Several questions about genetic engineering

[Eagle9]

TL;DR Summary

Are micropipets the best option?

Hello people

In future I am going learn biotechnology and genetic engineering at some moderate level, but now I simply need to be aware of some basic aspects of this field

Imagine that we need to insert some gene(s) in ape/chimpanzee and the length of these genes are at least 100 000 bp or even more (this is quite long, as far as I am aware the average gene length in human/primate is about 30 000 bp). What methods are the most suitable for this purpose?

1) Plasmids? But are they capable for carrying such long genes? Are they used for transferring the genes into animal’s organisms? As I know they are mainly used in bacteria and plants.

2) Viruses? Can they transfer long genes? Long gene must fit inside the virus after all and surely we cannot “inflate” virus.

3) Probably micropipets can bear much longer genes and directly insert them into nucleus, I am correct?

4) Can the Gene gun method be used in order to directly insert genes in ape’s brain? Imagine that we open ape’s scull and “fire” genes into brain (cortex)? Has anybody done this? Generally, which method is used if we want to insert a very long gene in nucleus? The nucleus in human cell is ∼10 μm in diameter, but we may want to insert the gene that has got 200 kbp length and its physical length will be much more than this 10 μm.

 

[jim mcnamara]

Could you help us out? You referenced some things I do not know, either because they are popular science or I missed them.

And I think you overlooked micelles (nanolipid particles) for DNA delivery.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947036/

Also consider that viral DNA already exists in the human genome - all mammals for that matter. So it got there somehow right?
Popular Science article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947036/

If you want reasonable answers, then citations would improve things. Otherwise we treat vague claims as some kind of speculation. PF does not support speculation.

Plus, do you know about CRISPR, for starters at least?

 

[BillTre]

Your questions are pretty undirected.
.
What I am writting is based on stuff I did many years ago, so there is probably much more current information out there.

It is not clear what your intended use is, permanent insertion into the genome, transient expression, permanent expression in some somatic (non-germline) cells, ...
The transformation methods differ significantly for these.
Germline (the reproductive cells) transformation usually involves going through a generation or transforming an egg, transforming it and raising it to your desired stage.

There are many different plasmids and viruses able to handle different sizes of inserts (probably the micelles also). There other thing, similar things also. I have grown yeast artificial chromosomes to grow large sections of Drosophila chromosomes and later isolate them.
Manipulating large pieces of free floating (unpackaged) DNA requires special techniques. Simply pipetting large peices of DNA will break the molecules into smaller pieces, destroying their function.

I don't think many people transform great apes.
Shooting something into their brain would involve brain surgery (which would be very similar to human brain surgery, not for the inexperienced). The identity of the cells transferred would not be controlled only the location. Not all the cells in an area would be transformed. Some of the cells would probably be killed.
There would be many problems working with apes (besides the ethics of working with an almost human). Long generation times and expensive housing would usually be prohibitive. Small generationally quick research animals (C. elegans worms, fruit flies, zebrafish (fish in general), and mice are popular in labs).
Bigger animals, like horses and cows, have probably been done by now, but they would be backed up by agricultural scale money and farms. Organisms with genetic markers and that can be raised and bred in captivity are useful.

Micropipets are used to put things like nucleic acids into cells, one at a time. Works well for large eggs. Smaller cells would be more of a challenge.

There are also electroporation methods. This involves putting a solution of nucleic acids around some cells and applying high voltage pulses. This causes small transient holes in the membrane through which the DNA or RNA enter the cell. Usually done in a cuvette (small container) in a machine, but has now also been done in situ in parts of living animals (voltage only affects cells near the electrodes). This can transforma local group of cells (maybe killing some). The actual site of insertion into the genome can vary from cell to cell, or it maybe just floating around, unattached to a chromosome, in a non-dividing cell. This is a level of variability within the animal

Some viruses have also be applied in vivo to local patches of cells for transformation.

Targeting where the DNA might go once injected is another issue.
@jim mcnamara's comment about crispr deals with n aspect of this.
A gene inserted in different locations can have drastically different results.
Usually several transformants are recovered and sorted through to find one with the desired expression of the gene.

Before you get into all these various details, you should be clear about what you want to do.
This will trim down the number of possible approaches.
You could also look up in journals what people have done this in the past and see how they did it.
The genetics and the raising of animals is often involved. For success, all these things have to mesh together.

 

[Eagle9]

Plus, do you know about CRISPR, for starters at least?

Frankly saying I have got very vague idea about it

Actually I am looking for some online–courses, could you please tell me if there are any about Biotechnology and Genetic engineering? On Coursera there are some courses, but perhaps you know better ones

A gene inserted in different locations can have drastically different results.

Imagine that we want to insert it in zygote.

Before you get into all these various details, you should be clear about what you want to do.
This will trim down the number of possible approaches.
You could also look up in journals what people have done this in the past and see how they did it.

Could you help us out? You referenced some things I do not know, either because they are popular science or I missed them.

Perhaps you are right, yes. What I am looking is the possibility to insert a relatively long gene(s) in zygote. So I want to know which method is the most suitable.

If you want reasonable answers, then citations would improve things

I do not always remember them in one case I have seen in youtube how gene was inserted in cell (perhaps it was zygote, I do not remember) by micropipets, but there was nothing said how difficult it is, how long/length gene we can insert. That’s why I want to know more about Biotechnology and Genetic engineering.

