Science

At https://en.wikipedia.org/wiki/Electroporation#Gene_electroporation

See under sub-header: Gene electroporation,

 also copy / pasted below:

Application of electric pulses of sufficient strength to the cell causes an increase in the trans-membrane potential difference, which provokes the membrane destabilization. Cell membrane permeability is increased and otherwise nonpermeant molecules enter the cell.^[49][50] Although the mechanisms of gene electrotransfer are not yet fully understood, it was shown that the introduction of DNA only occurs in the part of the membrane facing the cathode and that several steps are needed for successful transfection: electrophoretic migration of DNA towards the cell, DNA insertion into the membrane, translocation across the membrane, migration of DNA towards the nucleus, transfer of DNA across the nuclear envelope and finally gene expression.^[51] There are a number of factors that can influence the efficiency of gene electrotransfer, such as: temperature, parameters of electric pulses, DNA concentration, electroporation buffer used, cell size and the ability of cells to express transfected genes.^[52] In in vivo gene electrotransfer also DNA diffusion through extracellular matrix, properties of tissue and overall tissue conductivity are crucial.^[53]

In eukaryotic cells, the nuclear DNA is inside the nuclear envelope, traffic through which is tightly regulated by the nuclear pore complex which is generally too small for nuclear DNA to pass through,

especially because such DNA is packaged into chromatin fibers wrapped around nucleosomes etc. So the cargo is too large to pass through, from inside the nucleus, outside into the cytoplasm, through the narrow nuclear pores.

That is one reason why electroporation works best in one direction, and not in both :)

At the outer cell membrane, external DNA that passes through, into the cytoplasm, I suspect is not just leaking through, but transported either actively or passively, by a pump or channel respectively.

There must be selectivity at various boundaries, especially at the cell membrane and nuclear membrane that makes this much more uni-directional that one would imagine based just on principles of electricity and ion transport.

Note that generally the concentration of specific ions is unequal across various membranes, for various biological reasons (energy generation, prevent toxicity etc.). 

This is possible due to selectivity of membrane pumps and channels. 

So the same property of membrane selectivity must operate even during electroporation, albeit less effectively, due to the electrical zap during electroporation creating a brief imbalance.

Cheers,

TdM

 

On Sun, Jan 24, 2021 at 4:01 PM Nathan McCorkle <nmz787@gmail.com> wrote:

Short answer is, electroporation opens the cell, electrophoresis moves your DNA in, but also any other ions too. This applies similarly for the inner contents, they may move out... Given enough time. I think the key is concentration of exogenous DNA is great enough that it's just outside of the cell when the pores form, and your open time is just enough for the length of the molecule to move inside before the pores close.

As for the nucleus, I'm not sure I've seen anything on what happens to the nucleus, whether pores also form in there. It's an interesting question that I'm sure I could get sucked into a good day or more of searching and digging through references.

On Sun, Jan 24, 2021, 11:45 AM Andreas "Mega" Stuermer <andreas.t.stuermer@gmail.com> wrote:

Hi guys, 

A question came up that I found interesting. 

Electroporation is a standard method in vitro and in vivo. 

The DNA swims around next to the cells, and then the electrical pulses drag the DNA through the membrane. And the membranes might also have developed pores from the shock. 

Why isn't the DNA from the nucleus pulled out of the cell? Its backbone is negatively charged as well. I assume that the cell as a whole has a charge (the neagtively charged membrane builds a faraday-like cage??) and thus the cell is deformed, but the charge within the cell is homogenous? 

I mean, electroporation would not work if the nuclear DNA would just be dragged out of the cell so I assume that is what heppens. 

Would be cool to see if anyone has deeper insight

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Short answer is, electroporation opens the cell, electrophoresis moves your DNA in, but also any other ions too. This applies similarly for the inner contents, they may move out... Given enough time. I think the key is concentration of exogenous DNA is great enough that it's just outside of the cell when the pores form, and your open time is just enough for the length of the molecule to move inside before the pores close.

As for the nucleus, I'm not sure I've seen anything on what happens to the nucleus, whether pores also form in there. It's an interesting question that I'm sure I could get sucked into a good day or more of searching and digging through references.

