DNA Recombination for Genetic Engineering

Question 1

Naturally occurring genetic mutations

Answer 1

• May be silent with no observable variations in gene
expression
• May cause a dysregulation of gene expression
• May alter protein sequence
• With no altered function of that protein
• With altered function of that protein
• May abolish protein expression
• Can cause observable variations in physiology
• Can cause genetic diseases
• The phenotypes induced by these poorly functioning genes
can shed light on the physiological functions of these genes

Question 2

Cystic fibrosis is caused by

Answer 2

a genetic mutation
• Patients have
• Difficulty breathing
• Frequent lung infections
• Frequent diarrhea
• Caused by
• Defect in chloride ion
channel
• Thick sticky mucous clogs
the respiratory and
gastrointestinal tracts
• Inferred/learnt
• Cystic fibrosis gene is
recessive
• Has an important role in the
regulation of mucous

Question 3

X-linked hyper IgM syndrome is caused

Answer 3

by a genetic mutation
Patients have
• Normal or elevated IgM
• Low IgG or IgA
• Symptoms from ~6 months of age
• Suffer recurrent infections
Caused by
• Mutation in CD40L gene
• CD40L normally expressed on T cells and
interacts with CD40 on B cells
Inferred/learnt
• IgG and IgA essential in fighting infection
• CD40/CD40L interaction necessary to signal
class switching from IgM to IgG or IgA

Question 4

Genetic engineering

Answer 4

Involves genetic manipulation of the genome of an organism causing the expression/loss of expression of defined gene products
• Gain of function (transgenics)
• Introduction of a new gene (transgene) into an organism’s genome
• Loss of function (knock-out)
• Deletion of a gene from an organism’s genome
• Altered function (transgenics or knock-ins)
• Manipulate the level, timing or location of expression of a gene
• Broadly applicable to a range of organisms
• Can involve expression of genes from one species into a
different species
• e.g. jellyfish genes expressed in mammals

Question 5

Genetic engineering to treat vitamin A deficiency

Answer 5

vitamin A deficiency
• Affects >200 million people worldwide
• 500,000 children go blind per year
• Contributes to 2 million deaths
Rice
• Staple diet for around 4 billion people
• Milled rice lacks B-carotene (the
precursor of vitamin A)
Transgenic rice
• Engineered to express b-carotene
• Plant and bacterial transgene encoding
enzymes for b-carotene synthesis introduced

Question 6

Genetic engineering to create ‘gain-of-function’ animals

Answer 6

‘Transgenic’ is the term given to genetically engineered
gain-of-function animals
• Expression of gene in a way not normally found in nature
• Self-gene expressed in a different location
• Self-gene manipulated with a different promotor to drive
over-expression
• Gene from one organism expressed in another organism
• Modified self-gene designed to act as a ‘dominant negative’
gaining the function of suppressing the endogenous gene

Question 7

Question. X-linked hyper IgM syndrome can result in elevated IgM and lower IgG
or IgA. It is commonly caused by a naturally occurring mutation in which gene?
A. IgM
B. IgG or IgA
C.CD40L
D.CD40

Answer 7

C.CD40L

Question 8

Transgenic mice (gain of function mutants)

Answer 8

• Relatively easy
• Transgene of choice injected
into the nucleus of a fertilised egg (zygote)
• DNA becomes randomly
integrated
• Low frequency event

Question 9

Summary of steps used in early technology to

generate transgenic mice – transgene construct

Answer 9

Identify the promoter and gene to be manipulated
• Generate a ‘transgene’ by ligating the promoter and
gene of choice
The 5’ regulatory region (promoter region)
defines the expression pattern, i.e. where, when, how much the gene will be expressed

The coding region encodes the gene product, i.e. what protein will be expressed- The coding region can
also be a cDNA
injected by pronuclear injection

Question 10

Embryo transfer

Answer 10

Culture the injected zygotes overnight to determine
which survived the ordeal and divided to the 2-cell
stage
• Transfer of injected 2-cell embryos into pseudopregnant mother (previously mated with a
vasectomised male mouse)
• Approximately 20 days later mouse pups will be
born
• Identify the “transgenic” mouse using PCR of tail
snip
• Efficiency of procedure is «<100%

Question 11

Question: To generate a transgenic mouse that is able to express green fluorescent protein (GFP) in
B lymphocytes the following scenario is required:
A. Entire GFP gene from jellyfish → inject mouse zygote pronucleus → random transgene integration
into genome
B. Entire GFP gene from jellyfish → inject mouse zygote pronucleus → homologous recombination of
transgene into genome
C.BCR(Ig)H promoter -GFP coding region → inject mouse zygote pronucleus → random transgene
integration into genome
D. BCR(Ig)H promoter GFP coding region→ inject mouse zygote pronucleus → homologous
recombination of transgene into genome

