IV. RESULTADOS Y DISCUSIÓN
4.2. Comunicación interpersonal y productividad laboral de los trabajadores de la
Throughout these experiments (CBA x C57B110)F, mice were used as embryo donors, stud males, pseudopregnant females, vasectomised males and mature females for breeding. AU techniques performed on animals were licensed under the Animals (Scientific Procedures) Act 1986, license no. PIL 80/00831.
2.4.1 Transgenic media
The most commonly used embryo culture media is M l6 , which is very similar to Whitten’s
medium and is bicarbonate buffered. However, fertilised eggs do nor readily continue development beyond the late two-ceU stage in vitro. To overcome this, T6 media was used and the components
are given in Table 1. This medium was incubated at 3TC in 5% CO2, 95% air during use. For
collecting embryos, and for experiments in which the embryos are handled for prolonged periods outside the incubator (e.g. microinjection), HEPES buffer is added in place of the bicarbonate in order to maintain the correct pH. This medium with HEPES supplement is named M2 and the components are shown in Table 2. Stock E was adjusted to pH 7.4 with NaOH. The stock solutions for both M2 and T6 could be stored for 3 months at -20 “C. To make up 50ml of M2 the following
were mixed: 5ml stock A, 0.8ml stock B, 0.5ml stock C, 0.5ml stock D, 4.2ml stock E, 39ml sterile distilled water and 200mg bovine serum albumin (BSA). To make up 50ml of T6 the
TABLE 1: Preparation o f T6 M edia
STOCK A
COMPONENT
g/lOOml
(10
X conc.)
NaCl
4.721
KCl
0.110MgCl^.ôH^O
0.100NaH^PO^.lH^O
0.061
Sodium lactate (60%)
3.4 ml
Glucose
1.000Penicillin G
0.060
Streptomycin sulphate
0.050
STOCK B
COMPONENT
g/
100ml
(10X conc)
NaHCO,
2.100Phenol Red
0.010STOCK C
COMPONENT
g/lOmI
(100
X conc)
Sodium pyruvate
0.029
STOCK D
COMPONENT
g/lOml
(100 X conc)
TABLE 2: Preparation of M 2 Media
STOCK A
(10 X conc.)
COMPONENT
g/
100ml
NaCl
5.534
KCl
0.356
KH
2PO
40.162
MgSO^.TH^O
0.293
Sodium lactate(60% syrup)
3.4 ml
Glucose
1.000Penicillin G
0.060
Streptomycin sulphate
0.050
STOCK B
COMPONENT
g/lOOml
(10 X conc.)
NaHCO,
2.101Phenol Red
0.010STOCK C
COMPONENT
g/lOml
(100 X conc.)
Sodium pyruvate
0.036
STOCK D
COMPONENT
g/10ml
(100
Xconc.)
CaCl^.ZHzO
0.252
STOCK E
COMPONENT
g/lOOml
(10
Xconc.)
HEPES
5.958
following were mixed: 5ml stock A, 5ml stock B, 0.5ml stock C, 0.5ml stock D, 39ml sterile distilled water and 200mg BSA. The solutions were then filter-sterilised before aliquoting into sterile containers. Once prepared, M2 and T6 are stable at4"C for up to two weeks.
After about two years of using these media, it was decided to try a commercial product instead, as the quality of T6 and M2 was found to vary considerably from month to month. The substitute
for T6 used was Whitten’s medium, and for M2 the media names KSOM was used. Although (he
quality of these two products was found never to be as high as the best T6 or M2, it was very
consistent and proved to be satisfactory.
2.4.2 Preparation of DNA for microinjection
Linear DNA fragments were prepared using Glassmilk (see section 2.2.3) and redissolving the DNA in injection buffer instead of dHjO. The concentration was determined by mnning an aliquot of the solution through an agarose gel next to the Hindlll DNA marker. The concentration was then adjusted to Ing/ml, and the final solution was cleaned by centrifugation through a Spin-X column (Costar #8162).
2.4.3 Preparation of egg donors by superovulation
Naturally ovulating females will typically produce 6-10 eggs, so to reduce the number of females needed for each experiment the females used for this purpose were superovulated. This results in 20-30 eggs being produced from each 4-week old animal. The females, which are adjusted to a light period of 5am-7pm were given an intraperitoneal injection with 5IU of pregnant mare’s serum (PMS) at about 3pm, 3 days before the eggs are to be recovered. A second injection of 5IU human chorionic gonadotrophin (hCG) was given at about 1pm, the day before the experiment. Both hormones were obtained from Intervet Laboratories, as Folligon and Chorulon respectively. Following hCG injection, each female was placed in a cage with a stud F, male. The males used were between 2 and 5 months old. The number of viable, fertilised eggs obtained from a mating with a male older than 5 months was significantly reduced, so the stud males were replaced every 3 months. Copulation plugs were checked the following morning and these females removed for oviduct dis section. The oviducts were dissected into M2 medium and the eggs released by opening them with forceps. Cumulus cells were removed from the zygotes by adding hyaluronidase
to the M2 to a final concentration of 300ml/ml, and leaving the cells for 5 min. The eggs were then rinsed three times in M2, before being transferred to T6 and stored in the 37°C incubator (with 5%
CO 2). The drop of T6 was prevented from evaporating by a covering of parafin oil.
2.4.4 Pseudopregnant recipients
Pseudopregnant female mice, between 6 and 8 weeks of age, were prepared by mating females
in natural oestms with vasectomised males. Vasectomised males were prepared in the SPF facilty of the NIMR, and pseudoprenant females were ordered from the unit as required. The females were anaesthetised at O.Sdpc by an intraperitoneal injection of 0.35ml 2.5% avertin, or 0.35ml Hyp/Hyp. Microinjected embryos, at the one- or two-cell stage, were then transferred through the infundibulum into the oviducts, using a heat-polished pulled-capillary needle. Between 10 and 15 embryos were transferred into each oviduct.