Question

The following chart show the results of cloning nuclei from several cell lines (e.g. v6.5, v18.6, etc.) derived from two different genetic strains of mice (F1 and 129). Note: your answers require no prior knowledge of these strains or the specific cell lines listed)

Genetic Background	Cell Line	Passage Number	Activated Oocytes	Embryos Transferred (% Activated)	Dead (% Transferred)	Alive (% Transferred)	Survived Long Term (% Transferred)
F1	V6.5	5-6	149	21 (14%)	0	4	4 (19%)
F1	V6.5 sc84	8-9	46	6 (13%)	2	2	2 (33%)
F1	rtA2 SD-18	8-9	32	7 (22%)	0	1	1 (14%)
Total (F1)			227	34 (15%)	2	7 (21%)	7 (21%)
129	V18.6	5-6	107	19 (19%)	1	4	0
129	J1	11	145	26 (18%)	2	4	0
129high	J1	35	92	23 (25%)	10	0	0
129	LJG-13	8-9	74	8 (11%)	4	0	0
Total (129)			418	76 (18%)	17	8 (11%)	0 (0%)

1. What is the main conclusion that can be drawn based on the results of these data with respect to success of the cloning experiments?
2. Give three possible cell intrinsic differences between v6.5 cells and LJG-13 cells that could play a role in their different results in this assay.

For one of the three potential factors you mention above, describe a brief experimental plan to test whether this factor is responsible for the differences between v6.5 cells and LJG-13 cells in this assay. Include in this plan roughly how you would perform the experiment, what your readout would be and what you could conclude based on your results.

Answer 1

a.Full term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage.Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients(NT2).Pronuclear exchange in zygotes was used for comparision (NT1).Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. In the genetic strain of mice F1 ,the cell lines are V6.5,V6.5 sc84 and rtA2 SD-18 and activated Oocytes are 149,46 and 32 respectively.So by these cloning experiments according to the activated Oocytes the Embryos Transferred are increasing automatically.As the embryo transfer increases the percentage of alive and survive long time percentage are also increasing,The percentage of death is decreasing which is a sign and proof of successful cloning experiment based on those datas. But in the genetic strain of mice 129 we can see that number of activated Oocytes are increased ,so embryo transfer percentage Is also increased.In that case the percentage of survive long and percentage of alive are less than the percentage of dead and that is not a sign of successful cloning experiments.

b.i)In the genetic strain of mice F1 from cell line V6.5 has the passage number 5-6 whereas the the genetic strain of mice 129 from cell line LJG-13 has the passage number 8-9.

ii)In the genetic strain of mice F1 from cell line V6.5 has 147 activated Oocytes where as in 129 cloning nuclei from cell line LJG-13 has 74 a activated Oocytes.

iii)In F1 cloning nuclei from cell line V6.5 has Embryo transferred 21 that means 14% are activated where as in LJG-13 the number is 8 that means 11% are activated,Which is very much lees than the nuclei V6.5.

These are the three possible cell intrinsic differences between V6.5 cells and LJG-13 cells that could play a role in their different results in this assay.

Description:

Cloning allows the asexual reproduction of selected individuals .Cloning by nuclear transfer thus far has been reported only with freshly isolated cells .Here we apply a method to clone mice from widely available,with the V6.5 cell lines R1 ,29% reconstructed Oocytes and 8% embryos are developed to live born pups when transfer to surrogate mothers.We thus cloned 26 mice from R1 cells .Nuclei from V6.5 cell line E14 also shown.The late-passage ES cells can be used to produce viable cloned mice and provide a link between the technologies of V6.5 cell and animal cloning.It thus may be possible to clone from a single cell a large number of individuals over an extended period.

Mouse embryonic system cell lines exhibit unusual karyotypic stability.ES cell lines are derived from the inner cell mass of embryos at the blastocyst stage and can be cultured in vitro for many passages without becoming evidently aneuploid(13-15).These cells can be used to generate chimetric mice containing an ES cell contribution that is apparently unrestricted in terms of cell type(15,16).The heterologous cells are from diploid (16,17) or tetraploid(18-20) embryos.

Cloning mice from V6.5 cells also has practical implications for manipulating the genome.Gene targeting in V6.5 cells has been widely used to create manifold strains of mice with targeted mutations (21,22).If nuclei from V6.5 cell lines ,even after prolonged in vitro culture,could be used to produce viable,fertile cloned animals.

So here,from this assay I can conclude that with varied parameters,microinjection of V6.5 cell nuclei into enucleated Oocytes enables development to term and beyond.I cloned five live-born offspring from the cell line V6.5 and 9 from cell line V6.5 sc84 :respectively , 4 and 2 mice are survived.A single V6.5 cell nucleus thus can direct the null development of an individual.