sgRNA Transgene Mapping Project Overview

BioMG2801 sgRNA Transgene Mapping Project Overview The goal of this two-part project is to use traditional genetic mapping techniques to map the sgRNA ytg transgene that was inserted into the Drosophila genome. Part one is conducted in labs 2-6 and uses Cross Schemes 5 and 6 to determine if the sgRNA ytg transgene was inserted into chromosome 2 or chromosome 3. Once you determine which chromosome the transgene was inserted into, you will conduct the second part of the project. Part two is conducted in labs 7-11. For Part two you will design a 3-point cross (which you learned/will learn about in BioMG2800) to map the sgRNA ytg transgene to a specific location on the chromosome. A detailed overview and purpose of each step is outlined in this document (below table). You should read this overview in a lab-by-lab manner along with the lecture and other materials for a particular lab. You are welcome to read the entire document at any point, keeping in mind that additional details will be discussed in the lably lecture and readings. sgRNA transgene Mapping Summary timetable Lab 1 --- Lab 2 Set up Generation I of Cross Schemes 5 and 6 Lab 3 Clear Generation I parents of Cross Schemes 5 and 6 Lab 4 Set up Generation II of Cross Schemes 5 and 6 Lab 5 Clear Generation II parents of Cross Schemes 5 and 6 Lab 6 Examine Gen II Progeny analyze results to determine which chromosome the sgRNA transgene was inserted into. Design 3 pt cross as part of Assignment 6. Lab 7 Set up Gen I of 3-point Cross Lab 8 Clear Parents from Gen I of 3-point Cross Start Collection of Virgin Females (for 3 pt Cross Gen II) Lab 9 Set up Generation II of 3-point cross Lab 10 Clear parents from Generation II of 3-point cross Lab 11 Analyze results of sgRNA transgene 3-point mapping cross Lab 12 sgRNA Mapping Report Due by 2:40 pm your lab day

BioMG2801 Lab 2: Set up Generation I of the crosses to begin the sgRNA transgene mapping project. See the Lab 2 Lab manual for detailed protocols. In Lab 2 you will set up generation I of Cross Scheme 5 and Cross Scheme 6: • In generation I of cross 5, sgRNA ytg stock males are crossed to stock 103 females. Stock 103 contains a chromosome 2 marker, Curly ( Cy) , allowing us to track the second chromosome. Examination of the phenotypes present in the final generation of this cross scheme will allow us to determine whether the sgRNA ytg transgene has been inserted into chromosome 2. Cross Scheme 5 is diagrammed on the next page (Figure 1). • In generation I of cross 6, sgRNA ytg transgene males are crossed to stock 107 females. Stock 107 has a chromosome 3 marker, Drop ( Dr), allowing us to track the third chromosome. Examination of the phenotypes present in the final generation of this cross scheme will allow us to determine whether the sgRNA ytg transgene has been inserted into chromosome 3. Cross Scheme 6 is diagrammed on the next page (Figure 1). • The sgRNA ytg transgene has no phenotype of its own, so you need track the sgRNA ytg transgene in your crosses by using a closely linked phenotypic marker gene. You will track the sgRNA ytg transgene by following the closely linked y+ transgene. Flies that have wildtype body color (y+) have the sgRNA ytg transgene. Flies that have yellow body color (y) do not have the sgRNA ytg transgene. You could also follow the sgRNA ytg transgene by following the closely linked v+ transgene, but we will not do this simply because wildtype eye color and vermilion eye color are very hard to tell apart. • The results of Cross Schemes 5 and 6 should agree with each other. That is, if Cross Scheme 5 indicates the sgRNA ytg transgene IS on chromosome 2, Cross Scheme 6 should indicate the sgRNA ytg transgene is NOT on chromosome 3 (and vice versa). • Notes about Cross schemes 5 and 6 diagrammed in Figure 1 on next page: o The cross schemes are written in shorthand and only indicate the progeny type(s) that will be used in the next generation (not all progeny types are shown). You should diagram out these crosses yourself, making sure you understand all progeny that will be produced depending on if the sgRNA ytg transgene is located on chromosome 2 or 3. (You will be asked to diagram out the generation I progeny of Cross 6 as part of Assignment 3.) o In the second generation, two different genotypes are written for the male parent. The first genotype written places the sgRNA ytg transgene on chromosome 2, while the second genotype written places the sgRNA ytg transgene on chromosome 3. Both genotypes are written since you do not yet know the location of the sgRNA ytg transgene Remember, that is the purpose of these crosses!