Manipulating large pieces of free floating (unpackaged) DNA requires special techniques. Simply pipetting large peices of DNA will break the molecules into smaller pieces, destroying their function.

Micropipets are used to put things like nucleic acids into cells, one at a time. Works well for large eggs. Smaller cells would be more of a challenge.

So, micropipets still can be used for this purpose, right?

It is not clear what your intended use is, permanent insertion into the genome, transient expression, permanent expression in some somatic (non-germline) cells, ...

I do not know what is the difference between them. But I want to insert some gene in nucleus, this gene will be floating there (it probably will not attach to inner surface of the nucleus) and then this gene will be expressed. Actually that’s it. So it is permanent insertion, but is it transient expression or permanent expression I do not know.

I feel I need to fill gap in my knowledge in molecular biology

 

[BillTre]

Imagine that we want to insert it in zygote.

Same problem, unless a method is used to target an insertion site. Crispr and some other techniques could do this.

Perhaps you are right, yes. What I am looking is the possibility to insert a relatively long gene(s) in zygote. So I want to know which method is the most suitable.

Often mircopipettes are used (Crisspr stuff can be injected. Have also heard of gene guns being used for this.
Not sure about the length issues.
Find a research article about putting a gene of your desired size into the kind of zygote you are interested. Different techniques might be used for different species.

So, micropipets still can be used for this purpose, right?

Yes, for some purposes.

I do not know what is the difference between them. But I want to insert some gene in nucleus, this gene will be floating there (it probably will not attach to inner surface of the nucleus) and then this gene will be expressed. Actually that’s it. So it is permanent insertion, but is it transient expression or permanent expression I do not know.

Transformations are done for different purposes, with different techniques, and different results.
Insertion into the genome: puts the new DNA in the a chromosome by making covalent bonds into the middle of the chromosome's DNA. This ensures that the new DNA is properly sorted out to each daughter cell when the parental cell divides. If it were not attached to a chromosome, it would basically float around in the cell and partion to the daughter cells in a somewhat random manner (not Mendelian inheritance).
DNA not attached to a chromosome: partitioning to daughter cells somewhat random, inheritance not predictable. If the cell will not be undergoing any more cell divisions (like a neuron), then this doesn't matter that much since the DNA won't get lost in a cell division.
permanent insertion into the genome: this (once the insertion becomes stable) should not change in any cells that inherit it.
transient expression: sometimes it is only desired to express a gene for a short time , integration into genome not required. In dividing cells the DNA could dilute out, or be partitioned to only some cells during division.
permanent expression in some somatic (non-germline) cells: proper integration into the genome, but in somatic cells, not germline cells. Only germline cells make reproductive cells which are required to make a genetic line through breeding the transformant. If the whole organism is transformed except the germ cells, the organism will only produce un-transformed offspring, which is no good for genetics.
Often times, establishing a genetic line is the desired end. They are (IMHO) usually the most useful result. You have the exact same mutation to test time after time (and pass around to your friends), but its more work to establish a genetic line.
Some times the first organism raised with a mutation has some "unstability" in the genetic mutation. This gets resiolved in a few cell generations in the first organism, which can result in some mozaic-ness. Not all cells in the organism having the same mutation. Lines established form the first transformed organism, should be stable. This depends on the details of the technique/species you use.

I feel I need to fill gap in my knowledge in molecular biology

Taking an actual course, with a lab would probably be best thing for you.
In any biological field, familiarity with molecular techniques will be very useful, generally speaking.
My daughter is interested in wildlife field biology, but she has worked in a molecular genetics lab (as well as taking courses) so she is familiar with the many molecular the concepts and techniques, which I am sure will be of beneficial to her.
It similar to understanding computers in other fields (computer understanding is good for biology too of course).

 

[Eagle9]

BillTre
Well, thanks a lot for a detailed answer

Insertion into the genome: puts the new DNA in the a chromosome by making covalent bonds into the middle of the chromosome's DNA. This ensures that the new DNA is properly sorted out to each daughter cell when the parental cell divides. If it were not attached to a chromosome, it would basically float around in the cell and partion to the daughter cells in a somewhat random manner (not Mendelian inheritance).

DNA not attached to a chromosome: partitioning to daughter cells somewhat random, inheritance not predictable. If the cell will not be undergoing any more cell divisions (like a neuron), then this doesn't matter that much since the DNA won't get lost in a cell division.

permanent insertion into the genome: this (once the insertion becomes stable) should not change in any cells that inherit it.

Insertion into genome is better if we want permanent usage of certain gene, but it requires advanced genetic engineering technologies. From the other hand, if we simply inject DNA molecule containing desired gene into nucleus then this DNA alongside the genes should also have:

1) Replication origin site in order this DNA to be replicated (like all other chromosomes are replicated) and distributed in daughter cells.

2) Special sequence in order this DNA to be attached to microtubules (kinetochore).

But still I think that this is easier than genetic techniques, CRISPR or other

 

[BillTre]

An injected piece of DNA, not attached to a chromosome, will not distribute well in dividing cells. It may work in some divisions, but there will be cases when all copies go to one cell and the other cell comes up short.

Putting a centromere on a piece of DNA will make it somewhat like a yeast artificial mini-chromosome. It will partition properly at cell division, however it will be much larger in size.

But still I think that this is easier than genetic techniques, CRISPR or other

I think that Crispr manipulations are way easier than those involving artificial chromosomes.

 

[Eagle9]

BillTre
Thanks