Genetic Engineering Protocols
phoenixbio
Can anyone tell me if there is a published protocol, in laymen's
terms, that describes exactly how:
Specific genes can be introduced into the genetic code of a host
organism?
Specific genes can be 'turned off' in a host organism?
Specific genes can be 'turned on' in a host organism?
Any help is greatly appreciated.
Thanks,
Christiaan
c.ti...@gmail.com
Bryan Bishop
> Specific genes can be introduced into the genetic code of a host
> organism?
Ah, you're looking for gene vectors. In general, see this:
http://en.wikipedia.org/wiki/Vector_(biology) "A viral vector is a
virus that has been modified to transduct specific genetic material
into a cell, e.g., for gene therapy. A plasmid vector is made by
splicing a DNA construct into a plasmid. Various techniques are then
used to transfect the plasmid into the cell."
(1) Plasmids. http://en.wikipedia.org/wiki/Plasmids
"Plasmids are considered transferable genetic elements, or
"replicons", capable of autonomous replication within a suitable host.
Plasmids can be found in all three major domains, Archea, Bacteria and
Eukarya.[1] Similar to viruses, plasmids are not considered a form of
"life" as it is currently defined.[4] Unlike viruses, plasmids are
"naked" DNA and do not encode genes necessary to encase the genetic
material for transfer to a new host. Plasmid host-to-host transfer
requires direct, mechanical transfer by "conjugation" or changes in
host gene expression allowing the intentional uptake of the genetic
element by "transformation".[1] Microbial transformation with plasmid
DNA is neither parasitic nor symbiotic in nature, since each implies
the presence of an independent species living in a commensal or
detrimental state with the host organism. Rather, plasmids provide a
mechanism for horizontal gene transfer within a population of microbes
and typically provide a selective advantage under a given
environmental state. Plasmids may carry genes that provide resistance
to naturally occurring antibiotics in a competitive environmental
niche, or alternatively the proteins produced may act as toxins under
similar circumstances. Plasmids also can provide bacteria with an
ability to fix elemental nitrogen or to degrade calcitrant organic
compounds which provide an advantage under conditions of nutrient
deprivation.[1]"
So, plasmids do not incorporate into the target genome of the target
cells. They are a special class of gene vectors known as "expression
vectors" because they are not integrated into the target genome, and
instead float around inside the cell there and the DNA is transcribed
into mRNAs, thus "expressed" (and the mRNAs into the proteins and so
on). A particular plasmid that you might have heard about in the news
is a bacterial artificial chromsome (BAC):
http://en.wikipedia.org/wiki/Bacterial_artificial_chromosome
"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.[1][2] 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 kbp,
but can be greater than 700 kbp.[3] A similar cloning vector called a
PAC has also been produced from the bacterial P1-plasmid. BACs are
often used to sequence the genome of organisms in genome projects, for
example the Human Genome Project. A short piece of the organism's DNA
is amplified as an insert in BACs, and then sequenced. Finally, the
sequenced parts are rearranged in silico, resulting in the genomic
sequence of the organism."
By the way, this is why some of us have been looking into cheap
designs for electroporators, or the microfluidic electroporators in a
straw, etc. etc. No, I don't have a bibliography yet.
(2) Retrovirus infection, adenoviruses, etc. This is the one that
integrates into the host genome.
http://en.wikipedia.org/wiki/Viral_vector
"Viral vectors are generally genetically-engineered viruses carrying
modified viral DNA or RNA that has been rendered noninfectious, but
still contain viral promoters and also the transgene, thus allowing
for translation of the transgene through a viral promoter. However,
because viral vectors frequently are lacking infectious sequences,
they require helper viruses or packaging lines for large-scale
transfection. Viral vectors are often designed for permanent
incorporation of the insert into the host genome, and thus leave
distinct genetic markers in the host genome after incorporating the
transgene. For example, retroviruses leave a characteristic retroviral
integration pattern after insertion that is detectable and indicates
that the viral vector has incorporated into the host genome."