BioMG2801 Lab 4: Set up Generation II. See the Lab 4 Lab manual for detailed protocols. Lab 4 Cross Scheme 5 and 6 Generation II crosses: In Lab 4 you will continue Cross Schemes 5 and 6 to set up Generation II. Setting up Generation II of Cross 5: You will select the Curly winged male progeny from generation I (discarding the Glazed progeny). See Figure 2 on the next page. These male flies are heterozygous for your sgRNA ytg transgene (which you are following with the linked y + transgene) and a chromosome 2 marked with the dominant Cy allele. To set generation II you will cross these males to stock 112 females which are homozygous for the yellow and vermilion mutations . In the generation II progeny (observed in lab 6), you will analyze the progeny to determine if the sgRNA ytg transgene (followed using y+ ) assorts independently (or not) of the chromosome 2 (followed using Cy ). The key to this cross is the fact that male Drosophila do not have recombination during meiosis. If the sgRNA ytg transgene is located on chromosome 2 only the 2 parental phenotypes will occur: (1) wildtype body color with wildtype wing and (2) yellow body color with Curly wings. This is because the two homologs of chromosome 2 segregate into different gametes. Alternatively, if the sgRNA ytg transgene is located on chromosome 3, the transgene will assort independently of chromosome 2 and 4 types of progeny will be seen: (1) wildtype body color with wildtype wings; (2) wildtype body color with Curly wings; (3) yellow body color with wildtype wings; and (4) yellow body color with Curly wings. Setting up Generation II of Cross 6: You will select the Drop eyed male progeny from generation I (discarding the Stubble progeny). See Figure 2 on the next page. These male flies are heterozygous for your sgRNA ytg transgene (which you are following with the linked y + transgene) and for a chromosome 3 marked with the dominant Dr allele. To set generation II you will cross these males to stock 112 females which are homozygous for the yellow and vermilion mutations . In the generation II progeny (observed in lab 6), you will analyze the progeny to determine if the sgRNA ytg transgene (followed using y+ ) assorts independently (or not) of the chromosome 3 (followed using Dr ). The key to this cross is the fact that male Drosophila do not have recombination during meiosis. If the sgRNA ytg transgene is located on chromosome 3 only the 2 parental phenotypes will occur: (1) wildtype body color with wildtype eyes and (2) yellow body color with Drop eye shape. This is because the two homologs of chromosome 3 segregate into different gametes. Alternatively, if the sgRNA ytg transgene is located on chromosome 2, the transgene will assort independently of chromosome 3 and 4 types of progeny will be seen: (1) wildtype body color with wildtype eyes; (2) wildtype body color with Drop eye shape; (3) yellow body color with wildtype eyes; and (4) yellow body color with Drop eye shape. You should diagram out these crosses yourself (through the second generation), making sure you understand all progeny that will be produced depending on if the sgRNA ytg transgene is located on chromosome 2 or 3. You will need understand the expected patterns in order to analyze your results in lab 6.

BioMG2801 Setting up Generation II of Cross Scheme 5 (Cross 5-Cy): Lab 2 Generation I Cross 5: sgRNA your target gene ♂ y v / Y ; {[ y + attP][ v + sgRNA ytg ]} ⨉ 103 ♀ y v ; CyO, Cy / Gla (supplied) (supplied) ⬇ Lab 4 Generation II Cross 5-Cy: ♂ y v / Y ; [ y + attP][ v + sgRNA ytg ]/CyO, Cy OR ♂ y v / Y ; + / CyO, Cy ; [ y + attP][ v + sgRNA ytg ]/ + ⨉ 112 ♀ y v (supplied) ⬇ Lab 6 Generation III: Analyze various progeny Setting up Generation II of Cross Scheme 6 (Cross 6-Dr): Lab 2 Generation I Cross 6: sgRNA your target gene ♂ y v / Y ; {[ y + attP][ v + sgRNA ytg ]} ⨉ 107 ♀ y v ; Dr / TM6B, Sb Hu (supplied) (supplied) ⬇ Lab 4 Generation II Cross 6-Dr: ♂ y v / Y ; [ y + attP][ v + sgRNA ytg ] / + ; Dr / + OR ♂ y v / Y ; [ y + attP][ v + sgRNA ytg ] / Dr ⨉ 112 ♀ y v (supplied) ⬇ Lab 6 Generation III: Analyze various progeny Figure 2. Cross Schemes 5 and 6. In lab 4, you will select Curly winged male progeny to set up generation II of Cross Scheme 5 Drop eyed male progeny to set up generation II of Cross Scheme 6. Notes: (1) The crosses schemes only indicate the progeny type(s) that will be used in generation II (not all progeny types are shown). (2) In generation II, two different genotypes are written for the male parent. The first genotype written places the sgRNA ytg transgene on chromosome 2, while the second genotype written places the sgRNA ytg transgene on chromosome 3. Both genotypes are written since you do not yet know the location of the sgRNA ytg transgene. Remember, that is the purpose of these crosses!