On Sun, Jan 24, 2021, 11:45 AM Andreas "Mega" Stuermer <andreas.t.stuermer@gmail.com> wrote:

Hi guys, 

A question came up that I found interesting. 

Electroporation is a standard method in vitro and in vivo. 

The DNA swims around next to the cells, and then the electrical pulses drag the DNA through the membrane. And the membranes might also have developed pores from the shock. 

Why isn't the DNA from the nucleus pulled out of the cell? Its backbone is negatively charged as well. I assume that the cell as a whole has a charge (the neagtively charged membrane builds a faraday-like cage??) and thus the cell is deformed, but the charge within the cell is homogenous? 

I mean, electroporation would not work if the nuclear DNA would just be dragged out of the cell so I assume that is what heppens. 

Would be cool to see if anyone has deeper insight

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Hi guys, 

A question came up that I found interesting. 

Electroporation is a standard method in vitro and in vivo. 

The DNA swims around next to the cells, and then the electrical pulses drag the DNA through the membrane. And the membranes might also have developed pores from the shock. 

Why isn't the DNA from the nucleus pulled out of the cell? Its backbone is negatively charged as well. I assume that the cell as a whole has a charge (the neagtively charged membrane builds a faraday-like cage??) and thus the cell is deformed, but the charge within the cell is homogenous? 

I mean, electroporation would not work if the nuclear DNA would just be dragged out of the cell so I assume that is what heppens. 

Would be cool to see if anyone has deeper insight

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It's possible to get 100% isopropanol and it can perform really well - I can't recall the differences but you get better/worse performance for different salts, and it takes a lot longer to re-dissolve isopropanol-washed DNA, but it's cheap and it doesn't absorb water the way ethanol does. Worth considering anyway..

Also, look into biofuel ethanol as an alternative to denatured industrial ethanol? You get it from suppliers of ethanol indoor burners. It's handy for spirit burners, but because it's used in burn-ey and evaporation-prone gadgets they use different denaturants. At the very least, they don't add nauseating smells to it.

I was able to buy 96% ethanol in jerrycans from one such supplier fairly cheaply. I never used it for nucleic acid work so I cannot say whether it's suitable.

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9 Jan 2021, 19:01 by dko...@gmail.com:

Given the volumes needed for >98% ethanol for prep kits you'll get

hundreds to a thousand preps out of a liter. No idea what the additive

would do on a spin kit.

On Sat, Jan 9, 2021 at 1:49 PM Andreas "Mega" Stuermer

<andreas.t...@gmail.com> wrote:

Hi guys!

Question on purification of DNA with a DNA purification kit (or a maxiprep kit).

The principle is that DNA binds to the silica columns, and then ethanol run above it so it washes away impurities.

Here, pure ethanol that has not been denatured costs 30€+ per litre.. Because the government decided it's a good idea to punish also science with a heavy drinking tax on all alcohol. Infact, the chemical companies here I only see with 80€/L

They add to Ethanol 1g/100L Denatonium benzoate and 1% Methylethylketon.

Methylethylketon looks like it would evaporate easily, so that might not be an issue.

Denatonium benzoate - no idea if it binds to DNA or silica under the given condition.

If you want very pure DNA, what would you do? Try to get expensive ethanol?

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Yeah not many Wallmarts and Costco's around here with the 10cc but I
never tried.  Endo doing it electronic for probably this reason.  I
have no interest in utilizing more than I'm prescribed right now, it's
just that, this dose still hasn't relieved all issues.  I think
estradiol is the big issue at this point.  I guess continually
dropping adipose tissue is all I can do.  I want something more
androgenic though.

