissue-specific control on where to have the deletions.
Basically, the system has two components: an enzyme, Cre recombinase, that can recognize and splice specific DNA sequences, called LoxP sites. The LoxP site consists of 34 base pairs: an 8bp spacer region and two flanking palindromic sequences 13bp-long each.

Question 26

What is genome manipulation by guide RNA ?

Answer 26

Since ~2013, it has bee used in genomic engineering, under active development / refinement.
Often called CRISPR-Cas system.
A guide RNA is directing a (engineered) nuclease to act at locations defined by the sequence of the guide-RNA