BioMG2801 Cross Scheme 5 Lab 2 Generation I Cross 5: sgRNA your target gene ♂ y v / Y ; {[ y + attP][ v + sgRNA ytg ]} ⨉ 103 ♀ y v ; CyO, Cy / Gla (supplied) (supplied) ⬇ Lab 4 Generation II Cross 5-Cy: ♂ y v / Y ; [ y + attP][ v + sgRNA ytg ]/CyO, Cy OR ♂ y v / Y ; + / CyO, Cy ; [ y + attP][ v + sgRNA ytg ]/ + ⨉ 112 ♀ y v (supplied) ⬇ Lab 6 Generation III: Analyze various progeny to determine if sgRNA ytg transgene is located on chromosome 2 Lab 2 Generation I Cross 6: sgRNA your target gene ♂ y v / Y ; {[ y + attP][ v + sgRNA ytg ]} ⨉ 107 ♀ y v ; Dr / TM6B, Sb Hu (supplied) (supplied) ⬇ Lab 4 Generation II Cross 6-Dr: ♂ y v / Y ; [ y + attP][ v + sgRNA ytg ] / + ; Dr / + OR ♂ y v / Y ; [ y + attP][ v + sgRNA ytg ] / Dr ⨉ 112 ♀ y v (supplied) ⬇ Lab 6 Generation III: Analyze various progeny to determine if sgRNA ytg transgene is located on chromosome 3 Figure 3. Cross Schemes 5 and 6. In lab 6, you will analyze the progeny to determine if the sgRNA ytg transgene is on chromosome 2 or 3. Lab 7: 3-point Cross Generation I. See the Lab 7 Lab manual for detailed protocols. Using Cross Schemes 5 and 6, you previously determined on which chromosome the sgRNA transgene was inserted. You then designed a 3-point cross series (as part of Assignment 6) which will utilize recombination mapping to localize this sgRNA transgene more precisely, relative to two known markers on that chromosome. You will carry out Generation I of this recombination mapping cross scheme this lab.

BioMG2801 Lab 8: 3-point Cross: Clear parents of Generation I. Begin to collect virgin females in order to set up generation II. See the Lab 8 Lab manual for detailed protocols. In Lab 8 you will clear the parents from generation I of the 3-point cross you set up last lab. The parents must be cleared as we will be collecting virgin female progeny from this cross to set up generation II (we do not want to confuse parental flies with progeny flies). To set up generation II of the 3-point cross series it is necessary to collect virgin females. Recall that (i) After copulation, Drosophila females can store sperm for several labs; and (ii) you want to ensure that the progeny of these females are fathered by the proper males for generation II of your 3-point cross. If the females are not virgin, they will have mated with one of their brothers, rather than the male you require her to mate with for mapping purposes. You will spend this lab collecting virgin females of the correct genotypes and then cross these females to the appropriate males in lab 9. Lab 9: 3-point Cross: Set up Generation II. See the Lab 9 Lab manual for detailed protocols. In lab 9 you will set up generation II of the 3-point mapping cross. You will cross your collected virgin females to the appropriate males. Lab 10: 3-point Cross: Clear Generation II parents. This lab the parents from generation II of the 3-point cross will be cleared for you. Clearing the parents is crucial as you will be analyzing the generation II progeny phenotypes next lab and do not want the parents included in your data. Lab 11: 3-point Cross: Score Generation II progeny. See the Lab 10 Lab manual for detailed protocols. In lab 11 you will score the 3-point cross generation II progeny. The particular patterns you see in the data will allow you to map the sgRNA ytg transgene with respect to two known genes on that chromosome. You can read more about how to analyze your 3-point cross data in the “sgRNA mapping: 3-point cross analysis” assigned reading on Canvas. Please come to office hours with any questions you may have on your 3-point analysis. Lab 12: sgRNA ytg transgene mapping report due . Your 3-point cross sgRNA ytg transgene mapping report is due by 2:40 pm on your lab day this lab. You can read more about how to analyze your 3-point cross data in the “sgRNA mapping: 3-point cross analysis” assigned reading on Canvas. Please come to office hours with any questions you have on your report.