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Embryo-mania: Freezing mouse embryos. More on phallocentrism


(Idea for tuning Blog gun Shora )

Friends!

Today we will learn how to freeze mouse embryos, in a fast, (ultrarapid freezing), simple and for the whole family.
This system has the advantage of being faster than conventional freezing (10 minutes instead of two hours) and also avoids having to have a biocongelador. The main drawback is that it can only be used in relatively small cells, so often used to compacted morula stage, ie in embryos obtained on day 3 in the afternoon, considering day 1 on the appearance of vaginal plug, which is pregnancy diagnosis used in mice: The semen of the male has a sticky last fraction compact forming a plug in the vagina of the female, which may be visible to the morning after intercourse.

need the following materials:

Females Males
mouse
mouse HCG (human chorionic gonadotropin)
PMSG (equine chorionic gonadotropin)
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90% ethanol surgical material (normal scissors fine point, two-tooth forceps and two needle nose pliers)
binocular magnifier glass pasteur pipettes

siliconized tube system to handle the embryos into the pipette. Petri plates
Middle
M2 35mm tempered in hot plate at 37 ° C
fine hypodermic needle (no.)
Hyaluronidase. Middle
freezing. Middle
defrost Straws

Empajuelador

Bank of liquid nitrogen nitrogen insulated box Rack

liquid metal foil thickness 2mm

STEP 1: FOR EMBRYO SUPEROVULATION in morula stage animalario
-Hours: The light comes on at 7:00 and off at 21:00. This step is critical for coupling the daily hormonal rhythm of mice to which we are going to impose.
-Intraperitoneal injection of PMSG at 15:00 of the day-
-2 intraperitoneal injection of HCG at 15:00 on day 0
-are placed with males in the day 0, popping the cap 1 day ago
-sacrifice of the females on day 3 from 17:00. The embryos are at uterotubal union, so it will be necessary washed oviduct and uterine horn on each side.

STEP 2: PREPARATION OF THE MEANS OF FREEZING
"He uses the same means by which embryos are collected (usually M2). Penetrating cryoprotectants are added (low molecular weight alcohols such as glycerol or ethylene glycol) and non-penetrating (macromolecular lining the embryo and help your dehydration, such as sucrose, bovine serum albumin or fetal calf serum.
- An example would be, M2:
• 5% fetal bovine
· 0.25 M sucrose
· 3M ethylene glycol.
-is sterilized by filtration, and before use must be shaking, because the ethylene glycol has a tendency , to choose and stick to the walls of the bottle. Is kept in the refrigerator for a month, although it is better to have the shortest possible time.



STEP 3: PREPARING THE MIDDLE OF THAWING
"It's PBS with 5% fetal bovine serum, but in this case takes 0.5 M sucrose. Receive the same treatment and behaves like that of freezing.

STEP 4: METHOD OF FREEZING
few minutes before starting the procedure, place a sheet of aluminum of about 2mm in thickness in a polystyrene box filled with liquid nitrogen so that the distance between the surface of nitrogen and the plate is 50mm, and is held in a to stay fully horizontal. (With a rack or similar method)


Place a drop of about 75 ul of freezing it on a plate, and it was pipetted into the smallest possible volume 10-15 embryos. From that moment must have 5 minutes. During that time, to gently agitate the embryos. These must be incurred due to the hyperosmotic solution and float, and later (in about 3 min) fall to the bottom, at which point you can start empajuelar.



straw was loaded into a drop of medium without embryos as 15mm, 10mm then air, then drop half with embryos air again, and a half to reach the end of the straw. Open end is sealed with heated tweezers, and when they have met the 5 minutes since the embryos entered the freezing medium, place the straw in the iron on nitrogen.


Straws 2 minutes should remain on the plate, and then immersed in liquid nitrogen. From here, you can take to the bank and stay long periods of time.


STEP 5: METHOD OF THAWING:
a) Bring the thawing medium, and pour in 2 ml PBS.
b) Remove the straws from liquid nitrogen 20 seconds and let the vapors evaporate so it does not explode when heated.
c) Browse to the bathroom to 37 º for another 20 seconds
d) Dry the straw, cut the seal, resting on the thawing medium, where embryos must remain at least 5 minutes, stirring gently.
e) Wash in M2
f) To plate with droplets of KSOM * whether to grow or transfer immediately whether to do the latter, it increases the success of the operation.