"Viral vectors are a tool commonly used by molecular biologists to
deliver genetic material into cells. This process can be performed
inside a living organism (in vivo) or in cell culture (in vitro).
Viruses have evolved specialized molecular mechanisms to efficiently
transport their genomes inside the cells they infect. Delivery of
genes by a virus is termed transduction and the infected cells are
described as transduced. Molecular biologists first harnessed this
machinery in the 1970s. Paul Berg used a modified SV40 virus
containing DNA from the bacteriophage lambda to infect monkey kidney
cells maintained in culture."
There's: retroviruses, lentiviruses, adenoviruses, oncolytic viruses, etc.
(3) Restriction enzyme cuts of chromosomal DNA -> ligate in some new
gene strands -> ligate the strands back together so that you have a
full genome -> use somatic cell nuclear transfer (SCNT). But realize
that SCNT is mostly used in cloning of embryos, etc. I actually don't
have references for this method (oops- can somebody help me out?)
http://en.wikipedia.org/wiki/Somatic_cell_nuclear_transfer
"In SCNT, not all of the donor cell's genetic information is
transferred, as the donor cell's mitochondria that contain their own
mitochondrial DNA are left behind. The resulting hybrid cells retain
those mitochondrial structures which originally belonged to the egg.
As a consequence, clones such as Dolly that are born from SCNT are not
perfect copies of the donor of the nucleus."
There's also a few methods that Craig Venter has been pioneering or
publicizing, like his work in whole genome synthesis and genome
transplantation between bacteria. There are probably some better
resources that you can find out there, but here's one off the top of
my head-
http://www.nytimes.com/2007/06/29/science/29cells.html?_r=1
> Specific genes can be 'turned off' in a host organism?
Look up "gene silencing"- there are two main methods: (1) siRNA-based
gene silencing, and (2) miRNA-based gene silencing. There are also
other forms, of course. For instance, you can change the concentration
of certain metabolites in a cell's medium, and subsequently a genetic
regulatory network (GRN) will respond to that- such as the lac operon
in most genetic circuits that you see constructed these days, known as
the operon model of Jacob and Monod- but realize that this is mainly
for lower *expression* levels of specific genes. More on this: "The
lactose system (lacI-lacZYA) in E. coli induces lactose catabolism in
response to the signal allactose, a catabolic intermediate. The
circuit is expected to maintain a stationary basal level of
beta-galactosidase (the lacZ gene product) in the absence of
allolactose, to dynamically increase the expression level when the
level of allolactose increases, and to maintain a higher, stationary
level of expression in the presence of a stationary, inducing level of
allolactose. The circuit function requires both the basal and induced
states to be stabal. If the circuit is to operate in a variety of
environments, the function must be robust to environmental changes. If
the organism must act quickly to make use of metabolites, catabolism
needs to be induced quickly in response to the signal. (catabolic gene
circuit -- see also the ecoli tryptophan system trpR-trpLEDCBA)."
You can see some of my notes on inducible/repressible genetic circuits here:
http://heybryan.org/genetic-circuits.html
In the case of gene silencing, --
http://en.wikipedia.org/wiki/Gene_silencing
"Gene silencing is a general term describing epigenetic processes of
gene regulation. The term gene silencing is generally used to describe
the "switching off" of a gene by a mechanism other than genetic
modification. That is, a gene which would be expressed (turned on)
under normal circumstances is switched off by machinery in the cell.
Genes are regulated at either the transcriptional or
post-transcriptional level. Transcriptional gene silencing is the
result of histone modifications, creating an environment of
heterochromatin around a gene that makes it inaccessible to
transcriptional machinery (RNA polymerase, transcription factors,
etc.). Post-transcriptional gene silencing is the result of mRNA of a
particular gene being destroyed. The destruction of the mRNA prevents
translation to form an active gene product (in most cases, a protein).
A common mechanism of post-transcriptional gene silencing is RNAi.
Both transcriptional and post-transcriptional gene silencing are used
to regulate endogenous genes. Mechanisms of gene silencing also
protect the organism's genome from transposons and viruses. Gene
silencing thus may be part of an ancient immune system protecting from
such infectious DNA elements. Genes may be silenced by DNA methylation
during meiosis, as in the filamentous fungus Neurospora crassa."