Answer 11

C

Question 12

Gene knockouts (loss of function mutants)

Answer 12

Making genetically modified mice where genes are
deleted (or altered) in the genome is a very powerful tool for studying molecular function
• Create a mouse unable to express the protein encoded by gene X
• Study the phenotype created in the gene X-deficient mouse
• Be aware that the loss of protein X may cause a primary phenotype that is masked by secondary phenotypes
• Other genes may change expression pattern to compensate for the loss of protein X and give secondary phenotype

Question 13

The principle of generating gene knockout mice

Answer 13

Replacing the endogenous gene of interest with a mutant gene via homologous recombination
• Homologous recombination
• A natural process
• Requires DNA sequence homology
Utilises Embryonic Stem (ES) cells
• Must be maintained in a pluripotent state
• Must be able to transmit the modification to the next generation
(germline transmission)
• Need to select for ES cells that have undergone homologous
recombination over random integration (rare)

Question 14

Designing a targeting construct to make knock-out mice

Two drug-selectable markers are needed

Answer 14

HSV-tk :Selectable marker (HSV-tk gene)
Converts ganciclovir to toxic product IF present → cells die in ganciclovir
neoR: Selectable marker for neomycin resistance (neoR) interrupts an exon of the target gene
IF present → cells survive in neomycin
+ region of homology

Transfect targeting vector into ES cells
Culture ES cells in neomycin (G418) to select for those that have integrated the targeting construct
Culture neomycin-resistant cells in ganciclovir to select for ES cells that have undergone homologous recombination

Question 15

IF random integration →

Answer 15

HSV-tk gene present

Survive neomycin selection -Die under ganciclovir selection

Question 16

IF homologous integration

Answer 16

HSV-tk gene removed
Survive both neomycin selection
and ganciclovir selection

Question 17

Transgenic technology to understand B cell lymphoma

Answer 17

Any one of a number of genetic elements may have been responsible for inducing uncontrolled division
• One hypothesis was that the close proximity of the
(proto)oncogene c-MYC to the BCR(Ig)H chain locus may lead to its dysregulated expression in B cells
To test this hypothesis, transgenic mice expressing a fusion gene were made
0/16 transgenic littermate
mice developed lymphoma
11/15 transgenic mice
developed lymphoma

Question 18

Knock-out technology to understand the role of MHC class I (2M)
in lymphocyte developmen

Answer 18

• MHC class I expression was known to require
2-microglobulin (2M) co-expression
• Deletion of 2M therefore effectively deletes
MHC class I expression from cells
• Easier than knocking out all of the different
MHC class I genes

Question 19

Question: The generation of knock-out mice lacking 2Microglobulin (2M)
demonstrated for the first time that it had a crucial role in
A. MHC class I expression
B. MHC class II expression
C. CD8+ T cell development
D. CD4+ T cell development

Answer 19

CD8+ T cell development

Question 20

CRISPR-Cas is a two component system

Answer 20

Schematic example: CRISPR/Cas9-targeted double-strand break

1. Spacer RNA with homology for target DNA sequence -
2. Plasmid encoding Cas9 transfected into cell

Question 21

Cas9 generates blunt double-strand breaks

Answer 21

Cas9 proteins rely on RNA guidance for
targeting specificity
• In engineered CRISPR-Cas9 systems, Cas9
interacts with the backbone of the guide
RNA (gRNA)
• Complementarity pairing of the spacer
portion of the gRNA to a DNA target
sequence positioned next to a 5′
protospacer adjacent motif (PAM) results in
a blunt double-strand break (DSB)
• Cleavage performed by the two Cas9
nuclease domains; RuvC and HNH

Question 22

lternative repair mechanisms after double-strand breaks

Non-homologous end joining (NHEJ)

Answer 22

Error prone. Induces small insertion or deletion mutations (indels)
2. Large targeted deletions can be produced by simultaneous targeting of two sites
3. Alternatively, homology-independent targeted integrations can be directed to a single cut
site by providing donor DNA independently targeted for cutting

Question 23

Alternative repair mechanisms after double-strand breaks

Homology directed repair (HDR) pathway

Answer 23

• Donor template is provided
• Contains homology arms (grey
rectangles)
• Can be double-stranded
• Can be single-stranded
oligodeoxynucleotide (ssODN)
• Single nucleotide alterations possible
• Insertion of larger sequences possible
• Insertion of silent mutations (B) that Block
subsequent target site recognition by the
nucleases possible