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and biology.


little ago talking about the blunder of a professor of physiology on the definition of clitoris, and how dangerous are the findings biased. Actually, my biggest surprise came from the forum in which that statement was, as it is a fact that the inclination macho society has been projected in many areas, but is primed especially female sexuality, the pathophysiology of sociology. And

is no need to go back to Freud and origin "cancer" of the word hysteria ... is much closer than this: When someone tells a child that there are differences "down there", usually be something like this:

And that you have there is the tail. The girls have no tail but have a hole.

Do you find a bug in the previous discussion? Has anyone ever heard a child tell his genitals, saying he has no clitoris, but has a bag with two balls? It is equivalent.

And this is where I contradict the observers: "A penis," says this school of thought, is much more obvious to see that a clitoris, and that is why an explanation and " Vale. Well. And the teeth are much more obvious to see that the wheels, why not stop telling a child that has both.

But there's more: daddy puts

a seed in mom, and that seed grows and grows inside your tummy, even when it is ready, a baby born from it.

live the "moms-pot" enriched mitochondrial DNA!

And, even in something as seemingly trivial, has canceled all maternal contribution, aside from his role in nurturing machine. An embryo is formed from a zygote, a cell formed by half of the chromosomes of potato, and half of the chromosomes from mom. What is the difference? Well, mom is sighted, and puts their chromosomes in a single cell, full of nutrients, huge, and so protected, Dad (Which is also sighted) has to spend all their energy into making many cells and very mobile, so that at least one arrives. So far, the contribution of both is exactly the same.

But to be a dad, and single dad who "decide" the sex of the embryo (talking about mammals), as all eggs carry an X chromosome breast, while among the sperm of Dad, will you carry X and who carry Y.

only will mom and mother who shall transmit the mitochondrial DNA, which therefore propagates through the mother (grandmother, mother, daughter ...)

When egg and sperm fuse, also do their pronuclei, with your copy (each 50%) of the genetic material of the future embryo. Sexual reproduction has many advantages, such as invalid or replace damaged genes, while in other cases, the two versions of the same gene can undergo phenomena of dominance-recessive or codominance, to model the phenotype of future living.

However, in addition to energy reserves for the early stages of the embryo in the egg is also the "machinery" necessary to take advantage: the mitochondria. These remarkable organelles are not entirely dependent kernel for replication, but travel with their own genetic material and may be considered independently of the cell to which they belong, probably because of its origin (endosymbiont theory Lynn Margulis)

Since this genetic material is transmitted in full from one parent (the mother) is very suitable for tracking sources species, common ancestry and migration.

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anhydrobiosis is called the ability to survive on high dehydration as a result of drying atmospheric air. We observe this phenomenon

in many small aquatic animals (nematodes, rotifers beloides, tardigrades freshwater and terrestrial ...) that have the capacity to enter into a state of anhydrobiosis at any stage of the life cycle or only during certain stages of life, as with cladoraceas eggs (water fleas), the larvae of some flies, embryonic cysts coelenterates, etc.

In the case of frozen embryos of Artemia, one of the best studied cases, living desiccated persists does not mean that any water molecule. Proteins and other molecules are able to preserve what is called "structural water", ie molecules water trapped in the structures of other compounds, or are part of cohesive and adhesive bonding. This phenomenon is favored under conditions of high humidity, but still, anhydrobiotic forms contain less than 2% of water each hydrated state.

The most surprising feature of this vital state, is temporarily away signs of metabolic activity: These forms _{stop using O 2} , and accumulating waste products. But at any given time, under favorable conditions, the body can resume metabolism and "back to life."

In these organisms, it is often the accumulation of a disaccharide called trehalose, which prevents dehydration destabilize vital cellular structures, membranes, macromolecules ...

Animals anhydrobiosis state will tolerate many extreme conditions, as well as dehydration. And this state can be maintained for many years. Represents a highly useful evolutionary strategy, because these animals can be transported by air currents to other areas where conditions are more favorable, and once there, to establish a new colony to fall into the water.