On Thu, Jan 14, 2021 at 7:07 PM Frank <fgarcia0007@gmail.com> wrote:
>
> Btw in my experience CVS is the worst about filling testosterone prescriptions . They will take a 10 mL prescription and tell you that they have to give it to u in 1cc allotments because it's a controlled substance . they also charge more than any other pharmacy for that and everything else. You might want to consider getting a prescription but not using insurance to pay for it because it's really not that expensive especially if you use a coupon from good RX and then they cannot do anything but give you the full prescription since you are paying
>
> On January 14, 2021 6:44:01 PM Dakota Hamill <dkotes@gmail.com> wrote:
>
>> I'd talk for days about hormones. That website is awesome, thanks
>> Frank.  I ended up finding a urologist via CVS who runs a men's clinic
>> on the side but I can't seem to get him to take over my insurance
>> covered TRT because...I can't even get an appointment with him.  It's
>> super frustrating being on TRT after  4 endos, and the one who finally
>> agreed to treat me doesn't believe in AI's or hCG.
>>
>> I'm a keep this thread rolling with the hijack if that's ok Frank lol.
>> I don't know why there isn't a crowd-sourced diagnostic platform to
>> help people (not give medical advice) but give "ideas" about what to
>> test or what may be a root cause.
>>
>> https://imgur.com/a/FpWVqUe
>>
>> That was my development on TRT.  I basically went from 0 facial hair
>> and body hair my entire life to a beard in 10 months, I consider that
>> I'm finishing puberty at 33.  Psychologically it's fucked me.  Science
>> the only thing that kept me alive.
>>
>> I was diagnosed with normosmic idiopathic hypogonadotropic
>> hypogonadism but I don't think that's the case.  I agree with
>> hypogonadism without a known cause...but I don't think it's Kallmann
>> syndrome at all.  I KNOW there is something wrong with my sympathetic
>> nervous system because I've been in fight-or-flight mode since 5 years
>> old, and nothing horrific happened.
>>
>> I've been pouring sweat since 5/6 years old after a Chicken Pox
>> infection.  My hands are dripping liquid as I write this...and there's
>> no reason for that.
>>
>> Always had thermal dysregulation, always felt hot, feet and hands cold
>> sweat.  Went from skinny as a rail as a track athlete to obese (with
>> that visceral fat), insulin resistant, metabolic syndrome,
>> dyslipidemia, heart issues, dermatographia, spider veins everywhere,
>> massive stretch marks.
>>
>> I've just felt like I have been high on adrenaline/cortisol my entire
>> life, and never able to relax, and it took a toll.  But...my cortisol
>> always comes back normal, as did my catecholamines.
>>
>> Sympathetic nervous system will shut down GI movement, and I also had
>> horrible GI bleeding and constipation, with joint aches (first
>> testosterone was 171ng/dL with a 7 total estradiol, both below range
>> and bottom of range).
>>
>> TRT my GI bleeding went away within 48 hours after 5 years of bleeding
>> (never did a colonoscopy), joint pain went away (finally had
>> estrogen), lipids normalized, insulin normalized, blood sugar
>> normalized.
>>
>> Only thing is I still have hyperhidrosis for no reason, and I feel
>> anxious all the time and had a few bouts of mania/psychosis on TRT.
>> Idk if my thyroid is fucked or my adrenals are fucked or I have a
>> tumor somewhere since 5 years old or what.
>>
>> Well maybe someone smarter than me in this can help.  I've just felt
>> tired...forever.
>>
>> On Thu, Jan 14, 2021 at 5:09 PM Frank <fgarcia0007@gmail.com> wrote:
>>>
>>>
>>> To better answer the question that you actually asked, yes the testosterone that I've taken has always been intramuscular and by prescription although I have definitely taken my shot at getting testosterone without a prescription. I would not recommend it.
>>>
>>> there is a way to discover what doctors in your area are likely to prescribe testosterone to you . The Medicare payment database is made available through an organization called propublica . heres a link to the resource with testosterone selected. https://projects.propublica.org/checkup/drugs/8883 just sort results by state and look up your state and you'll see the top testosterone prescribing doctors.
>>>
>>> ------ Original Message ------
>>> From: "Tom De Medts" <tdmedts@gmail.com>
>>> To: "DIYbio" <diybio@googlegroups.com>
>>> Sent: 1/13/2021 3:23:43 PM
>>> Subject: Re: Re[2]: [DIYbio] How does one hire a bio lab technician for consulting?
>>>
>>> @ Dakota - Is your testosterone cypionate a prescription intra-muscular injection? And is it possible to procure this without a prescription?