Christiaan Tickle
Cory Tobin
> in humans, cows, and let's say mice and the name of that gene is ABC, then
> does that mean that the exact gene ABC is present in all three species or is
> it the case that what appears in all three species are analogs of each other
> and not exactly the same?
I'm going to break this question into two parts. First, about the
names. If humans have a gene called ABC and cows have a gene called
ABC there isn't necessarily any relationship. People working on one
species tend to not have any idea what people working on other species
have named their genes. Although, sometimes genes that are homologous
across species do have the same name. There has been a push recently
to unify some of the names, at least within some sub-fields (the cell
cycle community comes to mind), which has been somewhat successful.
The other part of the question regards the similarity of homologous
sequences. Two genes that are considered homologous across species
definitely don't have identical nucleotide or amino acid sequences. I
think the rule of thumb is that 25% nucleotide similarity is a strong
match (I recall hearing that number in a text book a while back).
> Also, I was looking for more specific descriptions of protocols for gene
> insertion and manipulation. Let's say that I want to splice a specific gene
> from a fire-fly into a fern (just as an example), what would I exactly be
> doing step by step in order to accomplish this?
The protocols for getting genes out of different species are pretty
uniform. They usually go something like 1) crush up tissue 2)
purify DNA 3) PCR with appropriate primers. Getting that DNA into
another species is a whole different story. For example, I do plant
biology and the protocols for getting genes into Arabidopsis are
different than tobacco which are different than soybean, etc. I would
love it if there was some uniform protocol for moving genes between
species (my life would be so much easier) but the reality is that it's
different for most species.
> I'm just very very new to this and would like to learn as much as I can.
You've come to the right place!
-Cory
Nathan McCorkle
if you know the gene sequence: isolate firefly DNA, PCR gene using primers specific and complementary to either end of the gene sequence(and possessing 5' restriction sites), ligate into a vector(possibly for Agrobacterium). This being a fern, I don't think Agrobacterium is known whether it will be of use to transform the plasmind with the gene into the fern. If Agrobacterium does infect ferns, then you would transform Agrobacterium with the ligated plasmid, grow them a bit, and then dip sporulating fern areas, and culture the spores selecting for a mutant by having antibacterial resistance coupled with your gene.
Other ways would be to get some known gene sequence, and ligate that to the flanking areas of your gene+antibiotic resistance gene... and do a PEG transformation of just the DNA, and hope that the known sequence flanking your gene, get's recombined with the genomic homologous sequences.
Could also probably find a virus that infects ferns, and throw your gene in there, or make fern mutants using chemical or radiation, search for your mutation, sequence around the mutation, and use the data generated as your homologous stretch of DNA for a try at homologous recombination.
and also you could isolate RNA, make a cDNA library, which you use to transform with and express in yeast (if it's firefly luciferase for example, you would "see" a result)
it really depends on what you are working from, and toward. There are a number of bioinformatic comparisons you could do against similarly functioning genes, if any of their sequences are known, and have homology, then you could try using the conserved region as a primer in your uncharacterized genome/organism.
Use pubmed and google scholar, they should be primary resources
homology = similarity between DNA sequences
Orthologs = evolutionarily related genes, with same enzymatic function
paralog = similar gene sequence, with possibly different functionality
http://en.wikipedia.org/wiki/Homology_(biology)#Orthology
take for example the amino acid decarboxylase enzymes (AADC) in mammals, it's got a conserved region homologous to plant tryptophan decarboxylase (TDC).... AADC can catalyse multiple different substrates, whereas TDC is tryptophan specific.
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Mackenzie Cowell
Bryan Bishop
> We have got to get some of these answers into the FAQ! Whoo hoo, great
> responses guys!
Okay, I added them.
http://openwetware.org/wiki/DIYbio/FAQ#Genetic_engineering
Although I didn't elaborate or write any text in paragraph form, which
might be useful there.