>>>
>>> I have stubborn abdominal visceral fat that just will not go away to the point that exercising with that objective has led to multiple ligaments tears of the knees,
>>> at least 3 vertebral disc wear requiring future surgery, bad low-back with very limited flexibility, displacement of cervical vertebrae etc.
>>>
>>> I've heard that testosterone injections generally help with healing better and faster, is this true?
>>>
>>> Also, my testosterone levels were normal in my recent blood test, but I wonder whether its levels vary just as levels of most female hormones cycle up and down? Perhaps my androgen levels
>>> seem OK in a snapshot, but not if followed as a time course?  Like some days, in the morning especially, my voice will be deeper. And other times, my  voice will sound less deep, a little more squeaky. As a non-medical biologist, I would not venture to hypothesize if these are all related or not.  So I'm just thinking aloud and throwing out these observations.
>>>
>>> Any suggestions / advice is welcome...
>>> @ Frank and Dakota, this is not at all an effort to hijack your discussion. Sorry if this is going off on a slight tangent, happy to start a new thread if you wish...
>>>
>>> Thank you, in advance.
>>>
>>> Cheers,
>>> TdM
>>>
>>>
>>> On Wed, Jan 13, 2021 at 1:09 PM Dakota Hamill <dkotes@gmail.com> wrote:
>>>>
>>>>
>>>> Seems like you have a solid reason.  Apologies for projecting the
>>>> notion of vanity on your decisions.   Outside of anti-aging clinics or
>>>> treating rare diseases I'm not sure who offers treatments like this
>>>> with stem cells.  How you separate who is legit from a quack, no idea
>>>> either.  I'm not even sure what FDA approved stem cell treatments
>>>> there are.
>>>>
>>>> I can see the allure of anabolic steroids, I'm just on testosterone
>>>> cypionate now and it's reversed all sorts of problems I've had my
>>>> entire life overnight.
>>>>
>>>> Steroids wouldn't help with your bone loss and teeth problems?
>>>> They're designed to help fight osteoporosis.
>>>>
>>>> On Wed, Jan 13, 2021 at 12:09 AM Frank <fgarcia0007@gmail.com> wrote:
>>>>>
>>>>>
>>>>> thanks for the message and for being concerned. I am familiar with body
>>>>> dysmorphia more as an observer than a suffer. waaaaay back when I was
>>>>> young I did my stint with body building and steroids. I'm lucky that it
>>>>> did not turn into an addiction for me. I have friends who did not fare
>>>>> so well. One of them , a radiologist with whom I attended medical school
>>>>> died at the age of 31 from a massive heart attack resulting from
>>>>> cardiomegaly.
>>>>> Today I am the poster child for just about every damn age-related
>>>>> degenerative condition, I had three artificial intervertebral disks in
>>>>> my neck (before they were approved) due too herniated disks. The veins
>>>>> in my legs are basically useless forcing me to wear compression socks
>>>>> always. I got Halux Valgus (bunions) in both feet almost overnight, and
>>>>> while i have no cavities,  I do have chronic aggressive periodontal
>>>>> disease  with bone loss.  There's more but i'll hold off.   two of my
>>>>> conditions have been shown to respond and even resolve with stem cell
>>>>> therapy. With periodontitis, stem cell injections right on the receding
>>>>> edge of the bone in a matrix that keeps them there for a little while
>>>>> significantly stimulates not just bone but all the lost tissues to grow.
>>>>> That treatment is actually in practice but it costs a fortune and I'm
>>>>> about to loose 4 teeth if i don't act soon,.
>>>>> the other which i have mentioned is more of a problem with even less of
>>>>> a solution but in which stem cells have shown remarkable efficacy..
>>>>> Believe me i have no thing about wanting to inject stem cells for
>>>>> vanity. I would love to not have to go through all this trouble, So no
>>>>> danger of me doing this just for th hell of it.
>>>>>
>>>>> To your other point, I am absolutely into anti aging but in a true way,
>>>>> not just superficially. If i had the money i would have a protocol for
>>>>> myself that attacks some of the key drives and pay people to  force me
>>>>> to do things like intermittent fasting, being in bed by midnight, and
>>>>> exercising consistently. that right there is has more impact than just
>>>>> about any single drug. Then I'd buy myself a plasmapheresis machine, and
>>>>> get my hands on  4 or 5 of the molecules in this list:
>>>>> https://docs.google.com/document/d/1c79BsB8ooKkefmKLNRwTj9F1grkyrEN0_vwwuR3gFmA/edit?usp=sharing
>>>>>
>>>>> so my metric for "youthfulness" would not be things like wrinkles but
>>>>> rather LDL/HDL ratio, GFR, C-reactive protein, HBAC, blood pressure,
>>>>> physical strength, cognitive performance,  etc.
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> ------ Original Message ------
>>>>> From: "Dakota Hamill" <dkotes@gmail.com>
>>>>> To: "diybio@googlegroups.com" <diybio@googlegroups.com>
>>>>> Sent: 1/12/2021 9:45:09 PM
>>>>> Subject: Re: [DIYbio] How does one hire a bio lab technician for
>>>>> consulting?
>>>>>
>>>>>> I think you have an interesting project idea from your previous posts
>>>>>> and literature research, but it seems like you'll run into a number of
>>>>>> issues.  I hesitate to ask the "why" of why you want to re-inject
>>>>>> adipocyte stem-cells back into yourself because usually people that
>>>>>> want to do something like this have reasons no one else can talk them
>>>>>> out of.
>>>>>>
>>>>>> Not judging though, because I have some serious pathological body
>>>>>> dysmorphia issues from a rare endocrine disorder, and I'm on
>>>>>> endocrinologist supervised treatment, but it never feels like enough.
>>>>>> I assume this is what it feels like to be anorexic or obsessed with
>>>>>> plastic surgery.  People see things about themselves no one else does.
>>>>>>
>>>>>> If you're dead set on it, you're probably going to have to do it
>>>>>> yourself.  That's going to make it difficult and expensive, and
>>>>>> dangerous.  You could die from an infection or immune response.  I
>>>>>> assume everyone has a "right" to self-treat themselves, but to what
>>>>>> extent another may be liable for manslaughter if it goes awry, I have
>>>>>> no idea.
>>>>>>
>>>>>> What's your end measurable of it "working"?  Never aging?  No
>>>>>> wrinkles? No cellulite? You're going to inject it all over your entire
>>>>>> body that's covered with fat tissue?
>>>>>>
>>>>>> Skip the headache/lawsuit/hospital bill of doing it yourself if you're
>>>>>> dead-set on it and talk to
>>>>>> https://www.bostonstemcell.com/stem-cell-therapy/fat-graft/  at least
>>>>>> you can sue a doctor if it goes awry.  $900-$6,000 and only one google
>>>>>> away!
>>>>>>
>>>>>> Good luck hope you find what you're looking for.
>>>>>>
>>>>>> On Tue, Jan 12, 2021 at 4:37 PM Frank <fgarcia0007@gmail.com> wrote:
>>>>>>>
>>>>>>>
>>>>>>>
>>>>>>>
>>>>>>> As you know I am attempting to produce an sample of viable fat stem cells suitable for personal medical use, on my own, and on a budget. At this point I think I understand the salient biological and medical issues but I am lacking hands on laboratory know-how.
>>>>>>> I looked into utilizing a community biospace but they are nonexistent close by or not interested in what I'm doing. Renting space in a biolab is too expensive.
>>>>>>> So I was thinking that a way to accomplish this might be to hire as a consultant an experienced lab technician who has done all of the parts of what I'm attempting to do.
>>>>>>> they would evaluate the protocol I'm gonna follow , tell me what to expect and about pitfalls, make sure i know all needed materials and equipment, and walk me through it. They could also tell me were I can use cheaper equipment alternatives .
>>>>>>>
>>>>>>> So my question to this group: are biolab tech folks hired for this sort of thing? Does someone doing stem cell culturing at a biotech company ever moonlight to make extra money? If so is there a preferred source for such a consultant like a forum or classified?
>>>>>>>
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>>>>>>> Learn more at http://www.diybio.org
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>>>>>>> To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/2fc2fe25-4aea-4fb4-ae67-c17f29e23096n%40googlegroups.com.
>>>>>>
>>>>>>
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>>>>>> Learn more at http://www.diybio.org
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>>>>>> To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/CAGdeWmQoad_QtQqANjRc5HocL_xg--A6N40cm645osLWaThmqw%40mail.gmail.com.
>>>>>
>>>>>
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>>>>> Learn more at www.diybio.org
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>>>>
>>>>
>>>> --
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>>>
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>>
>>
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>
>
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> Learn more at www.diybio.org
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Learn more at www.diybio.org
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